THE 
MICROTOMIST’S VADE-MECUM First Edition . . March, 1885. 
Second Do. . . April, 1890. 
Third Do. . . September, 1893. 
Fourth Do. . . September, 1896. THE 
MICROTOMIST’S VADE-MECUM 
A HANDBOOK OF THE METHODS OF 
MICROSCOPIC ANATOMY 
BY 
ARTHUR BOLLES LEE 
FOURTH EDITION 
PHILADELPHIA 
P. BLAKISTON, SON & 00. 
1012, WALNUT STREET 
1896 PREFACE 
The short period of three years that has elapsed since the 
publication of the last edition of this work has not brought 
with it any radical change in the methods of histological 
research. Such progress as has been realised has consisted 
rather in improvements in the detail of already-established 
methods than in the introduction of new methods or new 
reagents. Nevertheless, the present edition has undergone 
a most thorough revision—a revision indeed so thorough as 
to amount to extensive re-writing in many parts. 
It has seemed to me advisable in the interest of the be- 
ginner, and indeed in the interest of readers who are not 
beginners at all, to enter more fully than was hitherto done 
into the detail of the more important processes, to explain 
more fully the principles on which they are founded, and to 
add in many cases a critical estimate of their rationality and 
practical value. In consequence of this re-writing, and in 
spite of strenuous efforts to keep down the bulk of the work, 
it has turned out to be considerably increased. I regret, 
however, this increase the less, in so far as it is due rather to 
the ampler treatment that has been accorded to the more 
valuable methods than to increase in the number of processes 
described. The number of new processes described is in fact 
a smaller one than I have had to deal with in the preparation 
of any edition since the first. 
The classification of the various methods has received most 
careful attention, and has been in many cases greatly simpli- 
fied, whilst at the same time a large number of superfluous vi 
PREFACE 
processes have been rejected. Advice to the beginner con- 
cerning the choice of methods has been given wherever 
practicable, and I think that notwithstanding the abundance 
and complexity of the matters treated of, there can hardly be 
any risk that the student may be unable to see the wood for 
the trees. 
The chapters treating of Staining and of the Carmine and 
Haematei'n stains have had the great advantage of revision 
by Dr. Paul Mayer, who, it is superfluous to remind the 
reader, has made a speciality of this subject, with results 
brilliant alike in theory and in practice. Not indeed that the 
present English text has been directly revised by him, but 
that it has been prepared from a recent text so revised. 
Dr. Mayer was good enough to revise most carefully the 
three corresponding chapters prepared by me for the recent 
new edition of the Traite des Methodes Techniques de I’Ana- 
tomie Microscopique (Lee et Henneguy), and in the prepara- 
tion of the present English text I have closely followed the 
chapters so revised. 
No less obligation have I to express to Professor van 
Gehuchten, who with great kindness has thoroughly revised 
for me the three chapters entitled Neurological Methods. It 
occurred to me that my treatment of this complicated subject 
could not but gain greatly by the advice of an observer who 
is not only one of the foremost of the new school of neuro' 
logists but at the same time an instructed and capable 
cytologist, and therefore likely to sympathise with my feeling 
that it would be much to be deplored that the study of 
nervous anatomy should degenerate into a mere study of 
topographical relations, to the neglect of the inner mechanism 
of nervous elements. By Professor van Gehuchten’s advice 
I have entirely re-arranged the contents of these three 
chapters according to a scheme worked out by him, thereby 
effecting a great gain in cleaxmess of exposition. I cannot 
but acknowledge that the arrangement adopted in previous 
editions resulted in something like a chaos ; whilst the new PREFACE 
VII 
arrangement may, I think, fairly claim to be natural, logical, 
and easily comprehensible. By his advice too I have entirely 
re-written the account of the bichromate of silver impregna- 
tions of Golgi; the account as it now stands is, I believe, 
the only complete one that has appealed in the English 
language. 
I am under the greatest obligation to Professor van 
Gehuchten, as well as to Dr. Paul Mayer, for the generous 
assistance which enables me to affirm that the important 
subjects in question have been treated with all the requisite 
accuracy and thoroughness. 
The essential feature of the first edition was that it was an 
altogether exhaustive collection of all the methods of pre- 
paration that had up to that time been recommended as 
useful for the purposes of microscopic anatomy, and its 
primary intention that of being a work of reference for the 
instructed anatomist. Its character of a guide to the be- 
ginner was secondary only. It contained indeed a general 
introduction and much explanatory matter in the different 
chapters, but, on the whole, the didactic matter bore but an 
insufficient proportion to the historical matter. This has now 
been rectified. It has come to pass that during the repeated 
operations of revision to which the book has been subjected, 
the explanatory and didactic element has been continually 
increasing, whilst at the same time the historical element 
has been continually diminishing—diminishing, that is, in all 
parts of the book relatively to the former element, and in 
some parts absolutely (as may be seen, for instance, by com- 
paring the number of formulae given in the chapters on 
carmine and haematoxylin with the number given in former 
editions). On the one hand the book has been lightened by 
the jettison of much useless matter, and on the other hand 
there has been accorded to the matter that has been retained 
a far ampler share than before of explanation and detail. 
To such an extent, indeed, have the instructions to students 
and other explanatory matter been amplified that I am not viii 
PREFACE 
acquainted with any modern work on the subject that con- 
tains anything like so complete an account of the various 
fundamental operations of histological technique—fixing, im- 
bedding, staining, and the like. I only felt justified in 
claiming for the first edition that it “ went far to make up 
a formal treatise on the art.” Through the changes above- 
mentioned the book has come to assume altogether the 
character of a formal treatise, and now contains in due pro- 
portions both the grammar and the dictionary of the art. 
The rejection of superfluous matter above referred to re- 
lates chiefly to old methods that have been before the public 
for so long a time that there can be no doubt that they have 
no good claim to further survival, whilst recent methods, 
which may be considered to be still on their probation, have 
been treated with the accustomed fulness. 
Nyon, Switzeeland; 
September, 1896. CONTENTS 
PART i 
CHAPTER I. page 
Introductory.—The General Method . . . . .1 
CHAPTER II. 
Killing . . . . . . . .11 
CHAPTER III. 
Fixing and Hardening . . . . . .18 
CHAPTER IY. 
Fixing Agents : Mineral Acids and their Salts . . .21 
CHAPTER V. 
Fixing Agents : Chlorides, Organic Acids, and others . . 38 
CHAPTER YI. 
Hardening Agents . . . . . . .56 
CHAPTER VII. 
Clearing Agents . . . . . . .64 
CHAPTER VIII. 
Imbedding Methods.—Introduction . . . .71 
CHAPTER IX. 
Imbedding Methods : Paraffin and other Fusion Masses . 79 
Paraffin, 79 ; Soap, 94 ; Gelatin, 97. X 
CONTENTS. 
CHAPTER X. page 
Collodion (Celloidin) and other Imbedding Methods . . 99 
Collodion or Celloidin, 99 ; other Evaporation Masses, 113; 
Freezing Masses, 115. 
CHAPTER XI. 
Serial Section Mounting ...... 119 
Methods for Paraffin Sections, 119 ; Methods for Watery Sections, 
128; Methods for Celloidin Sections, 128. 
CHAPTER XIL 
Staining ........ 135 
CHAPTER XIII. 
Carmine and Cochineal Stains ..... 145 
Theory of Carmine Staining, 145 ; Aqueous Carmines, Acid, 149 ; 
Neutral and Alkaline, 153 ; Alcoholic Carmines and Cochineals, 
156. 
CHAPTER XIY. 
Hematein (Hematoxylin) Stains ..... 161 
Theory of Staining with Hsematoxylin, 161; Hmmatein (Hsema- 
toxylin), 163 ; Alumina-hsematein Lakes, 167; other Hsema- 
te’in Lakes, 173. 
CHAPTER XY. 
On Staining with Coal-tar Colours .... 179 
Basic, Acid and Neutral Coal-tar Colours, 179 ; Progressive and 
Regressive Stains, 181; General Directions for the Regressive 
Method, 182. 
CHAPTER XVI. 
The Coal-tar Chromatin Stains ..... 189 
Regressive Stains, 189; Progressive Stains, 197. 
CHAPTER XVII. 
Methylen Blue ....... 202 
CHAPTER XVIII. 
The Coal-tar Plasma Stains ...... 215 CONTENTS. 
xi 
CHAPTER XIX. page 
Otheb Oeganic Stains and Combinations .... 233 
Other Organic Stains, 233 ; Carmine combinations, 235 : Hsema- 
te'in Combinations, 237. 
CHAPTER XX. 
Metallic Stains (Impbegnation Methods) .... 241 
Silver, 243; Gold, 250; other Metallic Stains, 258. 
CHAPTER XXI. 
Examination and Peeseevation Media .... 262 
Aqueous Liquids, 262; Mercurial Liquids, 266; other Fluids, 
267 ; Glycerin Media, 270; Resinous Media, 277. 
CHAPTER XXII. 
Cements and Vabnishes ...... 284 
PART II 
SPECIAL METHODS AND EXAMPLES. 
CHAPTER XXIII. 
Injections : Gelatin Masses ..... 293 
Carmine, 296 ; Blue, 300 ; other Colours, 304. 
CHAPTER XXIY. 
Injections : othee Masses ...... 307 
White of Egg, Gum, 307; Glycerin, 308; Aqueous, 309 ; Cel- 
loidin, 310; other Masses, 310. 
CHAPTER XXY. 
Macebation and Digestion ...... 312 
Maceration, 312 ; Digestion, 320. 
CHAPTER XXVI. 
Cokbosion, Decalcification, and Bleaching . . . 322 
Corrosion, 322 ; Decalcification and Desilicification, 323; Bleach- 
ing, 328. XII 
CONTENTS. 
CHAPTER XXVII. page 
Embryological Methods ...... 331 
CHAPTER XXVIII. 
Cytological Methods ...... 354 
CHAPTER XXIX. 
Tegumentary Organs . . . . . .370 
CHAPTER XXX. 
Muscle and Tendon (Nerve-endings) .... 378 
Striated Muscle, 378 ; Tendon, 381 ; Smooth Muscle, 382. 
CHAPTER XXXI. 
Neurological Methods.—Introduction and Section Methods . 385 
CHAPTER XXXII. 
Neurological Methods. —Nerve-fibre Stains (Weigert and others) 405 
CHAPTER XXXIII. 
Neurological Methods.—Axis-cylinder and Protoplasm Stains 
(Golgi and others) . . . . . . 415 
Retina, 439; Inner Ear, 442. 
CHAPTER XXXIV. 
Some other Histological Methods ..... 444 
Connective Tissues, 444; Blood, 456 ; Glands, 461. 
CHAPTER XXXV. 
Some Zoological Methods ...... 466 
Tunicata, 467; Mollusca, 468; Arthropoda, 472 ; Vermes, 476 ; 
Echinodermata, 485 ; Coelenterata, 488 ; Porifera, 493 ; Pro- 
tozoa, 495. 
Appendix ........ 501 
Index ......... 507 THE MICROTOMIST’S VADE-MECUM. 
CHAPTER I. 
INTRODUCTORY. 
1. The General Method.—The methods of modern microscopic 
anatomy may be roughly classed as General and Special. 
There is a General or Normal method, known as the method 
of sections, which consists in carefully fixing the structures to 
be examined, staining them with a nuclear stain, dehydrating 
with alcohol, and mounting series of sections of the structures 
in balsam. It is by this method that the work is blocked-out 
and very often finished. Special points are then studied, if 
necessary, by Special Methods, such as examination of the 
living tissue elements, in situ, or in “ indifferent ” media; 
fixation with special fixing agents; staining with special 
stains; dissociation by teasing or maceration; injection; 
impregnation ; and the like. 
There is a further distinction which may be made, and 
which may help to simplify matters. The processes of the 
preparation of tissues may be divided into two stages, Pre- 
liminary Preparation and Ulterior Preparation. Now the pro- 
cesses of preliminary preparation are essentially identical in 
all the methods ; essential divergencies being only found in 
the details of ulterior preparation. By preliminary prepara- 
tion is meant that group of processes called by German 
anatomists Conservirungsmethoden, those namely whose object 
it is to get the tissues into a ht state for passing unharmed 
through all the ulterior processes to which it may be desired 
to submit them. Preliminary preparation comprehends the 
operations of (1) killing; (2) fixing; (3) the washing and 
other manipulations necessary for removing the fixing agent 2 
CHAPTER I. 
from the tissues, and substituting for it the preservative liquid 
or other reagents which it is desired to employ. 
2. Preliminary Preparation.—The first thing to be done with 
any structure is to fix its histological elements. (This state- 
ment applies equally to all classes of objects, whether it be 
desired to cut them into sections or to treat them in any other 
special way.) Two things are implied by the word “ fixing; ” 
first, the rapid hilling of the element, so that it may not have 
time to change the form it had during life, but is fixed in 
death in the attitude it normally had during life ; and second, 
the hardening of it to such a degree as may enable it to resist 
without further change of form the action of the reagents 
with which it may subsequently be treated. Too much stress 
can hardly be laid on this point, which is the most distinctive 
feature of modern histological practice; without good fixation 
it is impossible to get good stains, or good sections, or prepa- 
rations good in any way. 
The structure having been duly fixed by one of the pro- 
cesses described in the chapter on Fixing Agents, is washed 
in order to remove from the tissues as far as possible all traces 
of the fixing reagent. 
The kind of liquid with which washing out is done is not a matter of in- 
difference. If corrosive sublimate (for instance) or osmic acid, or a solution 
into which chromic acid or a chromate enters, have been used for fixing, the 
washing may be done with water. But if picric acid in any form has been 
used, the washing must be done with alcohol. The reason of this difference 
is that the first-named reagents (and, indeed, all the compounds of the heavy 
metals used for fixing) appear to enter into a state of chemical combination 
with the elements of tissues, rendering them insoluble in water; so that the 
hardening induced by these agents is not removed by subsequent treatment 
with water. Picric acid, on the other hand, produces only a very slight 
hardening of the tissues, and does not appear to enter into any combination 
whatever with their elements, as it is entirely removeable by treating the 
tissues with water or alcohol. If the removal be effected by means of water, 
the tissue elements are left in a soft state in which they are obnoxious to all 
the hurtful effects of water. Alcohol must therefore be taken to remove the 
picric acid and to effect the necessary hardening at the same time. Instruc- 
tions for washing out are given, when necessary, in the discussion of the 
different fixing agents in the following parts of this work. 
These operations having- been dnlv performed, two roads 
become open. The object may be further prepared by what 
may be termed the wet method, in which all subsequent INTRODUCTORY. 
3 
operations are performed by means of aqueous media. Or it 
may be further prepared by what may be termed the Dehydra- 
tion method, which consists in treatment with successive 
alcohols of gradually increasing strength, final Dehydration 
with absolute alcohol, imbibition with an essential oil or other 
clearing agent, and lastly either mounting at once in balsam 
or other resinous medium or imbedding in paraffin for the 
purpose of making sections. The dehydration method is the 
course which, is generally preferred. The chief reason for this 
lies in the great superiority of the dehydration methods as 
regards the preservation of tissues. The presence of water is 
the most important factor in the conditions that bring about 
the decomposition of organic matter, and its complete removal 
is the chief condition of permanent preservation. It is of 
course not intended here to suggest that wet methods of pre- 
paration should be altogether discarded. They have great 
value, they are even indispensable, for special ends; and all 
that is intended to be suggested is, that they should be re- 
garded not as general but as special methods. 
The further course of preliminary preparation by the de- 
hydration method is as follows : 
3. Dehydration and Preservation.—At the same time that the 
superfluous fixing agent is being removed from the tissues, or 
as soon as that is done, the water of the tissues must be removed. 
This is necessary for two reasons ; firstly, in the interest of 
preservation, the presence of water being the condition of all 
others that most favours post-mortem decomposition; and 
secondly, because all water must be removed in order to allow 
the tissues to be impregnated with the imbedding material 
necessary for section-cutting, or with the balsam with which 
they are to be finally preserved. (The cases in which aqueous 
imbedding and preserving media are employed are exceptional, 
and will be treated of in the proper places.) The dehydration 
is performed as follows :—The objects are brought into weak 
alcohol, and are then passed through successive alcohols of 
gradually increased strength, remaining in each the time 
necessary for complete saturation, and the last bath consist- 
ing of absolute or at least very strong alcohol. 
In dealing with extremely delicate objects, it may be necessary to take 
special precautions in order to avoid injury to them through the violent 4 
CHAPTER I. 
diffusion-currents that are set up in the passage from water to alcohol, or 
from one bath of alcohol to another of considerably different density. Some 
kind of diffusion-apparatus should he used in these cases. The objects may 
be placed in a tube plugged at one end and closed at the other by a 
diaphragm of chamois skin or other suitable membrane, the tube being 
then immersed in a vessel containing the grade of alcohol that it is desired 
to add to the liquid in the tube, and the whole allowed to remain until by 
diffusion through the diaphragm the two liquids have become of equal 
density. Or Cobb’s differentiator (Proc. Linn. Soc., N.S.W., v, 1890, 
p. 157; Journ. Boy. Mic. Soc., 1890, p. 821) may be employed. Or, more 
conveniently in most cases, the apparatus described and figured by Haswell 
{Proc. Linn. Soc., N.S.W., vi, 1891, p. 433 ; Journ. Boy. Mic. Soc., 
1892, p. 696). This consists of two wash-bottles connected in the usual way 
by tubing, and furnished, the one with an overflow-tube, and the other with a 
feeding-tube leading from a reservoir connected with it by means of a 
regulating tap or drop arrangement. The objects are placed in the first 
bottle ; some of the same liquid as that containing the objects is placed in 
the second bottle ; and alcohol of the grade that it is desired to add is led 
into it from the reservoir. The mixture of liquids therefore takes place in 
the bottle that does not contain the objects, and the mixture itself is 
gradually led over to the objects through the siphon-tube connecting the 
two bottles. Another apparatus for rapid dehydration, devised by Cheatle, 
will be found described in Journ. Patliol. and Bacteriol., i, 1892, p. 253, 
or Journ. Boy. Mic. Soc., 1892, p. 892. 
I would here call attention to the varied usefulness of the “ Siebdosen ” 
or sieve-dishes of Steinach, Zimmekmann, and Suchannek (vide Zeit.f. 
iviss. Mile., iv, 4, 1887, p. 433, and vii, 2, 1890, p. 159). They consist of a 
covered glass capsule into which is fitted a “ sieve ” made of a watch-glass 
pierced with holes and supported on legs. It is evident that the arrange- 
ment is very handy, not only for staining, washing out, treatment with 
vapours, &c., but for any operation in which it is desirable to have speci- 
mens supported in the upper layers of a quantity of reagent. They are sent 
out in a very neat form by Dr. Griibler. Faiechild’s perforated porcelain 
cylinders for washing seem to be a very neat idea. These are made small 
enough to be floated by the cork that closes them. See the descriptive 
paper in Zeit.f. wiss. Mile., xii, 3, 1896, p. 301. 
It is sometimes stated that it is necessary that the last alcohol bath 
should consist of absolute alcohol. This however is incorrect, a strength of 
90 per cent., or at all events 95 per cent., being sufficient in almost all cases. 
For the small amount of water that remains in the tissues after treatment 
with these grades of alcohol is efficiently removed in the bath of clearing 
agent, if a good clearing agent be employed. Oil of cedar will remove the 
remaining water from tissues saturated with 95 per cent, alcohol; oil of 
bergamot will “ clear ” from 90 per cent, alcohol, and anilin oil will clear 
from 70 per cent, alcohol. 
It is not generally necessary that pure ethylic alcohol be employed for 
dehydration ; methylated spirit will suffice in most cases. 
I am not aware of any substance that can entirely take the place of 
alcohol for dehydration and preservation. Acetone, and methylal, have been INTRODUCTORY. 
5 
lately (Parker, Zool. Anz., 403, 1892, p.376) substituted for alcohol in the 
dehyhration of methylen-blue preparations; but the great boon of an efficient 
substitute for alcohol in general work remains yet to be discovered. For- 
maldehyde (see under Fixing and Hardening Agents) is now known to be a 
most admirable medium for the preservation of museum specimens, being 
for that purpose in many cases greatly superior to alcohol; but experience 
is wanting as to how far it is available for the preservation of histological 
material, whilst of course, occurring as it does in the form of an aqueous 
solution, it can have no dehydrating effect. 
Considered as a mere dehydrating agent, alcohol fulfils its functions fairly 
well. But considered as a histological preservative agent, it is far less 
satisfactory. If tissues be left in alcohol for only a few days before sub- 
jecting them to the further stages of preparation, the injurious effects of a 
sojourn in alcohol will perhaps not be very disagreeably evident. But it is 
otherwise if, as must often be the case, they are put away for many weeks 
or months before the final preparation can be carried out. The dehydrating 
action of the alcohol being continuously prolonged, the minute structure of 
tissues is sometimes considerably altered by it; they become overhard and 
shrink, and become brittle, and their capacity for taking stains well becomes 
seriously diminished. Kultschitzky (Zeit. f. wiss. Mik., iv, 3, 1887, 
p. 349) has proposed to remedy this by putting up objects, after fixation and 
washing out with alcohol, in ether, xylol, or toluol. Flemming has lately 
(Arch./, mile. Anat., xxxvii, 1891, p. 685) advised putting up objects after 
fixation in a mixture of alcohol, glycerin, and water, in about equal parts, 
pointing out that objects thus preserved may be at any moment either pre- 
pared for sectioning by treatment with pure alcohol, or softened for dissec- 
tion or teasing by a little soaking in water, and that they do not become so 
hard and brittle as alcohol specimens, and retain their staining power much 
better. After extensive experience of this plan, I can highly recommend it, 
and would only further suggest that the action of the liquid seems to me to 
be in many cases much improved by addition of a little acetic acid (say 05 
to 075 per cent.). 
For material that is intended only for section-cutting, I 
find that by far the best plan is to clear and imbed at once in 
paraffin. This affords, as far as I can see, an absolutely 
perfect preservation. I have been working lately on some 
material that has been preserved in this way for over seven 
years. The preservation of the tissues, down to the finest 
details of cell-structure, appears to be perfect, and the stain- 
ing, though perhaps somewhat slowed, is as precise as when 
the specimens were first put up, or more so. The only notice- 
able defect is that the tissues are rather brittle, and do not 
cut well; but it is not certain that that is not owing to their 
having been over-hardened in the first instance. Cedar-wood 
oil is, I find, nearly, if not quite, as good as paraffin. 6 
CHAPTER I. 
4. Ulterior Preparation. Clearing.—The water having been 
thus sufficiently removed, the dehydration method proceeds 
as follows :—The alcohol is in its turn removed from the 
tissues, and its place taken by some anhydrous substance, 
generally an essential oil, which is miscible with the material 
used for imbedding. This operation is known as Clearing. 
It is very important that the passage from the last alcohol to 
the clearing agent be made gradual. This is effected by 
placing the clearing medium under the alcohol. A sufficient 
quantity of alcohol is placed in a tube (a watch-glass will do, 
but tubes are generally better), and then with a pipette a 
sufficient quantity of cleariug medium is introduced at the 
bottom of the alcohol. Or you may first put the clearing 
medium into the tube, and then carefully pour the alcohol on 
to the top of it. The two fluids mingle but slowly. The 
objects to be cleared being now quietly put into the super- 
natant alcohol, float at the surface of separation of the two 
fluids, the exchange of fluids takes place gradually, and the 
objects slowly sink down into the lower layer. When they 
have sunk to the bottom, the alcohol may be drawn off with 
a pipette, and the objects will be found to be completely 
penetrated by the clearing medium. (It may be noted here 
that this method of making the passage from one fluid to 
another applies to all cases in which objects have to be trans- 
ferred from a lighter to a denser fluid—for instance, from 
alcohol, or from water to glycerine. It is a more exact method 
than that of successive baths of mixture of alcohol and clear- 
ing agent.) 
It should also be noted here that this is the proper stage 
for carrying out minute dissections, if any such have to be 
done, a drop of clearing medium agent being a most helpful 
medium for carrying out such dissections in, as will be further 
explained later on (§ 8). 
5. Imbedding, and Treatment of Sections.—The objects are 
now imbedded. They are removed from the clearing medium, 
and soaked until thoroughly saturated in the imbedding 
medium. This is, for small objects, generally paraffin, 
liquefied by heat, and for large objects generally a solution 
of collodion or “ celloidin ” (in this latter case the clearing 
is omitted, the tissues beins’ imbedded direct from the INTRODUCTORY. 
7 
alcohol). The imbedding medium containing the object is 
then made to solidify, as described in the chapter on im- 
bedding processes, and sections are made with a microtome 
through the imbedding mass and the included objects. The 
sections are then mounted on a slide by one of the methods 
described in the chapter on Serial Section Methods, the 
imbedding material is removed from them (in the case of 
paraffin), they are stained in situ on the slide, dehydrated 
with alcohol, cleared and mounted in balsam or dam at*. Or 
they may be stained, washed, dehydrated, aud cleared in 
watch-glasses, and afterwards mounted as desired—the im- 
bedding medium being first removed if desirable. 
It is not always desirable to remove the imbedding mass; celloidin sec- 
tions stain well without being freed from it, and are usually even dehydrated, 
cleared, and mounted without removal of the mass, which becomes quite 
transparent in balsam. This plan has the advantage, which is a very im- 
portant one for large sections, of allowing the sections to remain during the 
whole of the manipulations protected by a supporting mass that holds all 
their parts together. 
The plan of staining sections on the side is of somewhat recent introduc- 
tion ; before it had been worked out the practice was to stain structures in 
toto, before cutting sections. In this case the object, after having been 
fixed, and washed out, is taken from the water, or while still on its way 
through the lower alcohols (it should not be allowed to proceed to the higher 
grades of alcohol before staining, if that can he avoided), and passed through 
a bath of stain (generally alcoholic borax-carmine or other alcoholic stain) of 
sufficient duration, then dehydrated with successive alcohols, passed through 
a clearing medium into paraffin, cut, and treated as above described, the 
sections m this case being mounted direct from the turpentine, naphtha, or 
other solvent with which the paraffin is removed. If aqueous staining 
media be applied (and it is sometimes very desirable for particular pur- 
poses to prepare specimens with some aqueous stain) the structures should 
either be stained in toto immediately after fixing and washing out, or 
sections may be stained on the slide, the objects being passed through suc- 
cessive baths of alcohol of gradually deci'easing strength before being put 
into the aqueous stain (a precaution which will not be necessary for chromic 
objects (see below, § 9) ). 
But it is generally advisable not to stain in bulk material that is intended 
to be sectioned; much time is saved by staining it as sections, with the 
further advantage that the staining can be much better controlled, and that 
many excellent stains can in this way be employed that are not available for 
staining in bulk. 
6. Resume of the Section-Method.—It was stated in the first 
edition of this work that “ the great majority of preparations 
are made by fixing either with sublimate or a picric acid com- 8 
CHAPTER I. 
bination, washing out with alcohol, staining with alcoholic 
borax-carmine, imbedding in chloroform paraffin, cutting with 
a sliding microtome, and mounting the sections in series in 
Canada balsam.” But histological practice has greatly 
changed since then, and I would now suggest the following : 
—Fix either in sublimate, or preferably in many cases in 
Flemming’s chromo-aceto-osmic mixture, or one of the other 
fixing agents recommended in later chapters; wash out; 
dehydrate; clear with oil of cedar-wood ; imbed in paraffin; 
mount sections on the slide with Mayer’s albumen medium; 
stain as desired ; and mount in balsam or damar. That, or 
something like that, is now the practice of many of the most 
advanced workers ; and I know of no method that seems to 
have equal claims to be considered a classical method of 
general morphological investigation. 
As regards the method to be used for imbedding, I take it that the paraffin 
method is the method par excellence for small objects (objects up to 5 or 7 
millimetres diameter); whilst the collodion or “cello'idin ” method is the 
method par excellence for large objects. 
As above said, I consider the practice of staining section material in bulk 
to be superannuated. 
7. Preparation of Entire Objects, or of Material that is not to 
be Sectioned.—The treatment of objects which can be studied 
without being cut into sections is identical with that above 
described, with the omission of those passages that relate to 
imbedding processes. Its normal course may be described 
as fixation, washing out, staining, treatment with successive 
alcohols of gradually increasing strength, final dehydration 
with absolute alcohol, clearing, and mounting in balsam. 
This method is usually preferred, as a general method, to the 
wet methods, for the reasons that have been given above 
(§ 2), and for some others, amongst which may be noted the 
greater transparency given to tissues by mounting them in 
media of high refractive index, such as balsam. 
In the 'preparation of entire objects or structures that are 
intact and covered by an integument not easily permeable by 
liquids, special care must be taken to avoid swelling from 
endosmosis on the passage of the objects from any of the 
liquids employed to a liquid of less density, or shrinkage from 
exosmosis on the passage to a liquid of greater density. This 
applies most specially to the passage from the last alcohol INTRODUCTORY. 
9 
into the clearing medium. A slit should be made in the 
integument, if possible, so that the two fluids may mingle 
without hindrance. And in all cases the passage is made 
gradual by placing the clearing medium under the alcohol, 
as above described. Fluids of high diffusibility should be 
employed as far as possible in all the processes. Fixing 
agents of great penetrating power (such as picrosulphuric acid 
or alcoholic sublimate solution) should be employed where 
the objects present a not easily permeable integument. 
Washing out is done with successive alcohols, water being 
used only in the case of fixation by osmic acid, or the chromic 
mixtures or other fixing solutions that render washing by 
water imperative. Staining is done by preference with 
alcoholic staining media. The stains most to be recommended 
are Grenadier’s borax-carmine, or one of Mayer’s new 
carminic acid or hacmatein stains (for all of which see Staining 
Agents). Anilin stains are rarely applicable to this class of 
preparations. Aqueous stains are more seldom used, though 
there are many cases in which they are admissible, and some 
in which they are preferable. 
8. Minute Dissections.—These are best done, if necessary, 
in a drop of clearing agent. I recommend cedar-wood oil for 
this purpose, as it gives to the tissues a consistency very 
favorable for dissection, whilst its viscosity serves to lend 
support to delicate structures. Clove oil has a tendency to 
make tissues that have lain in it for some time very brittle. 
The brittleness is, however, sometimes very helpful in minute 
dissections. Another property of clove oil is that it does not 
easily spread itself over the surface of a slide, but has a 
tendency to form very convex drops. This property also 
makes it frequently a very convenient medium for making 
minute dissections in. 
9. General Principles.—Following Paul Mayer, I gave in the previous 
editions the following reasons for employing for entire objects alcoholic 
rather than aqueous staining media. Since, in most cases, treatment with 
alcohol forms part of the fixing process, alcoholic solutions are logically 
indicated for staining. For by means of them it is possible to avoid the 
bad effects that follow on passing delicate tissues from alcohol into water, 
violent diffusive currents being thereby set up which sometimes carry away 
whole groups of cells; swellings being caused in the elements of the tissues; 
and, if the immersion in the aqueous medium be prolonged, as is generally 10 
CHAPTER I. 
necessary in order to obtain a thorough stain, maceration of the tissues 
supervening. But alcoholic staining fluids have still other advantages ; 
they are in general more penetrating; with them alone is it possible to 
stain through chitinous integuments ; and, if it be desired to stain slowly, 
tissues may be left in them for days without hurt. 
Applied to the case now under consideration, the preparation in toto of 
objects protected by not easity permeable investments, this doctrine is evi- 
dently a wise one. For such objects must necessarily be fixed by some highly 
penetrating but not permanently hardening agent such as picric acid, and 
must necessarily be washed out with alcohol; and it is a good maxim for 
tissues so fixed that an object that has once been in alcohol should not be 
allowed to go back into water, if that can possibly be avoided. 
But in the case of structures that have been well fixed in a strongly and 
permanently coagulating medium such as chromic acid, this precaution is 
much less necessary. Sections of tissues that have been fixed for twenty- 
four hours in Flemming’s solution may be passed with relative impunity 
from absolute alcohol into an aqueous stain, and from that back again direct 
into absolute alcohol. It is this property of tissues fixed in chromic solution 
that determines me to recommend the practice of staining sections, instead 
of staining objects in toto. As regards the quality of the stain, aqueous 
solutions are in general the best. No alcoholic carmine or hsematoxylin, for 
instance, will give a stain equal in precision and delicacy to those given by 
alum-carmine or Bohmer’s hematoxylin. In a recent publication Mayer 
himself states that he has somewhat cooled in his preference for alcoholic 
staining media. 
For an excellent exposition of the principles underlying the practice above 
recommended, the reader may consult with advantage the paper of Paul 
Mayer, in Mitth. Zool. 8tat. Neapel, ii (1881), p. 1, et seq. See also the 
abstract in Journ. Roy. Mic. Soc. (N.S), ii (1882), pp. 866—881, and that 
in Amer. Natural., xvi (1882), pp. 697—706, in which two last some im- 
provements are mentioned which have been worked out since the publication 
of Mayer’s paper. CHAPTER II. 
KILLING. 
10. In the majority of cases, the first step in the prepara- 
tion of an organ or organism consists in exposing it as rapidly 
and as completely as possible to the action of one of the 
Fixing Agents that are discussed in the next chapter. The 
organ or organism is thus taken in the normal living state ; 
the fixing agent serves to bring about at the same time, and 
with sufficient rapidity, both the death of the organism and 
that of its histological elements. 
But this method is by no means applicable to all cases. 
There are many animals, especially such as are of a soft con- 
sistence, and deprived of any rigid skeleton, but possessing a 
considerable faculty of contractility—such as many Coelen- 
terata, Bi-yozoa, and Serpulida, for instance—which if thus 
treated contract violently, draw in their tentacles or branchiae, 
and die in a state of contraction that renders the pre>erved 
object a mere caricature of the living animal. In these cases, 
special methods of killing must be resorted to. 
Sudden Killing. 
11. Heat. — Speaking generally, there are two ways of 
dealing with these difficult cases. You may kill the animal 
so suddenly that it has not time to contract; or you may 
paralyse it by narcotics before killing it. 
The application of Heat is a good means of killing suddenly. 
It has the great advantage of allowing of good staining sub- 
sequently, and of hindering less than any other method the 
application of chemical tests to the tissues. By it the tissues 
are fixed at the same time that somatic death is brought 
about. 
The difficulty consists in hitting off the right temperature, 12 
CHAPTER II. 
which is of course different for different objects. I think that 
a temperature of 80° to 90° C. will generally be amply suffi- 
cient, and that very frequently it will not be necessary to go 
beyond GO0 C. An exposure to heat for a few seconds will 
generally suffice. 
Small objects (Protozoa, Hydroids, Bryozoa) may be brought into a drop 
of water in a watch-glass or on a slide and heated over the flame of a spirit 
lamp. Por large objects, the water or other liquid employed as the vehicle 
of the heat may be heated beforehand and the animals thrown into it. 
As soon as it is supposed that the protoplasm of the tissues is coagulated 
throughout, the animals should be brought into alcohol (30 to 70 per cent, 
alcohol) (if water be employed as the heating agent). 
An excellent plan for preparing many marine animals is to kill them in 
hot fresh water. Some of the larger Nemertians are better preserved by 
this method than by any other with which I am acquainted. 
12. Slowly Contracting Animals. —Animals that contract but 
slowly, such A Icyoilium and Veretillum, and some Tunicates, 
such as Pyrosoma, are very well killed by throwing them into 
some very quickly acting fixing liquid, either used hot or cold. 
Glacial or very strong acetic acid (van Beneden’s method) is 
an excellent reagent for this purpose; it may be used, for 
example, with some Medusas. After an immersion of a few 
seconds or a few minutes, according to the size of the animals, 
they should be brought into alcohol of at least 50 per cent, 
strength. See “ Acetic acid ” and “ Tunicata.” Lemon juice 
employed in this way has given me very good results with 
small Annelids and Hirudinea. Corrosive sublimate is another 
excellent reagent for this purpose. 
Narcotisation. 
13. The secret of narcotisation consists in adding1 some 
anaesthetic substance very gradually, in very small doses, to 
the water containing the animals, and waiting patiently for it 
to take effect slowly." 
The Tobacco-smoke Method for Actinia? (Hertwig, Die Actinien, 
1879) used to be practised as follows :—A dish containing the animals in 
water is covered with a bell-glass, under which passes a curved glass or 
rubber tube, which dips into the water. Tobacco-smoke is blown into the 
water for some time, through the tube, and the animals are then left for 
some hours in order that narcotisation may become fully established. The 
animals are irritated from time to time by touching a tentacle with a needle. KILLING. 
13 
As soon as it is observed that an animal begins to react slowly, that is to 
say as soon as it is found that the contraction of the tentacle does not begin 
until a considerable time after it has been irritated by the needle, the nar- 
cotisation may be considered sufficient. A quantity of some fixing liquid 
sufficient to kill the animals before they have time to contract is then added 
to the water. 
A space of several hours is necessary in order to thoroughly narcotise an 
Actinia by this method. 
14. Nicotin in solution may be used instead of tobacco smoke 
(Andres, Atti R. Ac.cad. dei Lincei, v, 1880, p. 9; see Journ. 
Roy. Mic. Soe., N.S., ii, 1882, p. 881). Andres employs a 
solution of 1 gramme of nicotin in a litre of sea-water. The 
animal to be anaesthetised is placed in a jar containing half a 
litre of sea-water, and the solution of nicotin is gradually con- 
ducted into the jar by means of a thread acting as a syphon. 
The thread ought to be of such a thickness as to be capable of 
carrying over the whole of the solution of nicotin in twenty- 
four hours. 
15. Chloroform may be employed either in the liquid state 
or in the state of vapour. Korotneff (Mitth. Zool. Stat. 
JVeapel, v, Hft. 2, 1884, p. 229; Zeit. f. wiss. Mik., 2, 1885, p. 
230) operates in the following manner with Siplionophora. 
The animals being extended, a watch-glass containing chloro- 
form is floated on the surface of the water in which they are 
contained, and the whole is covered with a bell-glass. As 
soon as the animals have become insensible they are killed by 
means of hot sublimate or chromic-acid solution plentifully 
poured on to them. 
Liquid chloroform is employed by squirting it in small 
quantities on to the surface of the water containing the 
animals. A syringe or pipette having a very small orifice, so 
as to thoroughly pulverise the chloroform, should be employed. 
Small quantities only should be projected at a time, and the 
dose should be repeated every five minutes, until the animals 
are anaesthetised. 
I have seen large Medusae very completely anaesthetised in 
the state of extension in an hour or two by this method. 
Andres finds that this plan does not succeed with Actiniae, as 
with them maceration of the tissues supervenes before anaes- 
thesia is established. 14 
CHAPTER II. 
16. Ether and Alcohol may be administered in the same way. 
Andres has obtained good results with Actiniae by the use of 
a mixture (invented by Salvatore lo Blanco) containing 20 
parts of glycerin, 40 parts of 70 per cent, alcohol, and 40 parts 
of sea-water. This mixture should be carefully poured on to 
the surface of the Avater containing the animals, and alloAved 
to diffuse quietly through it. Several hours are sometimes 
necessary for this. 
Eisig employs alcohol in the same way. 
17. Methyl-alcohol.—Coei (Zeit. f. wiss. Mile., vi, 4, 1890, 
p. 488) prefers methyl-alcohol to all other reagents. It has, 
amongst other advantages, that of having but a slight action 
on albumins. Cori recommends a mixture composed of 10 c.c. 
methyl-alcohol (of 96 per cent, strength), 90 c.c. water (fresh 
or sea-water), and 0‘6 g. of sodium chloride (to be added only 
when fresh water is taken, the addition of the salt having for 
its object to prevent maceration). It may be well to add to 
this mixture a very few drops of chloroform (for Cristatella ; 
Zeit. f. wiss. Zool., lv, 1893, p. 626; Zeit.f. wiss. Mile., x, 4, 
1893, p. 475). 
18. Hydrate of Chloral, which was first recommended, I 
believe, by Foettinger (Arch, de Biol., vi, 1885, p. 115), gives 
very good results with some subjects. Foettinger operates 
by dropping ci’ystals of chloral into the water containing 
the animals. For Alcyonella he takes 25 to 80 centigrammes 
of chloral for each hundred grammes of water. It takes 
about three quarters of an hour to render a colony sufficiently 
insensible to alloAv of fixing. Foettinger has obtained satis- 
factory results with marine and fresh-water Bryozoa, with 
Annelida, Mollusca, Nemertians, Actiniae, and with Asteracan- 
thion. He did not succeed with Hydroids. 
I am bound to state that I have never had the slightest success with Ne- 
mertians. 
Yekwoen (Zeit.f. wiss. Zool., xlvi, 9, 1887, p. 99; see also Journ. Boy. 
Mic. Soc., 1888, p. 148) operates differently for fresh-water Bryozoa. He 
puts Cristatella for a few minutes into 10 per cent, solution of chloral, in 
which the animals sooner or later become extended. 
Kukenthal (Zeit. f. toiss. Mile., iv, 3, 1887, p. 378; Journ. Boy. Mic. 
Soc., 1888, p. 509) has obtained good results with some Annelids, by means 
of a solution of one part of chloral in 1000 parts of sea-water. KILLING. 
15 
The chloral method gives rise to maceration with some sub- 
jects, and has been found to distort nuclear figures. (This, I 
suspect, will probably be found to be the case with most of 
these methods.) 
19. Cocaine (Richard; Zool. Anz., 196, 1885, p. 332) has 
been found to g'ive good results. Richards puts a colony of 
Bryozoa into a watch glass with 5 c.c. of water, and adds 
gradually 1 per cent, solution of hydrochlorate of cocaine in 
water. After five minutes, the animals are somewhat numbed, 
and half a cubic centimetre of the solution is added, and the 
tentacles are caused to contract by irritating them, with a 
needle. Ten minutes later the animal should be found to be 
dead in a state of extension. 
This method is stated to succeed with Bryozoa, Hydrax 
and certain worms. It is the best method for Rotifers 
(Rousselet). 
It has been pointed ont (by Cohi, in the paper quoted above) that unfor- 
tunately when fixing agents, such as sublimate solution, are added to the 
animals, the cocaine is thrown down on them as a white precipitate. 
20. Hydroxylamin.—Hofer (Zeit. f.wiss. Mih., vii, 3, 1890, 
p. 318) has employed hydroxylamin as a paralysing agent 
with success with the most varied animal forms. Either the 
sulphate or the hydrochlorate of the base may be used. He 
recommends that the hydrochlorate be taken. This, as found 
in commerce, is usually contaminated with HC1. It should 
be dissolved in water (spring or sea-water, according to the 
habitat of the organisms—in no case distilled water) and the 
solution exactly neutralised by addition of carbonate of soda. 
A 1 per cent, solution should be made up, and further diluted 
for use. The organisms are placed in the diluted solution, 
which may be taken of a strength varying from 01 per cent., 
used for thirty minutes or less (as for Infusoria), to 025 per 
cent., used for from fifteen minutes to one hour [Hydra), 1 per 
cent., one half to two hours (Hirudo), or as much as ten to 
twenty hours [Helix and Anodonta). 
It should be remembered that hydroxylamin is an extremely 
powerful reducing agent. Care must therefore be taken not 
to treat the paralysed animals with easily reducible fixing 
agents, such as osmic acid, chromic acid, sublimate, chlorides 16 
CHAPTER II. 
of gold or platinum, &c., unless it have been possible first to 
sufficiently wash out the hydroxylamin with water. 
21. Chloride of Magnesium.—Tullberg (Arch. Zool. Exper. et Gen., 
x, 1892, p. 11; Journ. Roy. Mic. Soc., 1892, p. 435) has obtained some 
results with this salt. For Actiniae, a 33 per cent, solution of the salt is to 
be very slowly added to the water containing the expanded animal, until the 
vessel contains 1 per cent, of the salt (thus for one litre of sea-water 33 c.c. of 
the solution must be added). The addition must be made gradually ; but it 
must be effected within half an hour. Thirty minutes later the animal will 
be found to be anaesthetised, and maybe fixed. 
For terrestrial and fresh-water Invertebrates, rather stronger solutions 
should be used. 
Redenbaugh (Amer. Natural., xxix, 1895, p. 399; Journ. 
Roy. Mic. Soc., 1895, p. 385) has obtained good results by 
means of Sulphate of Magnesium, either added in crystals to 
the sea-water containing the animals, until a saturated solu- 
tion is obtained; or in the shape of a saturated solution into 
which they are thrown (Annelids). 
22. Poisoning by small doses of some fixing agent is sometimes a good 
method. Salvatore lo Blanco employs the following method for pre- 
serving Ascidise in an extended state. A 1 per cent, solution of chromic acid 
acidulated with acetic acid is poured on to the surface of the water contain- 
ing the animals, and allowed to diffuse slowly through it. The operation 
takes four or five days (v. Garbini, Manuale per la Technica Mod. del 
Microscopio, p. 168). 
Osmic acid, or Kleinenberg’s solution, is sometimes employed in the 
same way. 
I have seen Medusae killed in a satisfactory manner by means of crystals of 
corrosive sublimate added to the water containing them. 
Morphia, Curare, Strychnin, Prussic Acid, and other paralysing 
drug have also been employed. 
23. Asphyxiation may be sometimes successfully practised. 
Terrestrial Gastropods may be killed for dissection by putting 
them into a jar quite full of water that has been deprived of 
its air by boiling, and hermetically closed. After from 
twelve to twenty-four hours the animals are generally found 
dead and extended. The effect is obtained somewhat quicker 
if a little tobacco be added to the water. 
Good results are sometimes obtained with aquatic animals 
by simply leaving them to exhaust the oxygen of the water in 
which they are contained. I have sometimes succeeded with KILLING. 
17 
Holothuriae and other Echinoderms in this way; and Ward 
(see Amer. Nat., xxv, 1891, p. 398) has succeeded with 
Hydroids, Actiniae, and similar forms. If the animals be 
found to be imperfectly expanded when nai'cosis has set in, 
they may be got to expand by putting them back for a short 
time into pure sea-water; and as soon as they are expanded 
should be quickly thrown into some rapidly-killing reagent. 
24. Carbonic Acid Gas has been recommended (by Fol, Zool. 
Anz., 128, 1885, p. 698). The water containing the animals 
should be saturated with the gas. The method is stated to 
succeed with most Ccelenterata and Echinodermata, but not 
with Molluscs or Fishes. It has not been found successful 
at the Naples Zoological Station. I have had most excellent 
results with small Annelids and Hirudinea. It is not neces- 
sary to employ a generator for obtaining the gas. It suffices 
to take an ordinary “ soda-water” siphon, and squirt its con- 
tents into the water containing the animals. Narcotised 
animals recover very quickly on being put back into pure 
water. 
25. Marine Animals are sometimes successfully killed by 
simply putting them into fresh water. 
Warm Water will sometimes serve to immobilise and even 
kill both marine and fresh-water organisms. CHAPTER III. 
FIXING AND HARDENING. 
26. The Necessity of Fixing.—The meaning of the term 
“ fixing” has been explained above ( § 2). It only remains 
here to insist on the absolute necessity of the employment of 
fixing agents, and to briefly illustrate this necessity by a 
single example. If a portion of living retina be placed in 
aqueous humour, serum, or other so-called “indifferent” 
medium, or in any of the media used for permanent preserva- 
tion, it will be found that the rods and cones will not preserve 
the appearance they have during life for more than a very 
short time ; after a few minutes a series of changes begins 
to take place, by which the outer segments of both rods and 
cones become split into discs, and finally disintegrate so as to 
be altogether unrecognisable, even if not totally destroyed. 
Further, in an equally short time the nerve fibres become 
varicose, and appear to be thickly studded with spindle- 
shaped knots; and other post-mortem changes rapidly occur. 
If, however, a fresh piece of retina be treated with a strong 
solution of osmic acid, the whole of the rods and cones will 
be found perfectly preserved after twenty-four hours’ time, 
and the nerve-fibres will be found not to be varicose. After 
this preliminary hardening, portions of the retina may be 
treated with water (which would be ruinous to the sti’uctures 
of a fresh retina), they may even remain in water for days 
without harm ; they may be stained, acidified, hardened, 
imbedded, cut into sections, and mounted in either aqueous 
or resinous media without suffering. 
27. The Action of Fixing Agents consists in the coagulation 
of certain of the constituents of tissues, of their albuminoids, 
their gelatin, their mucin. Some fixing agents seem to have 
further the property of combining chemically with the tissues, FIXING AND HARDENING. 
19 
so that they cannot be easily removed from them by washing. 
This is a consideration of great importance in view of ulterior 
operations, and most particularly in view of staining. Chromic 
acid and its salts, osmic acid, the chlorides of palladium, of 
gold, and of iron, are reagents that seem to combine chemi- 
cally with the tissues, and render necessary a special after- 
treatment and special modes of staining, whilst picric acid, 
nitric acid, and corrosive sublimate do not appear to enter 
into that kind of combination, and can be entirely removed 
from the tissues by washing, and leave the tissues in a state 
in which they are susceptible of any kind of staining. 
Practically it amounts to this, that if you fix with a chromic 
or osmic mixture, you cannot stain with carmine, but can only 
stain with hasmatoxylin, if you wish to stain your objects in 
toto ; or you may make sections, and stain them with safranin 
or some other coal-tar colour. Whilst if you fix with subli- 
mate or a picric acid mixture, you may do as you like in the 
matter of staining. 
The after-treatment appropriate to each fixing agent is 
indicated in the special paragraphs. 
28. Choice of a Fixing Agent.—Indications concerning the 
proper fixing agent to employ for the different tissues and 
organs of the animal kingdom will be found in Part II. The 
following remarks are intended as hints for beginners only. 
The chief fixing agents for general work are Flemming’s 
mixture, Hermann’s 'platinum-chloride mixture, corrosive sub- 
limate, and Perenyi’s mixture. 
I recommend that Flemming’s mixture should be used 
wherever it is possible, as I believe it to be in general by far 
the best fixing agent yet invented, with the exception of 
Hermann’s mixture, which is unfortunately too expensive to 
be employed in large quantities. 
But it will not always be found possible to use it. Its low 
power of penetration, for instance, puts it out of court in the 
•case of very impermeable objects, such as are frequently 
offered by the Arthropoda. For these, picro-sulphuric acid 
may be recommended. 
For very small objects, such as may be mounted whole, 
osmic acid is nearly as good as Flemming’s mixture, and is 
frequently much more convenient to use. 20 
CHAPTER TH. 
For objects a little larger, and for much embryological 
Avork on objects that it is convenient to have stained in 
toto, corrosive sublimate may be recommended. I think it 
is in general a much better preseiwative of the forms of 
anatomical elements than either Altmann’s nitric acid or 
Klein enberg’s fluid. 
29. The Practice of Fixation.—Hints and Cautions.—See that 
the structures are 'perfectly living at the instant of fixation, 
otherwise will only fix pathological states or post-mortem 
states. 
Do all you can to facilitate the rapid penetration of the 
fixing agent. To this end, let the structures be divided into 
the smallest portions that can conveniently be employed, 
and if entire organs or organisms are to be fixed whole, let 
openings, as large as possible, be first made in them. 
The penetration of reagents is greatly facilitated by heat. 
You may warm the reagent and put it with the objects to be 
fixed in the paraffin stove, or you may even employ a fixing 
agent heated to boiling point (as boiling sublimate solution 
for certain corals and Hydroids, or boiling absolute alcohol 
for certain Arthropods with very resistant integuments). 
Let the quantity of fixing agent employed be at least many 
times the volume of the objects to be fixed. If this precaution 
be not observed the composition of the fixing liquid may be 
seriously altered by admixture of the liquids or of the soluble 
substances of the tissues thrown into it. For a weak and 
slowly acting fixing agent, such as picric acid, the quantity of 
liquid employed should be in volume about one hundred times 
that of the object to be fixed. Keagents that act very ener- 
getically, such as Flemming’’s solution, may be employed in 
smaller proportions. 
Be careful to use the appropriate liquid for washing out the 
fixing agent after fixation. It is frequently by no means a 
matter of indifference whether water or alcohol be employed 
for washing out. Sometimes water will undo the whole work 
of fixation (as with picric acid). Sometimes alcohol causes 
precipitates that may ruin the preparations. Instructions on 
this head are given in the paragraphs devoted to the different 
fixing agents. 
Use liberal quantities of liquid for washing. FIXING AND HARDENING 
21 
Change the liquid as often as it becomes turbid, if that 
should happen. 
The process of washing out is often greatly facilitated by 
heat. Picric acid, for instance, is nearly twice as soluble in 
alcohol warmed to 40° C. as in alcohol at the normal tempe- 
rature (Fol). 
30. Fixation of Marine Animals.—In the case of marine 
organisms it may be stated as a general rule that their tissues 
are more refractory to the action of reagents than are the 
tissues of corresponding fresh-water or terrestrial forms, and 
fixing solutions should in consequence be stronger (about two 
to three times stronger, according to Langerhans). 
Marine animals ought to b& freed from the sea-water adhe- 
rent to their surface before treating them either with alcohol 
or any fixing reagent that precipitates the salts of sea-water. 
If this be not done, the precipitated salts will form on the 
surfaces of the organisms a crust that prevents the penetra- 
tion of reagents to the interior, thus allowing maceration to 
be set up, and hindering the penetration of staining fluids. 
Fixing solutions for marine organisms should therefore be 
such as serve to keep in a state of solution, and finally remove, 
the salts in question. They should never he made with sea- 
ivater as a menstruum, as some workers have inconsiderately 
proposed. If alcohol be employed it should be acidified with 
hydrochloric or some other appropriate acid. Picro-sulphuric 
acid is a reagent that fulfils the conditions here spoken of. 
(On this subject see Paul Mayer, in Mitth. Zool. Stat. Neapel, 
ii (1881), p. 1 et seq. See also the abstract in Journ. Boy. 
Mic. Soc. (N.S.), ii (1882), pp. 866 — 881, and that in Amer. 
Natural., xvi (1882), pp. 697—706.) 
31. The Obligation of Hardening.—Methods of imbedding 
have now been brought to such a degree of perfection that 
the thorough hardening of soft tissues that was formerly neces- 
sary in order to cut thin sections from them is now, in the 
majority of cases, no longer necessary; by careful infiltration 
with paraffin or some other good infiltration mass, most soft 
objects can be satisfactorily cut with no greater an amount of 
previous hardening than is furnished by the usual passing of 
the tissues after fixing through successive alcohols in order to 22 
CHAPTER III. 
prepare them for the paraffin bath. But there are some 
exceptions. Such are, for instance, the cases in which it is 
desired to cut very large sections, such as sections of the 
entire human brain. Such an organ as this cannot be duly 
infiltrated with alcohol in a few hours, and it is doubtful 
whether it can be duly infiltrated with paraffin or any other 
imbedding mass in any reasonable time. And certain organs 
that are either extremely delicate or inaccessible, such as 
retina or cochlea, will require to be specially hardened in 
order to give the best results. The processes employed for 
hardening such specimens as these will be described when 
treating of the organs in question. In this chapter I confine 
myself to such general statements concerning the employment 
of the usual hardening agents as appear likely to be generally 
useful. 
32. The Practice of Hardening—Hints and Cautions.—Employ 
in general a relatively large volume of hardening liquid, and 
change it very frequently. The exact proportions may be 
made out by experiment for each reagent and each class of 
objects. If the volume of liquid be insufficient its composition 
will soon become seriously altered by the diffusion into it of 
the soluble substances of the tissues ; and the result may be 
a macerating instead of a hardening liquid. Further, as soon 
as, in consequence of this diffusion, the liquid has acquired a 
composition similar in respect of the proportions of colloids 
and crystalloids contained in it to that of the liquids of the 
tissues, osmotic equilibrium will become established, and dif- 
fusion will cease. That is to say, the hardening liquid will 
cease to penetrate. This means, of course, maceration of in- 
ternal parts. On the other hand, it appears that a certain 
slight proportion of colloids in the hardening liquid is favor- 
able to the desired reaction, as it gives a better consistency 
to the tissues by preventing them from becoming brittle. 
Hence the utility of employing a certain proportion of harden- 
ing agent. 
Hardening had better be done in tall cylindrical vessels, the 
objects being suspended by a thread at the top of the liquid. 
This has the advantage of allowing diffusion to take place as 
freely as possible, whilst any precipitates that may form fall 
harmlessly to the bottom. FIXING AND HARDENING. 
23 
Always begin hardening with a weak reagent, increasing 
the strength gradually, as fast as the tissues acquire a consist- 
ence that enables them to support a more energetic action of 
the reagent. 
Let the objects be removed from the hardening fluid as soon 
as they have acquired the desired consistency. 
As to the choice of a hardening reagent, if you wish, above 
all, for a rapid and energetic action, take chromic acid. If 
you wish for a more moderate and more equable action, take 
a chromic salt, or one of the compounds of which the chromic 
salts are the chief ingredients, or one of the platinum chloride 
mixtures. CHAPTER IV. 
FIXING AGENTS. MINERAL ACIDS AND THEIR SALTS. 
33. Osmic Acid.—The tetroxide of osmium (0s04) is the 
substance commonly known as osmic acid, though it does not 
possess acid properties. It is a substance that is exceedingly 
difficult to keep in use for any length of time. It is extremely 
volatile, and in the form of an aqueous solution becomes par- 
tially reduced with great readiness in presence of the slightest 
contaminating particle of organic matter. (It is generally 
believed that the aqueous solutions are reduced by light alone, 
but this is not the case : they may be exposed to the light 
with impunity if dust be absolutely denied access to them. 
It would even seem that the solutions are improved for some 
purposes by exposure to sunlight, vide infra, the remarks on 
gold chloride solutions. (Some observations communicated to 
me by Dr. Lindsay-Johnson go to prove that, if dust be 
avoided, solutions keep better in the light, with occasional 
“sunning,” than in the dark.) 
Great stress is laid by authors on the fact that the vapour of osmium is 
very irritating to mucous tissues. It is said that the slightest exposure to 
it is sufficient to give rise to serious catarrh, irritation of the bronchial tubes, 
laryngeal catarrh, conjunctivitis, &c. I have never myself suffered in this 
way, but there is no doubt that many persons do, and such susceptible sub- 
jects should be very careful in handling osmium in any form. 
34. How to keep the Solutions.—After having carefully tried 
several of the plans that have been recommended for keeping 
the working solutions free from dust, I have come to the con- 
clusion that the following is the most practical plan for 
preventing them from “ going bad ” :—The solution of osmic 
acid in chromic acid solution is not, like the solution in pure 
water, easily reducible, but may be kept without any special 
precautions. I therefore keep the bulk of my osmium in the 
shape of a 2 per cent, solution of osmic acid in 1 per cent. FIXING AGENTS. MINERAL ACIDS AND THEIR SALTS. 
25 
aqueous chromic acid solution. This solution serves for 
fixation by osmium vapours, and for making up solution of 
Flemming (or solution of Hermann) which is the form in 
which osmium is most generally employed. A small quantity 
of osmic acid may also be made up in 1 per cent, solution in 
distilled water, and kept carefully protected from dust for use 
in special cases. Those who have to do a great deal of fixing 
by means of the vapours may also keep a supply of the solid 
oxide for this purpose. 
Grubler and Co. now send out. osmic acid in tubes con- 
taining one tenth of a gramme. 
Cori (Zeit. f. wiss. Mik., vi, 4, 1890, p. 442) finds that 
solutions in distilled water keep perfectly if there be added to 
them enough permanganate of potassium to give a very 
slight rosy tint to the liquid. From time to time, as the 
solution becomes colourless, further small quantities of the 
salt should be added, so as to keep up the rosy tint. 
35. Regeneration of reduced Solutions.—Bristol (Amer. Nat., 
xxvii, 1893, p. 175; Journ. Roy. Mic. Soc., 1893, p. 564; Ref. 
Handbook Med. Sci., Sapp., p. 442) says that reduced solutions 
may be regenerated by oxidising them by means of peroxide 
of hydrogen. The reaction is stated to be identical with that 
which takes place in the bleaching of osmium-blackened tissues 
by peroxide. It is admitted that the tetroxide of osmium, 
0s04, is reduced by contact with organic matter into the 
deutoxide, 0s03. Then, 0s03 + 2H303 = 0s04 + 2H30. 
According to Bristol, for regenerating 100 c.c. of 1 per 
cent, solution of osmic acid (erratim 10 per cent in Journ. Roy. 
Mic. Soc.), ten to twenty drops of fresh peroxide solution should 
be added. 
Kolossow (Zed. f. ivins. Mik., ix, 1, 1892, p. 40) says that 
half-reduced solutions, so long as they have not lost their 
characteristic odour, may be regenerated by the addition of a 
little powdered potash-alum. 
36. Fixation by the Vapours.—Osmie acid is frequently em- 
played in the form of vapour, and its employment in this form 
is indicated in most of the cases in which it is possible to 
expose the tissues directly to the action of the vapour. The 
tissues are pinned out on a cork which must fit well into a 26 
CHAPTER IY. 
wide-mouthed bottle in which is contained a little solid osmic 
acid (or a small quantity of 1 per cent, solution will do). 
Very small objects, such as isolated cells, are simply placed on 
a slide, which is inverted over the mouth of the bottle. They 
remain there until they begin to turn brown (isolated cells will 
generally be found to be sufficiently fixed in thirty seconds, 
whilst in order to fix the deeper layers of relatively thick 
objects, such as retina, an exposure of several hours may be 
desirable). It is well to wash the objects with water before 
staining, but a very slight washing will suffice. For staining, 
methyl-green may be recommended for objects destined for 
study in an aqueous medium, and, for permanent preparations, 
alum-carmine, picro-carmine, or hsematoxylin. 
In researches on nuclei, it is possible and may be useful to 
employ the vapours of a freshly-prepared mixture of osmic 
and formic or acetic acid (Gilson, La Cellule, i, 1885, p. 96). 
The reasons for preferring the process of fixation by vapour of osmium, 
where practicable, are that osmium is more highly penetrating when em- 
ployed in this shape than when employed in solution, and produces a more 
equal fixation, and that the arduous washing out required by the solutions is 
here done away with. In many cases delicate structures are better pre- 
served, all possibility of deformation through osmosis being here eliminated. 
37. Fixation by Solutions.—When employed in aqueous solu- 
tions osmic acid is used in strengths varying from per cent, 
to 2 per cent. Solutions of A per cent, to 1 per cent, have 
been very largely used, but the tendency of modern practice 
seems to be towards weaker solutions and longer immersion. 
For Infusoria h per cent, for a few seconds ; for Porifera 
to yL per cent, for some hours; for Mollusca 1 to 2 per cent, 
for twenty-four hours ; for epithelia yL- to per cent, for an 
hour or two ; for meroblastic ova y per cent, for twenty-four 
hours; for medullated nerve-fibre y to 1 per cent, for from 
twenty minutes to two hours; for tactile corpusclesto 1 per 
cent, for twenty-four hours; for retina j to 2 per cent, for 
from ten minutes to twenty-four hours ; for nuclei T\j- to 2 per 
cent, for two or three hours. Such figures as these will serve 
to give a general idea of the practice, whilst more precise in- 
structions will be given when dealing with the tissues in detail. 
(The durations here quoted appear to me exaggerated, except 
for very voluminous specimens.) 
A little acetic or formic acid (05 to 1 per cent.) may fre- FIXING AGENTS. MINERAL ACIDS AND THEIR SALTS. 
27 
quently with advantage be added to the solutions just before 
using. 
If solutions made with pure water be used, they must be 
kept protected from the light during the immersion of tissues. 
This precaution is not necessary if Flemming’s or Hernjann’s 
solution be used. If the immersion is to be a long one the 
tissues must be placed with the solution in well-closed vessels, 
as osmium is very volatile. 
The principle of combining osmium solutions with alcohol is due to 
Ranvieb et Vigxal (Ranvier, Leg. d’Anat. Gen., “ App. term, des muscles 
de la vie org.,” p. 76 ; Vignal, Arcli. de Physiol., 1884, p. 181). They take 
equal volumes of 1 per cent, osmic acid and 90 per cent, alcohol (freshly 
mixed). They wash out in 80 per cent, alcohol, then wash with water and 
stain for forty-eight hours in picro-carmine or hsematoxylin. Viallanes has 
applied this method to the histology of insects. 
Kolossow {Zeit. f. wiss. Mile., v, 1, 1888, p. 51) has recommended a 0‘5 
per cent, solution of osmium in 2 or 3 per cent, solution of nitrate or acetate 
of uranium, as having a greatly enhanced penetrating power. 
He has more lately (op. cit., ix, 1, 1892, p. 39) recommended for the same 
reason a mixture of 50 c.c. absolute alcohol, 50 c.c. distilled water, 2 c.c. 
concentrated nitric acid, and 1 to 2 g. osmium. This mixture is said to keep 
indefinitely in a cool place. 
Manx (Zeit. f. wiss. Mile., xi, 4, p. 481) recommends a freshly-prepared 
mixture of equal volumes of 1 per cent, osmic acid solution and saturated 
solution of corrosive sublimate in normal salt solution (for nerve centres). 
38. After-treatment.—The excess of osniic acid must be well 
washed out before proceeding to any further steps in prepara- 
tion; water should be used for washing. Notwithstanding 
the greatest care in soaking, it frequently happens that some 
of the acid remains in the tissues, and causes them to over- 
blacken in time. To obviate this it is necessaiy to wash them 
out in ammonia-carmine or picro-carmine, or to soak them 
for twenty-four hours in a solution of bichixunate of potash 
(Muller’s solution or Erlicki’s will do), or in 05 per cent, 
solution of chromic acid, or in Merkel’s solution, or in a weak 
solution of ferrocyanide of potassium or cyanide of potassium. 
The treatment with bichromate solutions has the great advan- 
tage of highly facilitating staining with carmine or haema- 
toxylin. Max Schultze recommended washing, and mounting 
permanently in acetate of potash ; but I believe the virtues 
attributed to this method are illusory. Fol has recommended 
treatment with a weak solution of carbonate of ammonia. 28 
CHAPTER IV. 
But the best plan of all is to properly bleach the preparations 
(see “Bleaching”). This is perhaps most conveniently done 
(as recommended by Fol, Brass, and Overton) by means of 
j)eroxide of hydrogen, which regenerates the osmium to osmic 
acid. Overton (Zeit. f. wiss. Mik., vii, 1, 1890) finds that 
bleaching is completed in a few minutes in a mixture of 1 part 
commercial peroxide of hydrogen with 10 to 25 parts 70 per 
cent, alcohol. (The commercial peroxide, slightly acidulated 
with HC1, will keep well in the dark; but the mixture with 
alcohol must be made fresh for use.) CarazzTs peroxide of 
sodium may be found convenient for this purpose. Binet 
{Journ. de I’Anat. et de la Physiol., xxx, 1894, p. 449) has 
successfully used permanganate of potash. 
The same stains recommended for objects fixed by vapour will be found 
useful here, with the addition of ammonia-carmine, which is really very use- 
ful for strongly fixed specimens. For sections, of course in both cases 
safranin and the other nuclear anilin stains may be employed with ad- 
vantage. 
39. Characters of the Fixation with Osmic Acid.—In general 
osmic acid, especially when used in the form of vapour, 
preserves the forms of cells more faithfully than any other 
reagent. But there are some drawbacks over and above those 
before mentioned (§ 27). The penetrating power of the solu- 
tions is very low, so that if any but very small pieces of 
tissue be taken the outer layers become over-fixed before the 
action of the reagent has penetrated to the deeper layers. 
Over-fixed cells have a certain homogeneous, glassy or colloid 
look, owing to all their constituents having been raised by 
coagulation to so high an index of refraction that little or no 
detail is visible in them. They stain very badly, or not at 
all. Such cells are known as “ osmicated cells, osmirte Zellen.” 
There is no remedy for this state of things if once it has 
occurred. For this reason it is important to avoid using 
stronger solutions than is necessary. The danger of osmi- 
cation is lessened by using the osmic acid in conjunction 
with certain other reagents, such as chromic acid. But it is 
not thereby entirely removed ; Flemming’s mixture, especi- 
ally the strong formula, will readily osmicate superficial cells 
if care be not taken. For ordinary histological work osmica- 
tionof superficial layers is not of much consequence. But for 
cytological work care should betaken not to draw conclusions FIXING AGENTS. MINERAL ACIDS AND THEIR SALTS. 
29 
as to the structure of cells from osmicated specimens, and 
attention should be confined to cells four or five layers deeper 
down, which will generally be found to present the desired 
intensity of fixation. 
Osmic acid stains all fatty structures black ; it must therefore be avoided 
for tissues in which much fat is present ; or if not, the preparations must be 
subsequently very thoroughly bleached. 
40. Chromic Acid.—Chromic anhydride, Cr03, is found in 
commerce in the form of red crystals that dissolve readily in 
water, forming chromic acid, ETCr04. These crystals are very 
deliquescent, and it is therefore well to keep the acid in stock 
in the shape of a 1 per cent, solution. Care must be taken 
not to allow the crystals to be contaminated by organic matter, 
in the presence of which the anhydride is readily reduced into 
sesquioxide. 
Chromic acid is employed in solution either in water or in 
alcohol. 
The most useful strengths in which it is employed in aqueous 
solution are from OT to TO per cent, for a period of immersion 
of a few hours (structure of cells and ova). For nerve-tissues 
weaker solutions are taken, to d-th per cent, for a few 
hours. Stronger solutions, such as 5 per cent., should only 
be allowed to act for a few seconds. 
The object should be washed out with water before passing 
into alcohol or staining fluids. Long washing in water is 
necessary to prepare them for staining, except an anilin stain 
be used. It is possible to wash out in alcohol, and this may 
be useful in special cases, but in general I think the practice 
is not to be recommended. It is well to wash for many hours 
in running water. 
Tissues that have been fixed in chromic acid are best stained 
in aqueous solutions, as water does not appear to have an 
injurious effect on them ; the acid appears to enter into some 
chemical combination with the elements of the tissues, 
forming with them a compound that is not affected either 
physically or chemically by water. The best stain to 
follow chromic acid is hasmatoxylin, or, for sections, some 
anilin stain. But the previous washing out with water must 
be very thorough if good results are to be insured; it may 
take clays. 30 
CHAPTER IV. 
Chromic acid is not a very penetrating reagent, and for this 
reason, as well as for others, is seldom used pure, but plays an 
important part in the mixtures described below, of which the 
chief is certainly the mixture of Flemming. A chief objection 
to the use of chromic acid is that it precipitates certain of the 
liquid albuminoids of tissues in the form of filaments or net- 
works, which are often of great regularity, and simulate struc- 
tural elements of the tissues. This objection applies to all 
mixtures into which chromic acid enters. 
41. Action of light on alcohol containing chromic objects.—When objects 
that have been treated by chromic acid or a chromate are put into alcohol 
for hardening or preservation, it is found that after a short time a fine pre- 
cipitate is thrown down on the surface of the preparations, thus forming a 
certain obstacle to the further penetration of the alcohol. Previous washing 
by water does not prevent the formation of this precipitate, and changing 
the alcohol does not prevent it from forming again and again. It has been 
found by Hans Virchow (Arcli.f. mile. Anat., Bd. xxiv, 1885, p. 117) that 
the formation of this precipitate may be entirely prevented by simply 
keeping the preparations in the dark. The alcohol becomes yellow as usual 
(and should be changed as often as this takes place), but no precipitate is 
formed. If this precaution be taken, previous washing with water may be 
omitted, or at all events greatly abridged. 
The brownish-green colour of chromic objects may be removed by treat- 
ing them with peroxide of hydrogen (Unna, in Arch. f. mile. Anat., Bd. 
xxx, 1887, p. 47 ; cf. Journ. Roy. Mic. Soc., 1887, p. 1060; and see the in- 
structions for bleaching osmic acid preparations at the end of § 39). 
42. Chromic Acid and Spirit.—A mixture of 2 parts of -k 
per cent, chromic acid solution with one part of methylated 
spirit was much used by Klein in his investigations into the 
structure of cells and nuclei, and found to give better results 
than the ordinary reagents (including even osmic acid). 
Haematoxylin was used for staining. 
The addition of alcohol to augment the penetrating power 
of chromic acid seems to be a step in the right direction, and 
it is matter for surprise that such mixtures are not more used. 
The alcohol should be added to the acid in aqueous solution, 
as if strong alcohol be added to crystals of chromic anhydride, 
a very violent reaction is set up. The mixture should be kept 
in the dark. 
43. Chromo-acetic Acid (Flemming, Zellsbz. Kern u. Zellth., 
p. 382). 
Chromic acid . 0’2 to O'25 per cent. 
Acetic acid . . O’l per cent., in water. FIXING AGENTS. MINERAL ACIDS AND THEIR SALTS. 
31 
Flemming finds this the best reagent for the study of the 
achromatic elements of karyokinesis. (Flemming wrote this in 
1882, and 1 doubt whether it Avould now hold good.) Stain 
with hsematoxylin (the preparations are not favorable for 
staining with safranin or other coal-tar colours). 
I can recommend as a good fixing and hardening mixture 
for Annelids in general, and probably for other forms, the 
following fluid due to Ehlers (I do not know whether it has 
been published elsewhere) :—To 100 c.c. of chromic acid of 
0’5 to 1 per cent, add from 1 to 5 drops of glacial acetic acid. 
The proportion of acetic acid indicated is sufficient to counter- 
act any tendency to shrinkage due to the chromic acid. 
44. Chromo-formic Acid (Rabl, Morph. Jahrh., x, 1884, pp. 
215, 216).—Four or five drops of concentrated formic acid are 
added to 200 c.c. of 0 33 per cent, chromic acid solution. The 
mixture must be freshly prepared at the instant of using. 
Fix for twelve to twenty-four hours, wash out with water, 
harden in alcohol, stain with haematoxylin or safranin. For 
the study of karyokinesis. This is acknowledged to be one 
of the very best reagents for the purpose. 
45. Chromo-osmic Acid (Max Flesch, Arch. f. mik. Anat., xvi, 1879, 
p. 300).—This mixture (osmic acid 0T0, chromic acid 0'25, water lOO'O) origi- 
nally introduced for the preparation of the auditory organ of vertebrates, is 
of general application. It does not require to be kept in the dark. Objects 
may remain in it for twenty-four or thirty-six hours without risk of the 
osmic acid over-blackening them. Flemming found it to preserve nuclear 
figures well, but the preparations are pale, and difficult to stain well. He 
finds that the action of the mixture is improved (for nuclear figures) by the 
addition of acetic, formic, or other acid. This addition brings out the 
figures more sharply, and has the further advantage of allowing of a sharper 
stain with hsematoxylin, picro-carmine, or gentian violet. He recommends 
the following formula, which may be considered to have superseded Max 
Flesch’s. 
46. Chromo-aceto-osmic Acid (Flemming, Zellsubstanz, Kern 
und Zelltheilunq, 1882, p. 381) first or weak formula.— 
Chromic acid . . 0'25 per cent. 
Osmic acid . . 0'1 per cent. 
Glacial acetic acid . OT per cent. 
In water. 
The best results (as regards faithfulness of fixation) are ob- 
tained with this mixture when it is allowed to act for only a 
short time (about half an hour). 32 
CHAPTER IV. 
But it may, without inconvenience, be allowed to act for 
many hours or days, or according to some workers, even 
weeks or months. Wash out, very thoroughly, in water. 
Stain with haematoxylin, if you wish to stain in toto (staining 
in this way with other reagents is possible, but very difficult, 
and not be recommended). Stain sections with safranin, or 
other anilin, or with haematoxylin or Kernschwarz. 
To make up this mixture with the usual stock solutions, 
you take : 
Chromic acid of 1 per cent. . . 25 volumes. 
Osmic acid of 1 per cent. . . 10 ,, 
Acetic acid of 1 per cent. . .10 ,, 
Water 55 
If you keep your osmium in 2 per cent, solution in chromic 
acid of 1 per cent., as I have recommended, you will have to 
take only 20 vols. of chromic acid, 5 of your osmium solution, 
and 65 of water. See also the remarks on the deterioration 
of these solutions by keeping, in the next §. 
It has been already stated more than once that Flemming’s solution is, 
with the exception of Hermann’s solution, probably the very best fixing re- 
agent in general yet discovered. It has, however, been criticised. Faussek 
(Zeitschr. f. wiss. Zool., Bd. xlv, 1887, pp. 694, et seq.) found it totally in- 
applicable to the histology of the intestine of insects. He states that it 
caused the intima to disappear, and the cells to run together into a compact 
mass. Arnold {Arcli. f. mik. Anat., Bd. xxx, 1887, p. 205) states that it 
does not preserve cell-bodies faithfully. And A. Kotlarewsky (Mitth. d. 
naturf. Ges. Bern., 1887; cf. Zeit.f. wiss. Mik., iv, 3, 1887, p. 387) found 
that it preserved the forms of nerve-cells (spinal ganglia) less faithfully 
than any of the reagents tried. I have not, myself, been struck by any 
defect in the preservation of cytoplasmic structures in my preparations 
made by this reagent. 
It is not necessary in all cases to observe the exact proportions of the in- 
gredients in this mixture. Fol (Lehrb. d. vergl. mik. Anat., 1884, p. 100) 
recommends the following: 
1 per cent, chromic acid 25 vols. 
1 per cent, osmic acid . . . . . 2 ,, 
2 per cent, acetic acid . . . . . 5 ,, 
Water 68 „ 
That is to say, a mixture much weaker in osmium than Flemming’s. In 
the Traite des Methodes Techniques, &c., Lee et Henneguy, 1887, I recom- 
mended this mixture, as giving better results in general, but am now inclined 
to think that, at all events as regards fidelity of fixation, it is a step in the 
wrong direction. Fol’s formula has the advantage of allowing better 
staining with carmine, that is all. 33 
FIXING AGENTS. MINERAL ACIDS AND THEIR SALTS. 
A mixture still weaker than this in osmium, viz. with 1 vol. osmium solu- 
tion instead of 2, has been recommended by Cori (Zeit.f. wiss. Mik., vi, 1, 
1890, p. 441), but I still adhere to the opinion above expressed. 
47. Chromo-aceto-osmic Acid (Flemming, Zeit.f. wiss. Mik., 
1,1884, p. 349), second or strong formula.— 
1 per cent, chromic acid . .15 parts. 
2 per cent, osmic acid . 4 „ 
Glacial acetic acid . . . 1 ,, 
If 2 per cent, osmium solution should not be at hand, you may conve- 
niently make the mixture by taking— 
10 per cent, chromic acid . . . .15 parts. 
1 per cent, osmic acid . . . . . 80 „ 
Glacial acetic acid . . . . . . 10 „ 
Water . . . . . . . . 95 „ 
If this mixture be kept in stock in large quantities, it may- 
go bad, probably on account of the large proportion of 
organic acid contained in it. I therefore recommend that it be 
made up from time to time from stock solutions, in which the 
osmium is kept separate from the acetic acid. The propor- 
tions being as follows : 
Cr03 . . . . . . O’15 
Os 0-08 
Acid. acet. ..... TOO 
Aq 19-00 
You may make up and keep separately— 
(A) 1 per cent, chromic acid . .11 parts. 
Distilled water . . . 4 „ 
Glacial acetic acid . . . 1 „ 
and (B) a 2 per cent, solution of osmium in 1 per cent, 
chromic acid solution, and when required, mix four parts of 
A with one of B ; or, of course, if you prefer it, you may 
keep the osmic and chromic acid ready mixed in the propor- 
tions given, and add 5 per cent, of acetic acid at the moment 
of using. 
In any case, it is better not to make up very large quan- 
tities of the mixture at once, as osmium being very volatile it 
will be found that solutions that have been long in use no 
longer contain the proper proportion of that ingredient, and 
the hardening action being thus weakened the swelling action 
of the acetic acid will be insufficiently controlled. 34 
CHAPTER IV. 
. I am convinced that for some purposes there is ad\rantage 
in diminishing notably the proportion of acetic acid. 
Merk (Denkscli. d. Math. Naturw. Cl. d. K. Acad. d. Wiss. Wien, 1887 ; 
cf. Zeit.f. wiss. Mile., v, 2,1888, p. 237) proposes to make up separately (A) 
2 per cent, chromic acid 7'5 parts. 
Water . . . . . . . 3‘5 „ 
Acetic acid . . . . . . .1 „ 
and (B), some 1 per cent, osmium solution, and to mix for use 12 parts of 
A with 8 of B. But this plan leaves you in the old difficulty of keeping 
your osmium in aqueous solution. 
It does not appear necessary to observe the exact proportions of the ingre- 
dients of these mixtures, a certain latitude is allowable. Thus Carnot (La 
Cellule, 1, 2, 1885, p. 211) has employed a mixture one third stronger in 
osmium and twice as strong in chromic acid, viz. 
Chromic acid of 2 per cent, (or even stronger) 45 parts. 
Ostnic acid of 2 per cent . . . . 16 „ 
Glacial acetic acid , . . . . . 3 „ 
Podwyssozki recommends (for glands especially) the following modifi- 
cation : 
1 per cent. Cro.3 dissolved in 05 per cent, solution of 
corrosive sublimate ....... 15 c.c. 
2 per cent, osmium solution ..... 4 c.c. 
Glacial acetic acid . . . . . . 6 to 8 drops. 
The sublimate is said to augment the penetration of the osmium, but is 
unfavorable to staining. The proportion of acetic acid is reduced in order 
to avoid swelling of the tissue-elements (Ziegler’s Beitrdge z. path. Anat., 
i, 1886 ; cf. Zeit.f. wiss. Mile., iii, 3, 1886, p. 405). 
The strong formula was recommended by Flemming in the 
first instance merely for a very special purpose, the bunting for 
karyokinetic figures, and not for general purposes. Further 
experience has shown that it is applicable to general purposes, 
and is in many cases considerably superior to the iveak 
formula. But it is not suited to all objects, and Flemming 
has lately pointed out that some workers have used it for pur- 
poses for which it is not fitted. 
Arnold, in the place quoted in the last paragraph, says that it is to be 
avoided if yon wish to demonstrate the structure of certain nuclei (of 
wandering cells) ; and the other objections there quoted as applying to the 
weak formula are intended to apply more or less to the present formula. It 
will be well not to attach too much importance to them. Let delicate struc- 
tures he fixed for twenty-four hours or more, washed in running water for 
an hour, and in successive alcohols for twenty-four hours, sectioned, and 
stained with safranin or gentian violet, and there will be little complaint of 
defective preservation. FIXING AGENTS. MINERAL ACIDS AND THEIR SALTS. 
35 
The strong mixture does not brown tissues more than the 
weak mixture, but rather less. 
Pat is blackened by these mixtures ; but the blackened fat can be entirely 
dissolved out of the tissues by treating them for a few hours with turpentine 
that has been exposed to sunlight for an hour or two (see Flemming in 
Zeit.f. wiss. Mik., vi, 1, 1889, p. 39; and vi, 2, 1889, p. 178). 
The remarks on over-fixation made in § 39 apply to both 
these mixtures, and more especially to the stronger one. For 
staining see last §. 
48. Platino-aceto-osmic Acid (Hermann’s solution). This 
extremely important reagent is historically a modification of 
Flemming’s solution, platinum chloride being taken instead 
of chromic acid. See § 65. 
49. Nitric Acid (Altmann, Arch. Anat. u. Phys., 1881, p. 219). 
Altmann employs dilute nitric acid, containing from 3 to 3J per cent, pure 
acid. Such a solution has a sp. gr. of about 1'02 ; an araeometer may con- 
veniently he used to determine the concentration of the solution. Stronger 
solutions have been used, but do not give such good final results. After 
extensive trial I am convinced that Altmann’s solutionis much too weak 
a reagent for general work, and should he discarded. 
His (ibid., 1877, p. 115) recommended a 10 per cent, solution. Flemming 
at one time employed solutions of 40 to 50 per cent, for the ova of inverte- 
brates. This of course has the advantage of a very rapid fixing action. 
Nitric acid has the valuable property of hardening yolk without making 
it brittle. But, for general purposes at all events, the pure nitric acid solu- 
tions may be considered to be superseded by Perenyi’s chromo-nitric acid 
mixture (below, next §). 
50. Chromo-nitric Acid (Pekenyi’s formula, Zool. Anzeig., 
v, 1882, p. 459).— 
4 parts 10 per cent, nitric acid. 
3 parts alcohol. 
3 parts 0*5 per cent, chromic acid. 
These are mixed, and after a short time give a fine violet- 
coloured solution. 
The objects are immersed for four to five hours, and then 
passed through 70 per cent, alcohol (twenty-four hours), 
strong alcohol (some days), absolute alcohol (four to five days). 
They are then fit for cutting. The advantage of the process 
is, amongst others, that segmentation spheres and nuclei are 36 
CHAPTER IV. 
perfectly fixed, the ova do not become porous, and cut like 
cartilage. 
Chromo-nitric acid is not only an embryological I’eagent, 
and a very important one, but also an admirable one for 
general work. I have found it altogether excellent for pre- 
serving marine organisms, especially large forms. Strong 
alcohol need only be used if the objects are destined to be- 
sectioned. 
For a special formula for embryological purposes, see Part 
II, “ Embryological Methods.” 
51. Picro-ehromic Acid (Fol. Lehrh., p. 100).— 
Picric acid, sol. sat. in water . . . .10 vols. 
1 per cent, chromic acid solution . . . 25 „ 
Water . . . . . . . . 65 „ 
At the instant of using, you may add 0'005 of osmic acid, which makes 
the action more energetic. Wash with water (hot, nearly boiling water is 
best), and then with alcohol. Fol says, “ This reagent hardens tissues 
admirably, without hindering staining in any way ; but it is not very pene- 
trating and fixes slowly.” I find it gives very good results with Annelids. 
I have seen Fol’s formula, with the addition of a trace of acetic acid,, 
quoted as “ liquid of Haensel ”—I know not with what justification. 
52.—Chromic Acid and Platinic Chloride (Merkel’s solution ; 
from Mitth. Zool. Stat. Neapel, 1881, p. 11).—Equal volumes 
of P400 solution of chromic acid and P400 solution of platinic 
chloride (PtClJ. Objects should remain in it for several 
hours or even days, as it does not harden very rapidly. After 
washing out with alcohol of 50 per cent, to 70 per cent., 
objects stain excellently, notwithstanding the admixture of 
chromic acid. This is a very delicate and admirable fixative. 
If objects that have been fixed by osmium be put into it for 
some hours, blackening is effectually prevented. 
Salts. 
53. Bichromate and Cupric Sulphate Mixture (Ivultschitzky, 
Zeit.f. wiss. Milt., iv, 3, 1887, p. 348).—A saturated solution of bichromate 
of potash and sulphate of copper in 50 per cent, alcohol, to which is added 
at the instant of using a little acetic acid, five or six drops per 100 c.c. 
To make the solution, add the finely powdered salts to the alcohol in 
excess, and leave them together in total darkness, for twenty-four hours. 
Fix for twelve to twenty-four hours in the dark, otherwise the salts will 
be precipitated. Then treat with strong alcohol for twelve to twenty-four 
hours, and make sections. 
The rationale of this mixture is stated to he that it fixes tissues faithfully, FIXING AGENTS. MINERAL ACIDS AND THEIR SALTS. 
37 
without causing the production of tlie delusive reticular precipitates of 
albuminoids which we'have mentioned as being produced by chromic acid— 
that is the part played by the bichromate and sulphate; and that it also 
fixes faithfully the chromatin of nuclei—that is the part played by the 
organic acid. 
53a. For Lindsay-Joitnson’s Bichromo-osmic Mixture, which 
may be used with excellent results as a gentle fixative, see 
§ 97. 
54. The Chromates are useful as hardening rather than fixing agents. 
They have a very mild and even action on tissues, but are not at all pene- 
trating and act very slowly. They may still be found useful for fixing 
certain tissues, some of those of Mollusca for example. For mixtures that 
may be used for such a purpose, see the chapter on Hardening Agents. 
55. Cupric Sulphate.—Not of general utility. See “ Siphonophora.” 
56. Alum.—Alum has been used for fixing purposes. After an extended 
experience of it, I only quote it in order to recommend that it be avoided at 
all costs. CHAPTER V. 
FIXING AGENTS. CHLORIDES, ORGAN TO ACIDS, AND 0TI1EUS. 
Chlorides. 
57. Bichloride of Mercury (Corrosive Sublimate).—Corrosive 
sublimate is stated in tlie books to be soluble in about sixteen 
parts of cold and three of boding- water. It will probably 
be found that the aqueous solution contains about 5 per cent, 
of the sublimate at the temperature of the laboratory. It is 
more soluble in alcohol than in water, and still more so in 
ether. Its solubility in all these menstrua is augmented by 
the addition of hydrochloric acid, ammonious chloride, or 
camphor. With sodium chloride it forms a more easily 
soluble double salt; hence seawater may dissolve as much 
as 15 per cent., and hence the composition of the liquid of 
Lang. 
The simple aqueous solutions frequently deteriorate in even a short time 
through the formation of a pulverulent precipitate. The nature of this 
precipitate is unknown to me, and I have been unable to find any certain 
means of preventing its formation Thinking that it maybe due in part to 
ammonia derived from the air, I have lately been in the habit of adding a 
little nitric acid to my solutions, and certainly have found that they thus 
keep much better. The addition of one drop of nitric acid for each cubic 
centimetre of sublimate solution has already, 1 have since found, been re- 
commended by Fkenzel (see Mann, in Zeit. f. wiss. Mik., xi, 4, 1894, 
p. 480). In any case, for work in which it is desired to obtain as energetic a 
fixing action as possible, it is well to use only freshly made up solutions. 
For fixing, corrosive sublimate may be, and very frequently 
is, used pure; but in most cases a finer fixation will be ob- 
tained if it be acidified with acetic acid, say about 1 percent, 
of the acid. I find that a saturated solution in 5 'per cent, 
acetic acid is a very good formula for marine animals. Van 
Beneden has recommended a saturated solution in 25 per 
cent, acetic acid. 
It is sometimes advisable to take the most concentrated FIXING AGENTS. CHLORIDES, ORGANfC ACIDS, ETC. 
39 
solution obtainable. The cold saturated aqueous solution 
will suffice in most cases; but for some very contractile 
forms (coral polypes, Planaria), a concentrated solution 
in warm or even boiling water should be employed. For 
Arthropoda the alcoholic solution is frequently indicated. 
Delicate objects, however, may require treatment with weak 
solutions. Harting found solutions of 0'2 to 05 per cent, 
suitable for blood-corpuscles, and Pacini’s fluids are much of 
the same strength. For these see the chapter on Examination 
Media. 
Objects should in all cases be removed from the fixing bath 
as soon as fixed, that is, in other words, as soon as they are 
seen to have become opaque throughout, which is practically as 
soon as they are penetrated by the liquid. Small objects are 
fixed in a few minutes. I have found that a “ salivary ” gland 
of the larva of Chironomus is thoroughly fixed in three seconds. 
Wash out with water or with alcohol. I consider alcohol 
always almost preferable. Alcohol of about 70 per cent, may 
be taken. The extraction of the sublimate is hastened by the 
addition of a little camphor to the alcohol. Or, better, a little 
tincture of iodine may be added to the liquid, either alcohol or 
water, used for washing, and the liquid changed until it no 
longer becomes discoloured by the objects. It is important 
that the sublimate be thoroughly removed from the tissues, 
otherwise they become brittle. They will also become brittle 
if they are kept long in alcohol. 
It has been advised that solution of iodine in potassium 
iodide be taken instead of tincture of iodine for the washing 
out. But that is quite wrong, iodine in potassium iodide 
precipitates corrosive sublimate. I am much obliged to my 
friend Prof. Gilson for calling my attention to this point. 
It may happen that if the extraction of the excess of subli- 
mate from the tissues in bulk has been insufficient, crystals of 
sublimate may form in the sections after they have been 
mounted in balsam. This may easily be prevented by treating 
the sections themselves with tincture of iodine for a quarter 
of an hour before mounting. This will do away with the 
necessity of treating the tissues in bulk with iodine, which is 
frequently a very long process (unless it is desired to keep 
the material for a long time in alcohol before making the 
sections). 40 
CHAPTER V. 
You may stain in any way you like. Carmine stains are 
peculiarly brilliant after sublimate (owing, it has been said, 
to the formation of mercuric carminate). It is not necessary 
that the objects be thoroughly washed out before staining; 
the staining processes themselves may be made to constitute 
a part of the washing-out process. 
It must be remembered that the solutions must not be 
touched with iron or steel, as these produce precipitates that 
may hurt the preparations. To manipulate the objects, wood, 
glass, or platinum may be used; for dissecting them, hedge- 
hog spines, or quill pens. 
When properly employed, sublimate is for general work un- 
doubtedly a fixing agent of the very highest order. It is 
applicable to most classes of objects. It is perhaps less 
applicable, in the pure form, to Arthropods, as it possesses no 
great power of penetrating chitin. For cytological work it is, 
according to my experience, not to be trusted, and not to be 
recommended. 
58. Corrosive Sublimate (Lang’s formula, ‘ Zool. Anzeiger,’ 1878, i, 
p. 14). For Planaria.—Take— 
Distilled water . . . 100 parts by weight. 
Chloride of sodium . . 6 to 10 parts. 
Acetic acid . . . . 6 to 8 „ 
Bichloride of mercury . . 3 to 12 „ 
(Alum, in some cases . . £). 
Second formula (ibid., 1879, ii, p. 46).—Make a concentrated solution of 
corrosive sublimate in picro-sulphuric acid, to which has been added 5 per 
cent, of acetic acid. 
59. Other Simple Solutions.—A solution containing 5 g. sublimate, 
05 g. sodium chloride, and 100 c.c. water, has been quoted as “ solution of 
GrATTLE.” 
Keisee’s solution consists of 10 g. sublimate, 3 g. glacial 
acetic acid, and 300 g. distilled water (from Zeit. f. wiss. 
Mik., xi, 3, p. 378). 
M. Heidenhain has recommended a 0*5 per cent, solution 
of sodium chloride saturated while hot with sublimate. 
60. Gilson’s Mercuro-nitric Mixture.—I am indebted to Prof. 
Gilson for kindly sending the latest formula (1895), which is 
as follows: FIXING agents, chlorides, organic acids, etc. 
41 
Nitric Acid of 46° strength (this 
would be sp. gr. F456, or 80 per 
cent., nearly) . . . .78 c.c. 
Glacial acetic acid . . . . 22 ,, 
Corrosive sublimate . . . 95 to 100 grins. 
60 per cent, alcohol . . . 500 c.c. 
Distilled water .... 4400 ,, 
When required for marine animals add a few crystals of 
iodine, which will prevent the formation of precipitates of sea 
salts. If in any case the preparations should show a granular 
precipitate, due probably to an abundance of phosphates in 
the tissues, the precipitate may be removed by washing with 
water containing a little tincture of iodine (not iodide of 
potassium, which would precipitate the sublimate). 
I have tried this mixture and find that it affords in general 
a faithful and delicate fixation, and gives to tissues an 
excellent consistency. Objects may remain in it for a con- 
siderable time without hurt. Tissues are left in a state very 
favorable for staining. The liquid has a high degree of 
penetration. A treatment for a few days with it will serve 
to remove the albumen from the ova of Batrachians. This 
liquid may be i-ecommended to beginners, as it is very easy to 
work with. For some objects, as I have found, the pro- 
portion of sublimate may be increased with advantage. 
61, Rabl’s Picro-sublimate (Zeit. f. wiss. Mik., xi, 2, 1894, 
p. 165).— Sublimate, saturated solution in water, 1 vol.; a 
similar solution of picric acid, 1 vol. ; distilled water, 2 vols. 
Embryos may be left in it for tAvelve hours, washed for two 
hours in water, and brought into weak alcohol. 
62. Mann’s Piero-sublimate (op. cit., xi, 4, 1895, p.480).— 1 
per cent, of picric acid with or without 1 per cent, of tannin 
in a saturated solution of sublimate in normal salt solution. 
The same author’s Alcoholic Picro-sublimate (An at. Anz., 
8, 1893, pp. 441—443) consists of absolute alcohol 100 c.c., 
picric acid 4 grms., sublimate 15 grrns., tannin 6 to 8 grms. 
The tannin is added in order to prevent excessive hardening. 
The same author’s Mercurio-osmic Mixture has been given 
(§ 37). 
Yom Rath’s Picro-sublimate (Anat. Anz., xi, 9, 1895, p. 286). 42 
CHAPTER V. 
—Cold saturated solution of picric acid, 1 part; hot saturated 
solution of sublimate, 1 part; glacial acetic acid, to 1 per 
cent. Fix for several hours and bring direct into alcohol. 
The same author’s Picro-sublimate-osmic Mixture (loc. cit.) 
consists of the above with the addition of 10 per cent, of 2 per 
cent, osmic acid solution. 
63. Zenker’s Mixture (Miinchener vied. Wochenschr., 24,1894, p. 534; 
quoted from Mercier, Zeit. f. wiss. Mile., xi, 4, 1894, p. 471, where will be 
found minute instructions for using it). Five per cent, of sublimate and 5 
per cent, of glacial acetic acid, dissolved in solution of Muller. Fix for 
several hours, wash out with water, treat the tissues in bulk, or the sections, 
with alcohol containing tincture of iodine. 
63a. Foa’s Mixture {Quart. Journ. Mic. Sci., 1895, p. 287 ; Journ. 
JRoy. Mic. Soc., 1895, p. 486). Equal parts of saturated solution of 
sublimate in normal salt solution, and of liquid of Muller, or 5 per cent, 
solution of bichromate. 
The rationality of these mixtures is not apparent. The addition of the 
chrome salts is unfavorable to staining with carmine or hsematin. 
64. Chloride of Platinum (Platinic Chloride, PtClJ.—An 
extremely valuable reagent, originally introduced for the study 
of karyokinesis, but of general application. Rabl, to whom 
we owe the introduction of this agent, employed an aqueous 
solution of P300. The objects remain in it for twenty-four 
hours, and are then washed with water, hardened in alcohol, 
and sectioned. Rabl stained with Delafield’s hsematoxylin, or 
with safranin. 
The action of platinum chloride is similar to that of gold 
chloride, with the advantage that there is no blackening of 
the preparations. Rabl finds it give better results (for the 
study of karyokinesis) than any other reagent except chro- 
moformic acid (§ 44). It causes a slight shrinkage of the 
chromatin elements, a condition that renders the granules of 
Pfitzuer and the longitudinal division of the elements very 
distinctly visible (see Rabl’s well-known paper in Morph. 
Jahrb., Bd. x, 1884, p. 216). 
Platinum chloride is an extremely deliquescent salt, and for 
this reason had better be procured in solution. Ten per cent, 
solutions are found in commerce. 
For Merkel’s solution (chromo-platinic mixture) see ante, 
§ 52. FIXING AGENTS. CHLORIDES, ORGANIC ACIDS, ETC. 
43 
For Rabl’s Platino-sublimate Mixture, see Embryological 
Methods. 
65. Platino-aceto-osmic Mixture (Hermann, Arcli. f. mile. 
Ancit., xxxiv, 1889, p. 58).—The author obtained excellent 
results by substituting 1 per cent, platinic chloride for the 
chromic acid in Flemming’s strong formula for chromo-aceto- 
osmic acid (§ 47), the other ingredients either remaining as 
before, or the osmium being diminished one half. Tlius, 
1 per cent, platinic chloride 15 parts, glacial acetic acid 1 
part, and 2 per cent, osmic acid either 4 parts or only 2 parts. 
Hermann found that protoplasmic structures are thus better 
preserved than with the chromic mixture. It was noted 
above (§ 40) that a chief objection to the use of chromic acid 
is that it precipitates certain of the liquid albuminoids of 
tissues in the form of filaments or networks, which are often 
of great regularity, and simulate structural elements of the 
tissues. This 'platinum chloride docs not do. 
The after-treatment and staining should be the same as for 
objects treated with Flemming’s solution. 
The remarks in § 47, as to the deterioration of Flemming’s 
solution by evaporation of: osmium, apply with equal force to 
Hermann’s mixture. 
After considerable experience of this reagent I find that it 
lias the advantage of giving more colourless preparations, and, 
it may be in some cases, a more delicate fixation. But the 
fixation is certainly not in all cases superior, the hardening 
is less energetic, and in some cases not so energetic as may be 
desired. As this is the most expensive of all reagents, I am 
glad to find that for general work there is no valid reason for 
supposing that it ought to take the place of Flemming’s 
mixture. 
60. Palladium Chloride.—Palladium chloride has been recommended 
by experienced workers. It is used in solutions of 1’300, 1*600, or 1800 
strength, for from one to two minutes. Cattaneo recommends it as being 
the best of fixatives for Infusoria. Tissues are impregnated and coloured 
brown by it. For small objects one or two minutes will suffice for fixation. 
This salt is found in commerce in the solid state. To dissolve it, take 10 
grammes of the salt, one litre of water, and four to six drops of hydrochloric 
acid. Solution will be effected in twenty-four hours. 
Frenkel (Anat. Anz., viii, 1893, p. 538; Zeit. f. wiss. Mih., x, 2, 1893, 
p. 243) recommends for connective tissue a mixture of 15 parts 1 per cent. 44 
CHAPTER V. 
palladium chloride, 5 parts 2 per cent, osmic acid, and a few drops of acetic 
acid. 
07. Perchloride of Iron (Fol, Zeit.f. wiss. Zool., Bd. xxxviii, 1883, 
p. 491; and Lehrb. d. vergl. mile. Anat., p. 102).—Fed recommends 1 vol. 
of Tinct. Ferri Perclilor. P. B. diluted with 5 to 10 vols. of 70 per cent, 
alcohol. This reagent has too many defects to be recoinmendable except in 
very special cases. 
The tincture diluted with 3 to 4 vols. of either alcohol or water has been 
recommended for fixing medullated nerve by Platneu (Zeit. f. wiss. Mile., 
vi, 2, 1889, p. 187). 
Organic Acids, and other Agents. 
68. Acetic Acid.—The place of honour amongst organic 
acids considered as fixing agents appears rightfully to belong 
to this old-fashioned reagent. In the first edition of this 
work it was merely stated that acetic and formic acid “ are 
useful and well-known fixatives of nuclei. Flemming, who 
has made a special investigation of their action, finds (Zell- 
substanz, &c , p. 380) that the best strength is from 02 to 1 per 
cent. Strengths of 5 percent, and more bring out the nuclein 
structures clearly at first, but after a time cause them to swell 
and become pale, which is not the case with the weaker 
strengths ” {ibid., p. 103). It must now be stated that, thanks 
to v. BeNeden, the strong acid has become established as a 
most precious fixative of the most varied zoological objects. 
It is particularly applicable to very contractile objects, such 
as many Vermes, Ccelenterata, and Nudibranehs; it kills with 
the utmost rapidity, and has a tendency to leave them fixed 
in the state of extension. The modus operandi is in general 
as follows :—Pour glacial acetic acid in liberal quantity over 
the organisms, leave them until they are penetrated by it— 
which should be in five or six minutes, as the strong acid is a 
highly penetrating reagent—and wash out in frequent changes 
of alcohol of gradually increasing strength. Some persons 
begin with 30 per cent, alcohol, but this appears to me rather 
weak, and I think 70 per cent, or at least 50 per cent alcohol 
should be preferred. 
In the Traite des Moth. Teclin., 1887,1 stated that the reason 
why glacial acetic acid was not more used was that it did not 
faithfully preserve delicate histological and cytological detail. 
I now believe that if the instructions above given be followed. FIXING AGENTS. CHLORIDES, ORGANIC ACIDS, ETC. 
45 
in particular as regards the employment of the glacial acid, 
and the washing out with somewhat strong alcohol, even 
delicate detail will generally be found well preserved. I see 
no reason why other energetic reagents should not be com- 
bined with the glacial acetic acid if desired. Dr. Lindsay 
Johnson (in. litt.) has found that one of the best fixatives for 
retina is a mixture of equal parts glacial acetic acid and 2 per 
cent, osmic acid. S. Lo Bianco adds to the concentrated acid 
one-tenth of a 1 per cent, solution of chromic acid. He finds 
that even this small proportion of chromic acid serves to 
counteract in a marked degree the softening action of the 
acetic acid. 
69. Acetic Alcohol (Carnoy, La Cellule, t. iii, 1, 1886, p. 6; 
and ibid., 1887, 2, p. 276; v. Beneden et Neyt, Bull. Ac. roy. d. 
sci. de Belg., t. xiv, 1887, p. 218; Zacharias, Anat. Anz., iii, 
Jahrg., 1, 1888, pp. 24—27; v. Gehuchten, ibid., 8, p. 227). 
—Carnoy has given two formulae for this important reagent. 
The first is— 
Glacial acetic acid . . .1 part. 
Absolute alcohol . . . .3 parts. 
The second is— 
Glacial acetic acid . . 1 part. 
Absolute alcohol . . . .6 parts. 
Chloroform . . . . 3 ,, 
The addition of chloroform is said to render the action of 
the mixture more rapid. 
V. Beneden and Neyt take equal volumes of glacial acid 
and absolute alcohol. 
Zacharias takes— 
Glacial acetic acid . . .1 part. 
Absolute alcohol . . . .4 parts. 
Osmic acid ..... A few drops. 
Acetic alcohol is one of the most penetrating and quickly - 
acting fixatives known. It preserves nuclei admirably, and 
admits of admirable staining in any way that may be pre- 
ferred. It was imagined by all of the authors quoted for the 
study of karyokinesis in the ova of Ascaris,—proverbially one 
of the most difficult objects to fix,—but it is applicable to 
tissues in general. You may wash them out with alcohol and 
treat them afterwards in any way that may be preferred. It 46 
CHAPTER Y. 
will be well, however, to avoid treatment with water as much 
as possible. 
70. Formic Acid may be used dilute in the same wajr as acetic acid 
{supra, § 68). It is probable that it might also take the place of acetic 
acid in the concentrated form, but I am not aware of any experiments in 
this direction. 
71. Chloride and Acetate of Copper (Ripart et Petit's Liquid, 
Carnoy, La Biologie Gellulaire, p. 94). 
Camphor water (not saturated) . 75 grammes. 
Distilled water. . . . .75 „ 
Crystallised acetic acid ... 1 gramme. 
Acetate of copper . . . O'30 „ 
Chloride of copper . . . . O'30 „ 
This is a very moderate and delicate fixative. I consider 
that it has not sufficient hardening power for objects that are 
intended to be dehydrated and mounted in balsam, but is fre- 
quently excellent and sometimes indispensable for objects 
that are to be studied in as fresh a state as possible in aqueous 
media. Objects fixed in it stain instantaneously and perfectly 
with methyl green. Osmic acid may be added to the liquid 
to increase the fixing action. For cytological researches this 
is a most invaluable medium. 
72. Acetate of Uranium (Schenk, Mitth. a. d. Embryol. Inst. Wien, 
1882, p. 95 ; cf. Gilson, La Cellule, 1, 1885, p. 141).—This reagent is very 
similar in its properties to picric acid. It has a mild fixing action, and a 
high degree of penetration, which may make it useful for Arthropoda. It 
may be combined with methyl green, which it does not precipitate. 
73. Iodine.—Iodine possesses considerable hardening properties, and a 
very high degree of penetration. Kent (Manual of the Infusoria, 1881, 
p. 114 ; Journ. Roy. Mic. Soc. (N.S.), iii, 1883, p. 730) has found it to act 
in a manner almost identical with osmic acid, and in some instances even 
more efficiently (for fixing Infusoria). His instructions are as follows:— 
“ Prepare a saturated solution of potassic iodide in distilled water, saturate 
this solution with iodine, filter, and dilute to a brown-sherry colour. A very 
small portion only of the fluid is to be added to that containing the 
Infusoria.” 
Or you may use the solution of Lugol, of which the formula is as 
follows : 
Water ........ 100 parts. 
Iodide of potassium ..... 6 „ 
Iodine ........ 4 „ 
Iodine certainly kills cells very rapidly, without deforming them. Per- .FIXING AGENTS. CHLORIDES, ORGANIC ACIDS, ETC. 
47 
sonally I have found it very useful for the examination of spermatozoa. 
Unfortunately I am not acquainted with any nuclear stain that will work 
well with it. 
Very small objects may be instantaneously fixed by means of vapour of 
iodine. Crystals of iodine may be heated in a test-tube till the vapours 
are given off; then on inclining the tube the heavy vapours may be made to 
flow over the objects arranged on a slide. The slide should then be warmed 
to about 40° C. for 2 or 3 minutes in order to evaporate the iodine from the 
objects, which may then be mounted or otherwise treated as desired (Over- 
tox, Zeit.f. wiss. Mile., vii, 1, 1890, p. 14). 
74. Picric Acid.—Picric acid should always be employed in 
the form of a strong solution. (That is to say, strong solu- 
tions must always be employed when it is desired to make 
sections or other preparations of tissues with the elements in 
situ, as weak solutions macerate; but for dissociation prepara- 
tions or the fixation of isolated cells, weak solutions may be 
taken. Flemming finds that the fixation of nuclear figures is 
equally good with strong or weak solutions.) The saturated 
solution is the one most employed. (One part of picric acid 
dissolves in about 75 parts of cold water; in hot water it is 
very much more soluble.) Objects should remain in it for 
from a few seconds to twenty-four hours, according to their 
size. For Infusoria one to at most two minutes will suffice, 
whilst objects of a thickness of several millimetres require 
from three to six hours’ immersion. 
Picric acid should always be washed out with alcohol, as 
water is hurtful to tissues that have been prepared in it. For 
the same reason during all remaining stages of treatment, 
water should be avoided; staining should be performed by 
means of alcoholic solutions, the only exceptions to this rule 
being in favour of picro-carmine, which, probably on account 
of the picric acid contained in it, does not appear to exert so 
injurious an influence as other aqueous stains, and of methyl 
green, and some few other aqueous stains that are themselves 
weak hardening agents. It is one of the advantages of 
picric acid that, by sufficiently pi’olonged soaking, it can with 
certainty be entirely removed from any tissue by means of 
alcohol. 
It has been found by Jelinek (Zeit.f. wiss. Mik., xi, 2, 1894, 
p. 242) that the extraction of picric acid is greatly quickened 
by the addition of a base to the wash-alcohol. He recommends 
carbonate of lithine. A few drops of a saturated solution of 48 
CHAPTER V. 
the salt in water are added to the alcohol; a slight precipi- 
tate is formed. The objects are put into the turbid alcohol, 
which becomes clear and yellow in proportion as the picrin 
is extracted. Further quantities of carbonate are added from 
time to time until the colour has been entirely extracted from 
the tissues. 
Tissues fixed in picric acid can, after removal of the acid 
by soaking, be perfectly stained in auy stain. Mayer’s para- 
carmine, Grenadier's alcoholic borax-carmine, or Mayer’s 
hsemacalcium may be recommended for entire objects. 
The most important property of picric acid is its great 
penetration. This renders it peculiarly suitable for the pre- 
paration of chitinous structures. For such objects alcohol of 
70 per cent, to 90 per cent, should be taken for washing out, 
and staining should be done by means of Mayer’s cochineal 
or hiemacalcium. 
In very many if not most cases it is advantageous to employ picric acid 
in the manner suggested by Kleinenberg (see below), that is, in combination 
with sulphuric acid ; or with nitric acid, or hydrochloric acid, as suggested 
by P. Mayer (see below). 
75. Picro-sulphuric Acid (Kleinenberg, Quart. Journ. Mic. 
Sci., April, 1879, p. 208 ; Mayer, Journ. Roy. Mic. Soc. (N.S.), 
ii (1882), p. 867).—By picro-sulphuric acid, without any 
qualifying term, I understand a fluid made (following Mayer 
1. c.) as follows:—Distilled water, 100 vols.; sulphuric acid 
2 vols.; picric acid, as much as will dissolve (this will he about 
0’25 per cent.; as the picric acid is much less soluble in 
sulphuric acid solution than in water). This may also, in 
any case in which confusion is likely to arise, be called 
“ concentrated” or “ undiluted picro-sulphuric acid.” 
By “ liquid of Kleinenberg ” I understand a mixture sug- 
gested by Kleinenberg’ (l. c.), and best made by diluting- the 
concentrated picro-sulphuric acid prepared as above with three 
times its volume of water. 
(Kleinenberg also directed the addition of as much creasote as would mix. 
This was done with the idea of eliminating the swellings produced in some 
objects by the liquid, but it has been found not to have the effect attributed 
to it, and has been abandoned. For, (Lehrb., p. 100) states that the same 
end may be attained by adding one third vol. of 1 per cent, chromic acid.) FIXING AGENTS. CHLORIDES, ORGANIC ACIDS, ETC. 
49 
Of these two formulae the one commonly employed is that 
given by Kleinenberg,—the dilute mixture; undiluted picro- 
sulphuric acid being reserved for objects requiring special 
treatment, chiefly Arthropods. I may as well say at once 
that in my opinion this practice is erroneous, for I hold that 
Kleinenberg’s solution is much weaker than is desirable in the 
majority of cases, and should be reserved for special cases, 
such perhaps as that for which it was originally proposed, the 
embryology of the earthworm; and the concentrated solution 
should be the one taken for general work. This particularly 
applies to marine organisms. 
The treatment is the same in either case. “ The object to 
be preserved should remain in the liquid for three, four, or 
more hours ; then it should be transferred, in order to harden 
it and remove the acid, into 70 per cent, alcohol, where it is 
to remain five or six hours. From this it is to be removed 
into 90 per cent, alcohol, where it is to be changed until the 
yellow tint has either disappeared or greatly diminished.55 
Warm alcohol extracts the acid much more quickly than 
cold, without which weeks may be required to fully remove the 
acid from chitinous structures. I call attention here to what 
was said as to washing out under the head of picric acid, viz. 
that washing out must never he done with water. This is a 
most important point, and one that is not sufficiently attended 
to. You may stain as directed above for picric acid. You 
may, of course, stain sections with alcoholic solutions of 
safranin or the like. 
The advantages of picro-sulphurie acid as a fixing agent are, that it kills 
tissues very rapidly, that it has great penetrating power, that it can be 
totally soaked out of the structures with alcohol (it is much more easily 
removed from the tissues than pure picric acid), leaving them in a good con- 
dition for staining, and, in the case of marine organisms, that it effectually 
removes the different salts of sea-water that are present in them. 
It has many disadvantages. For vertebrata it should be used with caution, 
on account of the swelling caused by sulphuric acid in connective tissue. 
For structures that contain much lime it is not to be recommended, for it 
dissolves the lime and throws it down as crystals of gypsum in the tissues. 
(For such structures the picro-nitric or picro-hydrochloric acid is to be pre- 
ferred.) In numberless cases it produces swellings and maceration. For 
the preservation of delicate, watery organisms, such as Medusae, it is an 
abomination. For cytological researches it should he avoided, as its action 
on both cytoplasm and nuclei is frequently most injurious. On the whole, 
I find that for such objects as Arthropoda it is valuable on account of its 50 
CHAPTER Y. 
great penetrating power, the possibility of removing the acid entirely by 
washing, and the facility thereby given for staining in toto. But for 
general work, I consider that it is one of the most overrated reagents that 
ever came into favour through the prestige of authority. 
76. Picro-nitric Acid (Mayer, Mitth. Zool. Stat. Neapel, 1881, p. 5 ; 
Journ. Roy. Mic. Soc. (N.S.), ii, 1882, p. 868).— 
Water ........ 100 vols. 
Nitric acid (of 25 per cent. N205) . . . 5 „ 
Picric acid, as much as will dissolve. 
The fluid is used undiluted. 
The properties of this fluid are very similar to those of picro-sulphuric 
acid, with the advantage of avoiding the formation of gypsum crystals, and 
the disadvantage that it is much more difficult to soak out of the tissues. 
“ Mayer recommends it strongly, and states that with eggs containing a 
large amount of yolk material, like those of Palinurus, it gives better results 
than nitric, picric, or picro-sulphuric acid.” 
77. Picro-hydroehloric Acid (Mayer, ibid.).— 
Water 100 vols. 
Hydrochloric acid (of 25 per cent. HC1) . . 8 „ 
Picric acid, as much as will dissolve. 
The fluid is used undiluted. 
The properties of this fluid are similar to those of picro-nitric acid. 
78. Piero-chromic Acid. See ante, § 51. 
79. Picro-osmic Acid.—Flemming (Zells. Kern uJZellth., p. 381) has 
experimented with mixtures made by substituting picric for chromic acid in 
the ehromo-osmic mixtures (ante, §§ 46 and 47). The results are identical 
so far as regards the fixation (of nuclei) ; but staining is rendered more 
difficult. 
0. vom Rath (Anat. Anz., xi, 1895, p. 289) adds to 200 c.c. of saturated 
aqueous solution of picric acid, 12 c.c. of 2 per cent, solution of osmic acid, 
and 2 c.c. of glacial acetic acid. 
80. Picro-platinic and Picro-platin-osmie Mixtures.—0. tom 
Rath (1. c., pp. 282, 285) makes a picro-platinic mixture with 200 c.c. 
saturated aqueous solution of picric acid, 1 g. of platinic chloride (dissolved 
in 10 c.c. of water), and 2 c.c. of glacial acetic acid. 
The picro-platin-osmic mixture is made by adding to the foregoing 25 c.c. 
of 2 per cent, osmic acid. 
81. Picric Alcohol (Gage, Proc. Amer. Soc. Micr., 1890, p. 120; 
Journ. Roy. Mic. Soc., 1891, p. 418).—Alcohol (95 per cent.), 250 parts; 
water, 250 parts ; picric acid, 1 part. Fix for about 24 hours, wash out for 
a day in alcohol of 67 to 70 per cent., and then for a day or longer in alcohol 
of 75 to 82 per cent. FIXING AGENTS. CHLORIDES, ORGANIC ACIDS, ETC. 
51 
Other Fixing Agents. 
82. Alcohol.—For fixing, only two grades of alcohol are 
found generally useful—very weak alcohol on the one hand, 
and absolute alcohol on the other hand. Absolute alcohol 
ranks as a fixing agent because it kills and hardens with such 
rapidity that structures have not time to get deformed in the 
process by the energetic dehydration that unavoidably takes 
place. Dilute alcohol ranks as a fixing agent in virtue of 
being of such a strength as to possess a sufficiently energetic 
coagulating action and yet contain enough water to have but 
a feeble and innocuous dehydrating action. The intermediate 
grades do not realise these conditions, and therefore should 
not be employed alone for fixing. But they may be very 
useful in combination with other fixing agents (such as cor- 
rosive sublimate, chromic acid or nitric acid) by greatly 
enhancing their penetrating power; 70 per cent, is a good 
grade for this purpose. 
83. One-third Alcohol.—The one grade of weak alcohol that is 
found generally useful for fixing is one third alcohol, or 
Ranvier's Alcohol, known in France as “ Alcool au tiers,” 
which is the name given to it by Ranvier himself; in Ger- 
many as “ Drittelalcohol ” or “ Ranviersche alcohol dilutus ” ; 
in Italy as alcool al terzo.” It consists of two'parts of water 
and one part of alcohol of 90 per cent, (and not of absolute 
alcohol, as was stated by an oversight in the first edition— 
an error which I have seen copied in more than one place). 
See the Traits Technique of Ranvier, p. 241, et passim. 
Care should be taken that the alcohol is of the strength 
specified, as the effects of this reagent depend to a remark- 
able degree on its strength. 
Objects may be left for twenty-four hours in this alcohol; 
not more, unless there be no reason for avoiding maceration, 
which will generally occur after that time. You may con- 
veniently stain with picro-carmine, alum-carmine, or methyl 
green. 
This reagent is a very mild fixative. Its hardening action 
is so slight that it is seldom indicated for the fixing of objects 
that are intended to be sectioned. Its chief use is for ex- 
temporaneous and disassociation preparations. 52 
CHAPTER V. 
84. Absolute Alcohol.—This is also a very valuable reagent. 
It preserves very well the structure of nuclei, which is by no 
means the case with one-third alcohol. It has over the latter 
also the advantage of superior penetrating power, being 
indeed one of the most penetrating of known fixing agents. 
Mayer finds that boiling absolute alcohol is often the only 
means of killing certain Ai-thropoda rapidly enough to avoid 
maceration brought about by the slowness of penetration of 
common cold alcohol (especially in the case of Tracheata). 
It is important to employ for fixing a very large proportion 
of alcohol. Alum-carmine is a good stain for small specimens 
so fixed. For preservation, the object should be put into a 
weaker alcohol, 90 per cent, or less. 
Absolute alcohol is found in commerce. It is a product that it is almost 
impossible to preserve in use, on account of the rapidity with which it 
hydrates on exposure to air. Fol recommends that a little quicklime he 
kept in it. This absorbs part at least of the moisture drawn by the alcohol 
from the air, and has the further advantage of neutralising the acid that is 
frequently present in commercial alcohol. 
Another plan that I have seen recommended is to suspend strips of gelatin 
in it. It is stated that by this means ordinary alcohol may be rendered 
absolute. 
Ranvier adopts the following plan for preparing an alcohol absolute 
enough for all practical purposes. Strong (95 per cent.) alcohol is treated 
with calcined cupric sulphate, with which it is shaken up and allowed to 
remain for a day or two. It is then decanted and treated with fresh cupric 
sulphate, and the operation is repeated until the fresh cupric sulphate no 
longer becomes conspicuously blue on contact with the alcohol; or until, on 
a drop of the alcohol being mixed with a drop of turpentine, no particles of 
water can be seen in it under the microscope. The cupric sulphate is pre- 
pared by calcining common blue vitriol in a porcelain capsule over a spirit 
lamp or gas burner until it becomes white, and then reducing it to powder 
(see Proc. Acad. Nat. Sci. Philad., 1884, p. 27 ; Science Record, ii, 1884, 
p. 65; Journ. Roy. Mic. Soc. (N.S.), iv, 1884, pp. 322 and 984). 
85. Acidulated Alcohol (Paul Mayee, Mitth. Zool. Stat. Neapel, ii, 
1881, p. 7).—To 97 vols. of 90 per cent, alcohol, in which is dissolved a small 
quantity of picric acid, add 3 vols. pure hydrochloric acid. Leave the 
specimens in the mixture only just long enough to ensure that they are 
thoroughly penetrated by it. Wash out with 90 per cent, alcohol, the dis- 
appearance of the yellow stain of the picric acid being a sign that all the 
acid is removed. 
The use of this mixture is for the preparation of coarse objects it is in- 
tended to preserve in alcohol. The object of the acid is to prevent both 
that glueing together of organs by the perivisceral liquid, which is often 
brought about by the coagulating action of pure alcohol, and the precipita- FIXING AGENTS. OHLOEIDES, ORGANIC ACIDS, ETC. 
53 
tion on the surface of organs of the salts contained in sea-water, which is a 
hindrance not only to the penetration of the alcohol, but also to subsequent 
staining. 
Whitman (Journ. Boy. Mic. Soc. (N.S.), ii, 1882, p. 870) states that 
“ acid alcohol as above prepared loses its original qualities after standing 
some time, as ether compounds are gradually formed at the expense of the 
acid.” He also states that 70 per cent, alcohol may be taken instead of 90 
per cent, for washing out. 
86. Formaldehyde (Formol, Formalin, Formalose).—Formalde- 
hyde is the chemical name of the gaseous compound HCOH, 
obtained by the oxidation of methyl-alcohol. “ Formalin ” is 
the commercial name given by Schering & Co. to a 40 per cent, 
solution of this substance in water. “Formol” is the com- 
mercial name given to the same solution by Meister, Lucius, 
& Bruning. And “ Formalose ” is the name for the same 
solution adopted by an American firm. (These solutions may 
now be obtained from dealers in photographic chemicals.) 
As I have before pointed out (Ancit. Anz., xi, 8, 1895, p. 255), 
the already extensive literature which treats of the anatomical 
uses of formaldehyde is much confused by inaccurate use of 
these terms ; many writers use them indiscriminately. It is 
frequently impossible to discover from the statements of an 
author whether he means such or such a percentage of formal- 
dehyde, or such or such a percentage of the commercial 40 per 
cent, solution employed by him, the one being of course two 
and a half times stronger than the other. I think it must 
be admitted that the proper way of stating the strengths of 
these solutions is either to state them in terms of formaldehyde, 
and say so, or to say “formol, or formalin, diluted with so 
many volumes of water.” The present confusion is most 
inconvenient. 
Solutions of formaldehyde sometimes decompose partially or 
entirely with formation of a white deposit of paraform. Fish 
says that to avoid this the solutions should be kept in darkened 
bottles in the cool. The vapour of formaldehyde has a very 
irritating action on the conjunctiva and mucous membranes, 
but the effect is transitory, not so injurious as that of osmic 
acid. It is well not to soil the fingers with the solutions, as 
formaldehyde hardens the living skin very rapidly. 
It was discovered independently by F. Blum (Zeit. f. wiss. 
Mik., x, 3, 1893, p. 314) and by Hermann (Anat. Anz., ix, 4, 54 
CHAPTER Y. 
1893, p. 112) tliat formaldehyde possesses most remarkable 
hardening and preservative properties. 
Blum employed formol diluted with ten volumes of water 
(containing rather less than 4 per cent, of formaldehyde). 
He found this solution to penetrate rapidly, and to harden 
voluminous organs such as liver, kidney, brain, more rapidly 
than alcohol, and that sections were well preserved and sus- 
ceptible of good staining. 
Hermann used a solution containing O’5 to 1 per cent, of 
“ formalin ” (the context shows that 1 per cent, of formalde- 
hyde is what is meant, the solution being made by diluting 
Schering’s formalin with forty volumes of water). He found 
it harden very rapidly, with the remarkable result that the 
hardened organs preserve approximately the transparency 
of life, and that pigments are not discoloured. Since that time 
these observations have been amply confirmed, and there is 
no longer any doubt that for the preparation and preservation 
of museum specimens formaldehyde is the most valuable re- 
agent that has been discovered since the introduction of 
alcohol, to which it is for some purposes infinitely superior (for 
the employment of formaldehyde in museum work, see Blum, 
Zool. Anz., xviii, 3, 450). 
On account of the confusion in terminology above referred 
to, it is not at present possible to give precise instructions 
as to the strengths that have been employed by the diffe- 
rent authors for histological work. All that can be said is 
that they will almost certainly be found to lie between the 
limits of those indicated by Blum and Hermann, that is to say 
between O’5 per cent, and 4 per cent, if the formaldehyde be 
used pure. Only one writer (Hoyer, jun., Anat. Anz., ix, 1894, 
Erganzungsheft, p. 236 ; Zeit.f. wiss. Mih., xii, 1, 1895, p. 28) 
appears to have used concentrated solutions. He states that 
with such solutions tissues are better preserved than with weak 
ones, even better preserved than with corrosive sublimate. 
There is certainly some mistake here. I find that preparations 
fixed in 13'3 per cent, formaldehyde (formol with two volumes 
of water) have the cells enormously over-fixed and presenting 
the homogeneous aspect of osmicated cells. 
Experimenting further with weak solutions containing from 
2 per cent, to 4 per. cent of formaldehyde, I have found 
that like the stronger solution mentioned above, these too FIXING AGENTS. CHLORIDES, ORGANIC ACIDS, ETC. 
55 
give a homogeneous, colloid appearance to protoplasm, and 
have at the same time a marked swelling and vacuolating 
action. With the 2 per cent, solution the vacuolation is 
enormous. I have concluded that used pure formaldehyde is 
not at all fitted for cytological work, and should not be 
employed for that purpose, and I certainly should not think of 
using it myself, even for general morphological work. 
But preparations fixed in a mixture of 1 part of formol with 
two of 1 per cent, chromic acid, and with 4 per cent, of acetic 
acid added, or in a mixture of one part of formol with four of 
1 per cent, platinic chloride, and 2 per cent, of acetic acid 
added, give an excellent fixation, in some points superior to 
that of mixture of Flemming or of Hermann. 
I certainly think that mixtures of this sort will be found in 
some cases to give better results than osmio-chromic mixtures, 
on account of the superior penetration of the formaldehyde on 
the one hand, and on the other from the fact that formalde- 
hyde, whilst it hardens very rapidly does not over-harden. 
Mixtures with alcohol have also been recommended. 
Lavdowsky (Anat. Hefte, iv, 3, 1894, p. 355) gives the follow- 
ing two formulae : 
(1) Distilled water . . .20 parts. 
95 per cent, alcohol . 10 ,, 
Formol . . . . . 3 ,, 
Glacial acetic acid . . . O'5 ,, 
(2) Distilled water . . .30 parts. 
95 per cent, alcohol . .15 ,, 
Formol . . . . 5 ,, 
Glacial acetic acid . . 1 „ 
For the special hardening of the central nervous system with 
formaldehyde see further in Part II. CHAPTER VI. 
HARDENING AGENTS. 
87. Introductory.—Generalities concerning the necessity and 
the practice of hardening have been given in Chap. Ill, § § 
31 and 32. The present chapter will describe some reagents 
that are specially suited for slow hardening, and much less 
suited for that rapid hardening distinguished as fixing. It 
will at the same time describe the manner of employment for 
prolonged hardening of some of the reagents already described 
under the head of Fixing Agents. The very special question 
of the hardening of the central nervous system is merely 
touched on here, and receives further development in the 
chapters devoted to Neurological Methods in Part II. 
Mineral Acids. 
88. Chromic Acid.—Chromic acid is generally employed in 
strengths of -jtth per cent, to |per cent., the immersion lasting 
a few days or a few weeks, according to the size and nature 
of the object. Mucous membrane, for instance, will harden 
satisfactorily in a few days; brain will require some six 
weeks. 
Large quantities of the solution must be taken (at least 200 
grammes for a piece of tissue of 1 centimetre cube, Ranvier). 
In order to obtain the best results you should not employ 
portions of tissue of more than an inch cube. For a human 
spinal cord you should take two litres of solution, and change 
it for fresh after a few days. Six weeks or two months are 
necessary to complete the hardening. 
The solution should be taken weak at first, and the strength 
increased after a time. The objects should be removed from 
the solution as soon as they have acquired the desired con- 
sistency, as if left too long they will become brittle. (These HARDENING AGENTS. 
57 
precautions are peculiarly necessary in the case of chromic 
acid.) They may be preserved till wanted in alcohol (95 per 
cent.). It is well to wash them out in water for twenty-four 
or forty-eight hours before putting them into the alcohol. I 
think it is frequently useful to add a little glycerin to the 
hardening solution; there is less brittleness and, I think, less 
shrinkage. 
The reader’s attention is called to the statements made in 
§ 41 concerning the action of light on the alcohol containing 
chromic objects. 
Further directions for the employment of chromic acid will 
be given in the special paragraphs. Chromic acid is a most 
powerful and rapid hardening agent (by it, you may obtain 
in a few days a degree of hardening that you would hardly 
obtain it in as many weeks with bichromate, for instance.) It 
has the defect of a great tendency to cause brittleness. 
89. Chromic Acid and Spirit (Urban Pritchard, Quart. Journ. Mic. 
Sci., 1873, p. 427).—Chromic acid, 1 part; water, 20 parts ; rectified spirit, 
180 parts. Dissolve the chromic acid in the water first, and then add the 
spirit (violent action will ensue if the dry chromic acid be added directly to 
the spirit). The colour of the solution soon becomes brown. If, after a 
few days, it turns semi-gelatinous, it should be changed for fresh. From a 
week to ten days is required to harden such tissues as retina, cochlea, &c., 
for which this fluid is particularly well adapted. 
90. Chromo-osmic Acid (Max Flesch.) Chromo-aceto-osmic 
Acid (Flemming.)—Either of these mixtures may be used for 
prolonged hardening, and are admirable. The weak form of 
Flemming’s solution is the one that should generally be taken 
for hardening purposes. (See §§ 45 and 46.) 
For delicate objects perhaps eyen better results may be 
obtained by means of Chromic Acid and Platinic Chloride 
(Merkel’s Solution). See § 52 ante. 
91. Picro-chromic Acid.—This fixative may be found useful for 
hardening objects that are only penetrable with difficulty, some Tunicata, 
for instance. See § 51, ante. 
92. Osmic Acid.—Hardly used for anything but nervous tissue. See 
Neurological Methods in Part II. 
93. Nitric Acid.—Hardly used for anything but brain. See also 
Part II. 58 
CHAPTER VI. 
i.Salts. 
94. Bichromate of Potash.—Perhaps the most important of 
all known hardening agents, sensu stricto. It hardens slowly, 
much more so than chromic acid, but it gives an incompar- 
ably better consistency to the tissues, and it has not the same 
tendency to make them brittle if the reaction be prolonged. 
They may remain almost indefinitely exposed to its action 
without much hurt. 
The strength of the solutions employed is from 2 to 5 per 
cent. As with chromic acid, it is extremely important to 
begin with weak solutions and proceed gradually to stronger 
ones. About three weeks will be necessary for hardening a 
sheep’s eye in solutions gradually raised from 2 to 4 per cent. 
Spinal cord requires from three to six weeks ; a brain, at least 
as many months. 
After hardening, the objects should be well soaked out in 
water before being put into alcohol. They had better be kept 
in the dark when in alcohol (see above, § 41). If you wish to 
have a good stain with carmine, especially ammonia-carmine, 
which is admirable for portions of nervous system so hardened, 
you should not put the objects into alcohol at all, even for a 
second, until they have been stained. 
You may stain either with carmine or haematoxylin. 
Bichromate objects have an ugly yellow colour which cannot he removed 
by soaking in water. It is said that it can be removed by washing for a 
few minutes in a 1 per cent, solution of chloral hydrate. Gierke, however, 
says that this treatment is prejudicial to the preservation of the tissues. 
Prof. Gilson writes me that alcoholic solution of sulphurous anhydride 
(S02) is very convenient for the rapid decoloration of bichromate objects. A 
few drops suffice. See also § 41, and “ Bleaching.” 
95. Muller’s Solution.— 
Bichromate of potash . . . 2—2£ parts. 
Sulphate of soda ... 1 part. 
Water ..... 100 parts. 
The duration of the reaction is about the same as with the 
simple solution of chromic salts. 
This fluid was very highly in vogue for many years, but 
seems lately to be much less used. I fancy that the supe- 
riority of this mixture over the simple bichromate solution HARDENING AGENTS. 
59 
is not illusory, and is due to the formation in it of a trace of 
free chromic acid. Fol says that for mammalian embryos, for 
which it has been recommended, it is worthless. 
96. Erlicki’s Solution (Warschauer med. Zeit., xxii, Nos. 15 
and 18).— 
Bichromate of potash . . . 2-5 parts. 
Sulphate of copper . . . 1*0 part. 
Water ..... 10O0 parts. 
Here the addition of the cupric sulphate is intelligible. 
This salt is itself a hardening agent of some energy, and may 
well serve to reinforce the somewhat slow action of the bi- 
chromate. As a matter of fact, “ Erlicki ” hardens very much 
more rapidly than either simple bichromate or Muller’s solu- 
tion. A spinal cord may be hardened in it in four days at 
the temperature of an incubator, and in ten days at the 
normal temperature (Fol, Lelirb. d. vergl. mik. Anat., p. 106). 
I believe it to be one of the best hardening agents known for 
voluminous objects. Human embryos of several months may 
be conveniently hardened in it. 
Nerve-centres that have been hardened in Erlicki’s fluid frequently con- 
tain dark spots with irregular prolongations, simulating ganglion-cells. 
These were at one time taken to be pathological formations, but they are 
now known to consist of precipitates formed by the action of the hardening 
fluid. They may be removed by washing with hot water, or with water 
slightly acidified with hydrochloric acid, or by treating the specimens with 
0”5 per cent, chromic acid before putting them into alcohol (Tschisch, 
Virchow’s Arch., Bd. xcvii, p. 173; Edinger, Zeit.f. wiss. Mik., ii, 2, p. 
245 ,• Loewenthal, Rev. med. de la Suisse romande, 6me annee, i, p. 20). 
97. Bichromate and Platinic Mixture (Lindsay .Johnson’s 
Mixture). Latest Formula, 1895, communicated by Dr. 
Lindsay Johnson.— 
Bichromate of potash (2-5 per cent.) . 70 parts. 
Osmic acid (2 per cent.) . . 10 ,, 
Platinic chloride (1 per cent.) . . 15 ,, 
Acetic or formic acid . . . . 5 ,, 
It is not well to take the platinum chloride stronger than 
here given, as too strong solutions have a tendency to crys- 
tallize out on the tissues. Henneguy, who has worked a great 
deal with this reagent, and recommends it highly, says 
(.Leqons sur la Cellule, Paris, Carre, 1896, p. 61) that it is well 60 
CHAPTER VI. 
only to add tlie acetic or formic acid just before using, as 
it frequently provokes a spontaneous reduction of the osmium 
and platinum to such an extent that the mixture becomes 
quite black. 
This mixture was imagined for the preliminary hardening 
of retina, being allowed to act for two hours only, and then 
being followed by final hardening in pure bichromate solution. 
But it has proved applicable to other structures, and gives 
excellent results. The function of the osmic acid in the 
present formula is to enhance the hardening energy of the 
mixture. Dr. Lindsay .Johnson writes me that “ it greatly 
reduces the length of time necessary for hardening, three 
days being the time from removal of the organ to its being in 
celloidin under dilute spirit.5’ 
“ If the osmium has a tendency to blacken, this may he entirely prevented 
and a beautiful delicate chestnut-brown deepening towards Bartholozzi-red 
tint obtained by adding ten parts of 5 per cent, solution of nitrate of uranium, 
which forms a layer of uranium on the top of the reduced platinum and 
osmium (one or both).” 
It has already been pointed out, No. 53a, that this mixture 
may be used for fixing, in some cases with the best results. 
Henneguy, l. c., says it contracts the more spongy sorts of 
protoplasm less than mixture of Flemming, and that it does 
not produce the delusive precipitates that that mixture fre- 
quently does produce. 
I am convinced that for some purposes (e. g. delicate 
researches on protoplasmic structures) it will be found well to 
diminish the proportion of organic acid. 
98. Bichromates and Alcohol.—Mixtures of either bichro- 
mate with alcohol may be employed, and have a more rapid 
action than the aqueous solution. Thus Hamilton takes for 
hardening brain a mixture of 1 part methylated spirits with 
3 parts of solution of Muller (see the chapter on the Central 
Nervous System in Part II; see also Kultschizky’s Mixture, 
ante, § 53). Preparations should be kept in the dark during 
the process of hardening in these mixtures. 
99. Bichromate of Ammonia.—A review of the literature of the sub- 
ject shows that this salt is in considerable favour, for what precise motive is 
not apparent. Its action is very similar to that of the potassium salt. Fol 
says that it penetrates somewhat more l’apidly, and hardens somewhat more HARDENING AGENTS. 
61 
slowly. It should be employed in somewhat stronger solutions, up to 5 per 
cent. 
100. Neutral Chromate of Ammonia is preferred by some anatomists. 
It is used in the same strength as the bichromate. Klein has recommended 
it for intestine, which it hardens, in 5 per cent, solution, in twenty-four 
hours. 
Chlorides and others. 
101. Platinic Chloride Mixture (Merkel’s Solution).—The 
formula of this admirable reagent has been given above, § 52. 
It is an admirable hardening medium for delicate objects. 
Merkel states that he allowed from three to four days for the 
action of the fluid for the retina; for Annelids Eisig employs 
an immersion of three to five hours, and transfers to 70 per 
cent, alcohol ; for small leeches Whitman finds “ one hour 
sufficient, and transfers to 50 per cent, alcohol.” 
Whitman recommends, for the hardening of pelagic fish 
ova, a stronger mixture (due, I believe, to Eisig), viz.: 
0’25 per cent, solution of platinum chloride . 1 vol. 
1 per cent, solution of chromic acid . . 1 „ 
The ova to remain in it one or two days (Whitman, Methods 
in Micro. Anat., p. 153). 
102. Palladium Chloride (F. E. Schultze, Arch. mik. Anat., iii, 1867, 
p. 477).—This reagent was recommended by Schultze partly as giving to 
tissues a better consistency than chromic acid or Muller’s solution, and partly 
on account of a special faculty for penetrating organs rich in connective 
tissue that he attributes to it. It is an impregnation reagent, staining 
certain elements of tissues in various tones of brown. For the somewhat 
lengthy details of the manner of employing it, the reader is referred to the 
paper quoted. 
103. Chloride of Zinc is only employed for brain, see post, Part II. 
104. Picric Acid taken alone is a weak hardening agent, little used. It 
should be employed in saturated solution. But it is a useful ingredient in 
mixtures, serving to enhance the penetrating power. See Picro-ehromic 
Acid, ante, § 51; also Gage’s Picric Alcohol, § 81. 
105. Acetate of Lead.—Both the neutral acetate (sugar of lead) and 
the basic acetate have been used for hardening nerve tissues. AnnaKotla- 
sewsky found that nerve-cells hardened in 10 per cent, solution of sugar of 
lead were admirably preserved. See her “ Inaug.-Diss.” in Mitth. cl. naturf. 
Ges. Bern., 1887, and Zeit. f. wiss. Mik., iv, 3, 1887, p. 387. 62 
CHAPTER VI. 
106. Iodine may be used in combination with alcohol, and render service 
through its great penetrating power. See the method of Betz,post, Part II. 
107. Pyridin.—Pyridin has been recommended as a hai'dening agent 
(by A. de Souza). It is said to harden, dehydrate, and clear tissues at the 
same time. They may be stained after hardening hv anilin dyes dissolved 
in the pyridin, or passed through water and stained by the usual processes. 
It is said to harden quickly, and to give particularly good results with brain. 
See Comptes Rendus hebd. de la Soc. de Biologie, 8 ser., t. iv, No. 35, p. 
622; Zeit. f. wiss. Mile., v, i, 1888, p. 65; Journ. Roy. Mic. Soc., 1888, 
p. 1054. 
108. Alcohol.—When used alone, alcohol is inferior as a 
hardening agent to most of the reagents discussed above; 
but when judicially employed to complete the action of a 
good fixing agent, it renders most valuable services. 90 to 
95 per cent, is the most generally useful strength. Weaker 
alcohol, down to 70 per cent., is often indicated. Absolute 
alcohol is seldom advisable. You ought to begin with weak,, 
and proceed gradually to stronger, alcohol. Large quanti- 
ties of alcohol should be taken. The alcohol should be fre- 
quently changed, or the tissue should be suspended near the 
top of the alcohol, in order to have the tissue constantly sur- 
rounded with pure spirit (the water and colloid matters 
extracted from the tissue falling to the bottom of the vessel). 
Many weeks may be necessary for hardening large specimens. 
Small pieces of permeable tissue, such as mucous membrane, 
may be sufficiently hardened in twenty-four hours. 
109. Formaldehyde (Formol, Formalin, Formalose).—This 
important reagent has been in part described, ante No. 86. 
Considered as a hardening agent, sensii stricto its most im- 
portant use is for hardening nervous tissue. This will be 
considered in Part II. 
Blum (l. c., No. 86), found solutions containing 4 per 
cent, of formaldehyde hardened voluminous pieces of liver, 
kidney, stomachal mucosa, brain, &c., considerably quicker 
than alcohol, the preservation being excellent. 
Hermann (1. c., No. 86) found that such a large organ as 
a calf’s heart was hardened by a 05 to 1 per cent, solution 
in twelve to twenty-four hours. Entire eyes are so hardened 
in the 1 per cent, solution in twenty-four hours that they may 
be cut in two with a sharp knife like an apple. Hermann found HARDENING AGENTS. 
63 
this disadvantage, that tissues hardened in formaldehyde 
solution suffer when they are put into alcohol for the purpose 
of dehydration. The paper in question contains interesting 
observations on the property formaldehyde has of preserving 
the natural colours and transparent and life-like aspect of 
tissues. 
Blum (Anat. Anz., ix, 1894, p. 229), recapitulating, says 
that very voluminous pieces of material are hardened quickly 
and without shrinkage. The tissues stain well. Cells and 
nuclei preserve their forms; karyokinetic figures are fixed. 
Mucin is not precipitated, but remains transparent; fat is 
not dissolved. Micro-organisms retain their specific staining 
reactions. 
As to the degree and hind of hardening obtained by for- 
maldehyde the authors are not so explicit as could be wished. 
As far as I can see myself, the hardening obtained is gentle 
and tough, giving an elastic and not a brittle consistency. 
It probably varies greatly with different tissues. 
For prolonged hardening, considerable volumes of liquid 
should be taken and the liquid should be renewed from time 
to time. For the formaldehyde fixes itself on the tissues 
with which it comes in contact, deserting the solution, which 
thus becomes progressively weaker. 
For the employment of formaldehyde in hardening nervous 
tissue, see Part II. CHAPTER VII. 
CLEARING AGENTS. 
110. Introductory Remarks.—Clearing agents are liquids 
one of whose functions it is to make microscopic preparations 
transparent by penetrating amongst the highly refracting 
elements of which the tissues are composed, the clearing liquids 
themselves having an index of refraction not greatly inferior 
to that of the tissues to be cleared. Hence all clearing agents 
are liquids of high index of refraction. The same substances 
have also a second function, which consists in getting rid of 
the alcohol in which pi’eparations are generally preserved, and 
facilitating the penetration of the paraffin used for imbedding, 
or the balsam or other resinous medium in which preparations 
are, in most cases, finally mounted. Hence all of the group of 
bodies here called “ clearing agents ” must be capable of 
expelling alcohol from tissues, and must be at the same time 
solvents of Canada balsam and the other resinous mounting 
media. The majority of clearing agents are essential oils. 
111. The Practice of Clearing.—It is important to note again, 
notwithstanding some repetition, the manner of employing 
these agents. The old plan was to take the object out of the 
alcohol and float it on the surface of the clearing medium in a 
watch-glass. This plan was faulty, because the alcohol escapes 
from the surface of the object into the air quicker (in most 
instances) than the clearing agent can get into it; hence the 
object must shrink. To avoid or lessen this cause of shrinkage, 
clearing is now generally done by the method suggested by 
Griesbrecht, which consists in putting the clearing medium 
under the alcohol containing the object. This is done in the 
following manner. Take a short glass tube, and put into it 
enough alcohol to contain the objects (a watch-glass will often 
do well, but a tube is safer). With a pipette carefully put CLEARING AGENTS. 
65 
under the alcohol a sufficient quantity of clearing medium (or 
carefully pour the alcohol on to the clearing medium). Then 
put the objects into the alcohol. They will sink down to the 
level of separation of the two liquids at once; and after some 
time they will be found to have sunk to the bottom of the 
clearing medium. They may then be removed by means of a 
pipette, or the supernatant alcohol drawn off and the prepara- 
tions allowed to remain until wanted. They should not be 
considered to be perfectly penetrated by the clearing medium 
until the wavy refraction-lines caused by the mixture of the 
two liquids at their surface have ceased to form. 
The penetration of all clearing media may be hastened by 
using them warm. 
It frequently happens that the essential oil with which ob- 
jects are being treated in a watch-glass or on a slide becomes 
cloudy after a short time, and fails to clear the tissues. This 
is owing to a combination between the essential oil and mois- 
ture, derived, I think, rather from the air than from the ob- 
jects themselves. The cloudiness can usually be removed by 
warming (as pointed out by Hatchett Jackson, Zool. Anzeig., 
1889, p. 630), but this remedy is not always successful, for in 
certain states of the atmosphere the cloudiness will persist, 
notwithstanding continued warming. It is for this reason 
that I advise that clearing be done, whenever possible, in 
shallow well-corked tubes, under which conditions the pheno- 
menon rarely occurs. 
112. Classification of Clearing Agents.—For an account of 
Stieda’s experiments with essential oils, see previous editions. 
Neelsen and Schiefferdecker (Arch.f. Anat. u. Phys., 1882, 
p. 206) examined a large series of ethereal oils (prepared by 
Schimmel and Co., Leipzig), with the object of finding a 
not too expensive substance that should combine the properties 
of clearing quickly alcohol preparations, not dissolving out 
anilin colours, clearing celloidin without dissolving it, not 
evaporating too quickly, and not having a too disagreeable 
smell. 
Of these, the following three fulfil the conditions and can 
be recommended :—Cedar-wood, Origanum, Sandal-wood. 
To these should be added the others recommended in the 
following paragraphs. 66 
CHAPTER VII. 
It would be important to possess a list of the exact indices of refraction of 
the substances used for clearing. I have, unfortunately, not been able to 
obtain sufficient information of a trustworthy nature for the compilation of 
such a list. Cedar oil has nearly the index of crown glass (this is true of the 
oil in the thick state to which it is brought by exposure to the air, not of the 
new, thin oil, which is less highly refractive), it therefore clears to the same 
extent as Canada balsam. Clove oil has a much higher index, and therefore 
clears more than balsam. Tui-pentine, bergamot oil, and creasote have much 
lower indices, and therefore clear less. 
113. Choice of a Clearing Agent.—Special directions for 
clearing are given when necessary under the heads of the 
different organs and tissues. It will suffice here to advise the 
beginner to keep on his table the following :—Oil of cedar, for 
general use and for preparing objects for imbedding in paraffin, 
see “ Imbedding Methods—Paraffin ” ; clove oil, for making 
minute dissections in cases in which it is desirable to take 
advantage of the property of that essence of forming very 
convex drops on the slide and of imparting a remarkable 
brittleness to soft tissues, and for much work with safranin, &c.; 
oil of bergamot which will clear from 90 per cent, alcohol, and 
which does not extract coal-tar colours; carbolic acid, for 
rapidly clearing very imperfectly dehydrated objects. 
For special clearers for celloidin sections see tf Collodion 
(Celloidin) Imbedding Methods.” 
114. Cedar Oil (Neelsen and Schiefferdecker, op. cit., 
§ 112).—For finest cedar-wood oil, price per kilo varies from 
fifteen to twenty shillings, say about sevenpence halfpenny per 
ounce for small quantities, or about the price of clove oil. Veiy 
thin, colour light yellow or greenish, odour slight (of cedar- 
wood), evaporates slowly, is not changed by light, is miscible 
with chloroform balsam, and with castor oil. Clears readily 
tissues in 95 per cent, alcohol, without shrinkage; does not 
extract anilin colours, Celloidin sections are cleared in five to 
six hours. 
Cheap, but requires an inconvenient length of time for the 
clearing of celloidin sections. 
The observer should be careful as to the quality of the 
cedar oil he obtains. I have examined the clearing properties 
of a sample obtained from the celebrated firm of Kousseau, 
Paris. This sample was absolutely colourless. It totally CLEARING AGENTS. 
67 
failed to clear absolute alcohol objects after many days. I 
always use the thickened oil as supplied for use with immersion 
objectives. 
The authors think that a laboratory supplied with cedar oil 
and origanum oil is fully equipped for all possible cases (the 
origanum oil being used merely to take the place of cedar- 
wood oil for the special case of celloidin sections). 
Cedar oil is very penetrating, and for this and other reasons 
is, in my experience, the very best of all media for preparing 
objects for paraffin imbedding. I find it to be less hurtful to 
cells and delicate tissue-structures than any other medium 
known to me. 
115. Clove Oil.—Samples of clove oil of very different shades 
of colour are met with in commerce. It is frequently recom- 
mended that only the paler sorts should be employed in 
histology. A word of explanation is here necessary. Doubt- 
less it is, in general, best to use a pale oil, provided it be 
pure, but it is not always easy to obtain a light-coloured oil 
that is pure. Clove oil passes very readily from yellow to 
brown with age, so that in choosing a colourless sample you 
run great risk of obtaining an adulterated sample, for clove 
oil is one of the most adulterated substances in commerce. 
Two important properties of clove oil should be noticed here. 
It does not easily spread itself over the surface of a slide, but 
has a tendency to form very convex drops. This property 
makes it a very convenient medium for making minute dis- 
sections in. The second property I wish to call attention to 
is that of making tissues that have lain in it for some time 
very brittle. This brittleness is also sometimes very helpful 
in minute dissections. 
These qualities may be counteracted if desired by mixing 
the clove oil with bergamot oil. 
Clove oil has, I fancy, the highest index of refraction of all 
the usual clearing agents ; it clears objects more than balsam. 
It dissolves celloidin (or collodion), and therefore should not 
be used for clearing sections cut in that medium, without 
special pi-ecautions; Notwithstanding the opinion of Schieffer- 
decker, I consider this to be one of the best of clearing agents, 
and very valuable on account of the properties to which 
attention has been called above. New clove oil washes out 68 
CHAPTER Vlt. 
anilin colours more quickly than old. It is well to possess 
trustworthy samples of both new and old oil. 
116. Cannel Oil.—Greatly resembles clove oil, but is in general thinner. 
An excellent medium, which I particularly recommend. 
117. Oil of Bergamot.—Schiefferdecker (Arch. Anat. u. 
Phys., 1882 [Anat. Abth.], p. 206) finds that this oil has 
many good qualities ; it clears 95 per cent, alcohol prepara- 
tions and celloidin preparations quickly, does not attack anilin 
colours, but the strong odour is disagreeable ; it is as dear as 
oil of cloves, twice as dear as oil of origanum, and three times 
as dear as oil of cedar. He considers its action preferable to 
that of oil of cloves, but all things considered, gives the palm 
to cedar and origanum. I think that this is a very valuable 
medium, and though I do not agree with Schiefferdecker in 
thinking its action superior to oil of cloves, I think it should 
always be kept at hand. 
Bergamot oil is, I believe, the least refractive of these 
essences, having a lower index than even oil of turpentine. 
Suchannek (Zeit. f. iviss. Alik., vii, 2, 1890, p. 158) says 
that bleached, colourless bergamot oil will not take up much 
water, whereas a green oil will take up as much as 10 per 
cent. 
Van der Stricht (Arch, de Biol., xii, 1892, p. 741) says 
that bergamot oil will, with time, dissolve out the fatty 
granules of certain ova. 
118. Oil of Origanum (Neelsen and Schiefferdecker, Arch. 
Anat.u.Phys., 1882, p. 204).—Price per kilo 15 marks ( = 15s.). 
Thin, light brown colour, odour not too strong, agreeable, 
does not evaporate too quickly, is not changed by light, is 
miscible with chloroform balsam and with castor oil. Ninety- 
five per cent, alcohol preparations are cleared quickly, and so 
are celloidin sections, without solution of the celloidin. Anilin 
colours are somewhat extracted. 
For work with celloidin sections care should be taken to 
obtain 01. Origani Cretici (“ Spanisches Hopfenol ’*), not 01. 
Orig. Gallici (v. Gieson ; see Zeit. f. iviss. Alik., iv, 4, 1887, 
p. 482). Specimens of origanum oil vary greatly in their 
action on celloidin sections, and care should be taken to obtain 
a good sample. CLEARING AGENTS. 
69 
Squire, in his Methods and Formulae, &c., p. 81, says that 
origanum oil (meaning the commercial product) is nothing 
but oil of white thyme more or less adulterated, and that 
the product sold as 01. Origani Cretici is probably oil of 
marjoram. 
119. Oil of Thyme.—Fish (Proc. Amer. Mic. Soc., 189S, 
Zeit. f. wiss. Mik., xi, 4, p. 503), following Bumpus, says that 
for most of the purposes for which origanum oil has been re- 
commended, oil of thyme will do just as well if not better. 
After one distillation of the crude oil of thyme it is of a red- 
dish-brown colour, and is called the red oil of thyme ; when 
again distilled it becomes colourless, and is distinguished as 
the white oil. The red oil is just as efficient as the white for 
clearing. 
120. Sandal-wood Oil (Neelsex and Schieffebdecker, l. c., § 118).— 
Yery useful; but its high price is prohibitive. 
121. Turpentine.—Generally used for treating sections tliat have been 
cut in paraffin, as it has the property of dissolving out the paraffin and 
clearing the sections at the same time ; but many other reagents (see No. 
126) are preferable for this purpose. If used for alcohol objects it causes 
considerable shrinkage, and alters the structure of cells more than any other 
clearing agent known to me, unless used in the thickened state, a method 
which is much liked for some purposes in Germany. Thickened turpentine 
(“Yerhartzes Terpentinol ” of German writers) is prepared by exposing 
rectified turpentine in thin layers for some days to the air. All that is 
necessary is to pour some turpentine into a plate, cover it lightly so as to 
protect it from dust without excluding the air, and leave it until it has 
attained a syrupy consistency. Turpentine has, I believe, the lowest index 
of refraction of all the usual clearing agents except bergamot oil; it clears 
objects less than balsam. 
122. Carbolic Acid.—Best used in concentrated solution in 
alcohol. Clears instantaneously, even very watery prepara- 
tions. This is a very good medium, but it is better avoided 
for preparations of soft parts which it is intended to mount 
in balsam, as they generally shrink by exosmosis when placed 
in the latter medium. It is, however, a good medium for 
celloidin sections. 
123. Gage’s Mixture (Proc. Amer. Soc. Micr., 1890, p. 120; 
Journ. Roy. Mic. Soc., 1891, p. 418).—Carbolic acid crystals 
melted, 40 c.c.; oil of turpentine, 60 c.c. 70 
CHAPTER VII. 
124. Creasote.—Much the same properties as carbolic acid. 
Beech-wood creasote is the sort that should be preferred for 
many purposes,—amongst others, for clearing celloidin 
sections, for which it is a very good medium. 
125. Anilin Oil.—This is a rather important reagent on account of its 
ability to clear excessively watery objects. Common anilin oil will readily 
clear sections from 70 per cent', alcohol, and with certain precautions (for 
which, see the paper of Suchannek quoted below) objects may be cleared 
from watery media without the intervention of alcohol at all. This pro- 
perty renders anilin valuable in certain cases as a penetrating medium for 
preparing for paraffin imbedding. For ordinary work the usual commercial 
anilin will suffice; and it is immaterial whether it be colourless or have 
become brown through oxidation. For difficult work, it is well to use a 
perfectly anhydrous oil. For directions for preparing this see Suchannek, 
Zeit.f. wiss. Mik., vii, 2, 1890, p. 156, or the last edition of this work. 
Anilin is chiefly used for clearing celloidin sections, and is sometimes 
found very valuable for this purpose. 
126. Xylol, Benzol, Toluol, Naphtha, Chloroform.—Too volatile 
to be recommendable as general clearing agents, but very 
useful for celloidin sections or for paraffin sections. I find 
naphtha is quite good enough for removing paraffin from 
sections destined to be afterwards passed through alcohol 
and stained ; but that it should not be used for objects destined 
to be passed direct into balsam, as it frequently precipitates 
resinous media. Of the three first mentioned liquids, benzol 
is the most volatile, then toluol, and xylol is the least volatile, 
in the proportion of 4 : 5 : 9 (Squire, Methods and Formulas-, 
p. 20). Chloroform is injurious to some delicate stains. CHAPTER VIII. 
IMBEDDING METHODS INTRODUCTION. 
127. A Word on Microtomes.—It is no part of the purpose of this 
work to discuss instruments, yet a word on this subject may be helpful to 
the student. The freezing microtome so generally employed in England is 
less than any other form adapted to the wants of the zoologist. Very thin 
sections can be obtained by it more readily than with any other microtome, 
but they are of little use when obtained. The relations of the parts of the 
organs are deranged by the freezing and by the thawing, and the aqueous 
nature of the process prevents it from being readily applicable to the mount- 
ing of series of sections. The microtome of the zoologist, therefore, must 
be an imbedding microtome. 
Now there are two methods of imbedding in general use—the paraffin 
method and the celloidin method. In the paraffin method the object is cut 
dry, with the knife set square ; whilst in the celloidin method the object is 
usually cut wet, and in a softer and more elastic state than paraffin objects, 
and always with an obliquely-set knife. It so happens that the most precise 
and beautiful microtomes that have been constructed are designed in view 
of the paraffin method, and cannot be applied, or at all events are much less 
adapted, to work with celloidin objects. A thoroughly equipped laboratory 
should therefore possess two microtomes, one for paraffin work, and one for 
celloidin material, or other material that has to be cut in the wet way. If 
the anatomist cannot afford two instruments, he will perhaps do well not to 
choose one of those that are adapted only for paraffin, but to choose an all- 
round instrument, one that without being absolutely of the highest attain- 
able precision in paraffin work will yet give sufficiently good results in that 
way, and will also cut in the wet way. 
Amongst microtomes fulfilling these conditions various forms will be 
found almost equally convenient. Zeiss makes a good one ; Schanze, of 
Leipzig, makes a good one ; Reichert, of Vienna, makes a good one. All 
these are relatively cheap, and, being at the same time perfectly efficient for 
easy work, may be recommended. Amongst more precise instruments of 
this class the first place in order of date belongs to the Thoma sliding micro- 
tome. This is made in several sizes by R. Jung, Meehaniker in Heidelberg. 
For zoological and general histological work I recommend the medium size 
(No. 2a or 4), with the newest Naples object-holder and newest form of 
knife and knife-holder. 
This instrument is described in Journ. Boy. Mic. Soc. (N.S.), vol. iii, 72 
CHAPTER VIII. 
p. 298 ; the new Naples object-holder (which I consider essential for the 
zoologist) is described and figured p. 915. 
The Beckek microtome is in many respects an improvement on the 
Thoma model. It is essentially on the same principle, but possesses a 
mechanical arrangement for moving the knife-carrier; that is, the knife- 
carrier is not only guided by a mechanical arrangement, as in the Thoma 
model, but is put in motion by mechanism. This, I think, is certainly an 
advantage. Another improvement is that the slides are made of glass in- 
stead of metal; this allows one to dispense with the use of oil to the slides, 
which in the Thoma model gives rise to inequality in the thickness of sections. 
A minor point is that the instrument is somewhat cheaper than the Thoma 
form. It is made by Aug. Becker, Gottingen. Descriptions of two forms 
(Spengel and Schielferdecker) will be found in Journ. Roy. Mic. Soc., 1886, 
pp. 884 and 1084. The Naples object-holder can be fitted to the Becker 
microtome. 
The instruments above described are “ all-round ” microtomes ; by which 
is meant that they may be used either with a square-set knife or an obliquely- 
set knife, and will cut either celloidin sections or frozen preparations (if a 
freezing apparatus be added to them) just as well as paraffin sections. They 
will not, according to my experience, cut series of paraffin sections with any- 
thing like the same infallible i-egularity, certainly not with the same rapidity 
as the instruments next to be mentioned. But they give excellent results, 
and in view of their adaptability to celloidin or other semi-soft preparations, 
I think that one of them, the Becker by preference, should be the instru- 
ment chosen by the worker who desires not to be entirely confined to the 
paraffin method, and who cannot conveniently possess more than one 
microtome. 
All the instruments mentioned hitherto are sliding microtomes, that is 
instruments in which the object to be cut is a fixture during cutting, and 
the knife is moved on a slide and is only attached to its holder at one end. 
This arrangement will not allow the highest possible accuracy to be obtained 
with paraffin objects or any other hard objects. For with hard objects the 
knife is free to tilt slightly on meeting the object, instead of cutting its 
way through it. This defect is fatal to the attainment of perfectly cut 
series of sections of equal thickness throughout. For the highest class of 
work it is necessary to employ a microtome constructed on the opposite 
principle, namely one in which the knife is a fixture, and fixed at both ends 
as near as possible to the cutting point; the object being moved against it. 
The following instruments are constructed on this principle, and for accurate 
cutting of paraffin sections are undoubtedly superior to any sliding micro- 
tome ; they also work incomparably quicker. 
The Cambridge rocking microtome (furnished by the Cambridge Scientific 
Instrument Company, Carlyle Road, Cambridge, price £4 4s., or by Messrs. 
Swift and Son, or by Jung) is only adapted for cutting paraffin sections (Mr. 
Swift has shown me an arrangement for inclining the knife so as to give it 
the position required for cutting celloidin ; but I feel pretty sure that this 
will prove a failure in practice). This instrument is extremely simple and 
extremely rapid, and, what is more important, cuts more level series of 
sections than any other microtome I am personally acquainted with. It IMBEDDING METHODS—INTRODUCTION. 
73 
should be fitted with an adjustable object-holder, allowing of precise orienta- 
tion of the object. This, I believe, has been done in the newest instruments. 
Or the object-holder of Henneguy and Yignal (Compt. Bencl. Soc. Biol., 
1885, p. 647), may be added to it. (This, as well as the entire instrument, 
is manufactured in France by Dumaige, 24, Rue Saint-Merri, Paris, or 
Messrs. Swift on request will furnish such an arrangement, or it may be 
obtained, with or without the entire instrument, from Jung, of Heidelberg). 
See also in Zeit. f. wiss. Mik., iv, 4, 1887, p. 465, the description of an 
object-holder adapted to the rocking microtome by Hasselaee ; further, the 
price list of Jung; also a paper in Zeit.f. wiss. Mik., vii, 2, 1890, p. 165. 
It has been objected to this instrument by Schiefferdecker (see Zeit.f. 
iviss. Mik., ix, 2, 1892, p. 171, a description and criticism of the instrument 
as made by Jung) that it does not cut plane sections, but sections having 
the form of segments of a cylinder. This is true ; but it does not therefore 
follow, as Schiefferdecker concludes, that the instrument is inapplicable to 
many morphological purposes, and especially to embryological research. In 
practice, the slight deviation of the sections from a plane figure is found to 
be quite inappreciable, and therefore unimpoi-tant. And if I rightly under- 
stand, this slight defect has been overcome in the new model (1895). 
Rather more costly (£8 15s.) is the Minot microtome made by E. Zimmer- 
mann, Mechaniker, 21, Emilien Strasse, Leipzig. A description and figures 
of this instrument will be found in Zeit.f. wiss. Mik., ix, 2, 1892, p. 176, 
or in Journ. Boy. Mic. Soc., 1889, p. 143. It is worked on the sewing- 
machine principle : the knife is fixed as in the Cambridge instrument, and 
the object is made to impinge on it by means of a rotary motion given to a 
wheel by the hand, and converted by a crank and slide lever into a vertical 
one given to a slide carrying the object. This microtome cuts with very 
great rapidity, and those who have worked with it speak very highly of it. 
It is said that owing to the construction of the slide, which is subject to un- 
compensated wear and tear, its work is liable to fail in accuracy. The 
object-holder does not appear to be so scientifically constructed as the Naples 
one. Like the Cambridge instrument, this microtome is only adapted for 
paraffin work, and for this reason and the others stated above I do not feel 
satisfied that it should be preferred to the Becker or Thoma by those who 
have to be content with a single instrument. 
The most beautiful of all these instimments is the Reinhold-Giltay. 
It is constructed on essentially the same principle as the Minot, but the 
detail has been further elaborated, with the result of obtaining an instru- 
ment that is at the same time more precise in operation and more resistant 
to wear and tear, all working parts being compensated throughout. I have 
not had the advantage of working with this machine myself, hut I have 
seen it in operation, and can testify that with a common razor I have seen 
it cut perfectly continuous ribbons of sections of one micron in thickness, 
from a block of paraffin half an inch square. The sections were all of them 
entire and without any defect, further than that some of them were some- 
what compressed, a fault which is, of course, the expression of insufficient 
keenness in the knife, not of any want of accuracy in the machine. 
The Reinhold-Giltay has an arrangement for allowing the cutting of 
collodion material. I am unable to say whether this is a success. It is made 74 
CHAPTER VIII. 
by J. W. Giltay, Delft, and costs about twenty pounds. A description will 
be found in Zeit. f. wiss. Mik., ix, 4, 1893, p. 445, and in Journ. Boy. Mic. 
Soc., 1893, p. 706. 
128. Imbedding Methods.—The processes known as Im- 
bedding Methods are employed for a twofold end. Fii’stly, 
they enable us to surround an object, too small or too delicate 
to be firmly held by the fingers or by any instrument, with 
some plastic substance that will support it on all sides with 
firmness but without injurious pressure, so that by cutting 
sections through the composite body thus formed, the included 
object may be cut into sufficiently thin slices without dis- 
tortion. Secondly, they enable us to fill out with the im- 
bedding mass the natural cavities of the object, so that their 
lining membranes or other structures contained in them may 
be duly cut in situ ; and, further, they enable us to surround 
with the supporting mass not only each individual organ or 
part of any organ that may be present in the interior of the 
object, but each separate cell or other anatomical element, 
thus giving to the tissues a consistency they could not other- 
wise possess, and ensuring that in the thin slices cut from 
the mass all the details of structure will precisely retain 
their natural relations of position. Such a process of im- 
bedding is at the same time practically a process of hardening 
in so far as it gives to tissues a degree of firmness that conld 
otherwise only be obtained by the employment of chemical 
processes such as prolonged treatment with chromic acid and 
the like. 
These ends are usually attained in one of two ways. Either 
the object to be imbedded is saturated by soaking with some 
material that is liquid while warm and solid when cold, 
which is the principle of the processes here called Fusion 
Imbedding Methods; or the object is saturated with some 
substance which whilst in solution is sufficiently fluid to 
penetrate the object to be imbedded, whilst at the same time, 
after the evaporation or removal by other means of its solvent, 
it acquires and imparts to the imbedded object sufficient firm- 
ness for the purpose of cutting. The collodion process suffi- 
ciently exemplifies this principle. If a piece of soft tissue be 
dehydrated, and soaked first in ether and then in collodion, 
and if the ether contained in the collodion be allowed slowly 
to evaporate, the tissue and surrounding mass of collodion will IMBEDDING METHODS INTRODUCTION. 
75 
acquire a consistency such as to admit of thin sections being 
cut from them. The methods founded on this principle are 
here called Evaporation Imbedding Methods. 
In any of these cases the material used for imbedding is 
technically termed an “ imbedding mass ”—Einbettungsmasse 
—masse d’inclusion. Imbedding methods are spoken of by 
French writers as methodes d’inclusion, or methodes d’enrob- 
age. 
As before stated, the method most generally employed, 
and the one which may be considered the normal anatomical 
method, is the paraffin method. 
129. Imbedding Manipulations.—Before proceeding to de- 
scribe in detail the more important imbedding methods, it is 
necessary to give an account of the manipulations of the pro- 
cess of imbedding in general. 
Imbedding in a melted mass such as paraffin is performed 
in one of the following ways. A little tray or box or thimble 
is made out of paper, some melted mass is poured into it; at 
the moment when the mass has cooled so far as to have a 
consistency that will not allow the object to sink to the 
bottom, the object is placed on its surface, and more melted 
mass poured on until the object is enclosed. Or the paper 
tray being placed on cork, the object may be fixed in position 
in it whilst empty by means of pins and the tray filled with 
melted mass at one pour. The pins are removed when the 
mass is cold. 
In either case, when the mass is cold the paper is removed 
from it before cutting. 
To make paper trays, proceed as follows. Take a piece of 
stout paper or thin cardboard, of the shape of the annexed 
figure (Fig. 1); thin (foreign) post-cards do very well indeed. 
Fold it along the lines a a and b b', then along c c' and cl d', 
taking care to fold always the same way. Then make the 
folds A A', B B', C C, D D', still folding the same way. To 
do this you apply A c against A a, and pinch out the line 
A A', and so on for the remaining angles. This done, you 
have an imperfect tray with dogs’ ears at the angles. To 
finish it, turn the dogs’ ears round against the ends of the box, 
turn down outside the projecting flaps that remain, and pinch 
them down. A well-made post-card tray will last through 76 
CHAPTER VIII. 
several imbeddings, and will generally work better after 
having* been used than when new. 
Fig. 2. 
Fig.1. 
To make paper thimbles, take a good cork, twist a strip of 
paper several times round it so as to make a projecting collar, 
and stick a pin through the bottom of the paper into the 
cork. For work with fluid masses, such 
as celloidin, the cork may be leaded at 
the bottom to prevent it from floating 
when the whole is thrown into spirit or 
other liquor for hardening (Fig. 2). 
Leuckhart’s Imbedding Boxes are made 
of two pieces of type-metal (Fig. 3). 
Each of these pieces has the form of 
a carpenter’s “ square ” with the end of 
the shorter arm triangularly enlarged 
outwards. The box is constructed by 
placing the two pieces together on a 
plate of glass which has been wetted 
with glycerin and gently warmed. The 
area of the box will evidently vary 
according to the position given to the pieces, but the height 
can be varied only by using different sets of pieces. In 
Fig. 3. IMBEDDING METHODS—INTRODUCTION. 
77 
such a box the paraffin may be kept in a liquid state by 
warming now and then over a spirit lamp, and small objects 
be placed in any desired position under the microscope 
(Journ. Roy. Mic. Soc. [N.S.], ii, p. 880). 
Selenka has described and figured a simple but perhaps 
more efficacious apparatus having the same object. It con- 
sists of a glass tube, through which a stream of warm water 
may be passed and changed for cold as desired, the object 
being placed in a depression in the middle of the tube (see 
Zool. Anz., 1885, p. 419). A modification of this method is 
described by Andrews in Amer. Natural., 1887, p. 101 ; cf. 
Zeit. f. wiss. Mih., iv, 3, 1887, p. 375. 
For small paraffin objects the following procedure is very 
useful. The object is removed from the paraffin solution, the 
superfluous fluid is removed by means of blotting-paper, and 
the object placed on a cylinder of paraffin. A piece of stout 
iron wire is now heated in the flame of a spirit lamp, and with 
it a hole is melted in the end of the cylinder; the specimen is 
pushed into the melted paraffin, and placed in any desired 
position. The advantages of the method lie in the quickness 
and certainty with which it can be performed. 
I strongly recommend the reader not to neglect this simple 
method, which is capable of sometimes rendering services 
which no other method can. Those who have to do work 
with objects so small that their position can only be made out 
with the aid of a powerful lens ought to know how to arrange 
an object with a heated needle under a dissecting microscope, 
or on the object-carrier of the microtome. 
[In the first edition this procedure was attributed to 
Kingsley. It appears to have been first published by Born, 
see “ Die Plattenmodellirmethode,” in Arch. f. mih. Anat., 
1883, p. 591.] 
There remains the watch glass method. Melt paraffin in a 
watch-glass, and throw the object into it; or place the object 
in the watch-glass, add solid paraffin, and heat. After the 
mass has hardened, cut ont a block containing the object 
(this is of course applicable to other masses, such as celloidin). 
If paraffin be used you may, instead of cutting out a block, 
turn out the whole mass of paraffin by simply warming rapidly 
the bottom of the glass, but I find it is far safer to cut out a 
block. To facilitate the removal of the mass some persons 78 
CHAPTER VIII. 
lubricate the watch-glass before pouring in the mass. To do 
this a drop of glycerin should be smeared over it and wiped off 
with a cloth until hardly a trace of it remains. But this is 
not necessary. 
As regards the merits of the watch-glass process, I wish to 
say that, as regards small objects at all events, I consider it 
the very best ‘process of any. 
130. Choice of a Method.—Amongst the very various methods 
of imbedding that have been proposed two are pre-eminently 
important—the paraffin method for small objects, and the 
celloidin or collodion method for large objects. 
The subject of the respective merits of paraffin and celloidin 
still affords matter for discussion to some persons. The case, 
hoAvevei*, seems to be a very simple one. Celloidin does not 
afford by a long way the thinnest sections that are obtainable 
with small objects. For such objects it is, therefore, not equal 
to the demands made by modern minute anatomy, and paraffin 
must be taken. On the other hand, paraffin (as at present 
employed) will only cut very thin sections with fairly small 
objects; with objects of much over half an inch in diameter 
you cannot get with paraffin thinner sections than you can 
with celloidin; and if you try to cut in paraffin objects of still 
greater size, say an inch and upwards, it will frequently 
happen that you will not get perfect sections at all, blocks of 
paraffin of this size having a tendency to split under the 
impact of the knife; so that for very large objects celloidin 
gives better results. 
I have not been able to satisfy myself that the preservation 
of the tissues is better in celloidin sections than in paraffin 
sections; so that—convenience apart—the case remains as 
above stated,—paraffin for small sections, celloidin for large 
ones. 
To this must be added aqueous masses, such as gum or 
gelatin, for very special cases. 
It will be the purpose of the next chapter to describe the 
paraffin method, and to mention some other masses that can 
be employed in a similar manner, the celloidin method and 
the other methods that remain to be described being treated 
of in the following Chapter. CHAPTER IX. 
IMBEDDING METHODS—PARAFFIN AND OTHER FUSION MASSES., 
131. Penetration or Clearing.—The first stage of the paraffin 
method consists in the penetration .or infiltration of the 
object by some substance which is a solvent of paraffin. 
The process may be called a clearing process, since the chief 
substances used for infiltration are also “ clearing ” agents. 
The process of penetration or clearing should be carefully 
performed with well-dehydrated objects in the manner de- 
scribed in § 111. 
Penetration liquids being merely liquids that are, on the 
one hand, miscible with alcohol, and on the other hand good 
solvents of paraffin, are as numerous as could be wished. 
Amongst them may be mentioned essence of turpentine, clove 
oil, bergamot oil, creasote, benzol, xylol, toluol, naphtha, oil 
of cedar wood, chloroform, and anilin oil. 
Turpentine penetrates well, and mixes readily with paraffin. 
I do not, however, recommend it, because in my experience 
it is of all others the clearing agent that is the most hurtful 
to delicate structures. 
Clove oil penetrates well, and preserves delicate structures 
well; but it mixes very slowly with paraffin, and quickly 
renders tissues brittle. 
Benzin has been recommended by Brass (Zeit. f. wiss. Mik., 
ii, 1885, p. 301). 
Toluol (or toluen) has been recommended by Holl (Zool. 
Anz., 1885, p. 223). 
Naphtha has been recommended by Webster {Journ. Anat. 
and Physiol., xxv, 1891, p. 278). For large specimens it has 
the advantage of being very cheap. Dr. Webster writes me 
that a quality known as “ Persian naphtha ” is best for fine 80 
CHAPTER IX. 
work, but the common pure naphtha is sufficient for ordinary 
work. 
Field and Martin (Zeit. /. Wiss. Mik., xi, 1, 1894, p. 10) 
recommend a light petroleum known as “ petroleum-tether.” 
Xylol is said by M. Heidenhain (Kern und Frotoplasrna, 
p. 114) to be a cause of shrinkage in cells ; he employs oil of 
bergamot. 
Chloroform mixes well with paraffin, and after evaporation 
in a paraffin bath (in the manner described in the next para- 
graph) leaves behind a pure and very homogeneous paraffin, 
having but little tendency to crystallise. But it is deficient 
in penetrating power, so that it requires an excessive length 
of time for clearing objects of any size ; and it must be very 
thoroughly got rid of by evaporation in the paraffin bath, or 
by successive baths of paraffin, as if the least trace of it 
remains in the paraffin used for cutting it will make it soft. 
The process of removal requires a very long time, in some 
cases days. Chloroform ought therefore to be reserved for 
small and easily penetrable objects. 
Cedar-wood oil is, according to my continued experience, 
for the reasons stated by me in Zool. Anz., 1885, p. 563, in 
general the very best clearing agent for paraffin imbedding. It 
penetrates rapidly, preserves delicate structure better than 
any clearing agent known to me, does not make tissues brittle, 
even though they may be kept for weeks or months in it, and 
has the great advantage that if it be not entirely removed from 
the tissues in the paraffin bath it will not seriously impair the 
-cutting consistency of the mass; indeed, I fancy it sometimes 
improves it by reudering it less brittle. I always use the 
thickened oil as supplied for use with immersion objectives. 
In some difficult cases anilin oil is indicated (see § 125). 
132. The Paraffin Bath. — The objects having been duly 
“ penetrated ” or “ cleared/’ the next step is to substitute 
melted paraffin for the penetrating or clearing medium. 
Some authors lay great stress on the necessity of making 
the passage from the clearing agent to the paraffin as gradual 
-as possible,by means of successive baths of mixtures of clearing 
agent and paraffin kept melted at a low temperature, say 35° C. 
With oil of cedar or toluol, at all events, this is not necessary. 
.All that is necessary is to bring the objects into melted paraf- IMBEDDING METHODS—PARAFFIN, ETC. 
81 
fin kept just at its melting-point, and keep them there till they 
are thoroughly saturated; the paraffin being changed once or 
twice for fresh only if the objects are sufficiently voluminous 
to have brought over with them a notable quantity of clearing 
agent. 
The practice of giving successive baths first of soft and then 
of hard paraffin appears to me entirely illusory. 
It is important to keep the paraffin dry—that is, protected 
from vapour of water during the bath. 
It is still more important to keep it as nearly as possible at 
melting-point. If it be heated for some time to a point much 
over its normal melting-point, the melting-point will rise, and 
you will end by having a harder paraffin than you set out 
with. And as regards the preservation of tissues, of course 
the less they are heated the better. 
The duration of the bath must, of course, vary according to 
the size and nature of the object. An embryo of the size of a 
pea ought to be thoroughly saturated after an hour’s bath, or 
often less, if cedar oil has been used for clearing. In any 
case, the preparations should be cooled (see below, § 135) as 
soon as saturated. If left for many hours in a warm bath, as 
is sometimes done, delicate structures may be seriously 
injured. Indeed, the great point to be attended to in paraffin 
work of the finer order is to minimise the action of heat. It 
is therefore important both to employ a paraffin of the lowest 
melting-point that will give good sections (see below, § 142), 
and to abbreviate the warm bath as much as possible. 
If chloroform or other volatile agent be taken, choice may 
be made of two methods : either, as in Giesbrecht’s method, 
the chloroform containing the object is heated to the melting- 
point of the paraffin, and the paraffin gradually added, and 
the mass kept at the melting-point of the pure paraffin until 
all the chloroform is driven off; or, as in Biitschli’s method, 
the objects are simply passed direct from chloroform into a 
solution of paraffin in chloroform, in which they remain until 
thoroughly impregnated (half to one hour), and which is then 
evaporated at the melting-point of the paraffin. Biitschli 
recommends a paraffin solution melting at 35°. (Such a solu- 
tion is made of about equal parts of chloroform and paraffin 
of 50° melting-point.) Or, in the case of larger objects, 
instead of evaporating the chloroform (which is often a very 82 
CHAPTER IX. 
long process, as tlie chloroform must be completely driven off, 
or the mass will remain too soft for cutting), Biitschli simply 
transfers them from the bath of paraffin solution to a bath of 
pure paraffin. 
Giesbrecht’s method (Zool. Anz., 1881, p. 484), more fully 
stated, is as follows : 
Objects to be imbedded are saturated with absolute alcohol 
and then brought into chloroform (to which a little sulphuric- 
ether has been added if necessai*y, in order to prevent the 
objects from floating). As soon as the objects are saturated 
with the chloroform, the chloroform and objects are gradually 
Avarmed up to the melting-point of the paraffin employed, and 
during the warming small pieces of paraffin are by degrees 
added to the chloroform. So soon as it is seen that no more 
bubbles are given off from the objects, the addition of paraf- 
fin may cease, for that is a sign that the paraffin has entirely 
displaced the chloroform in the objects. This displacement 
having been a gradual one, the risk of shrinkage of the tissues 
is reduced to a minimum. 
133. Stoves and Water-baths.—It is important that the paraffin 
should not be exposed to a moist atmosphere whilst it is in the liquid state. 
If a water-bath he used for keeping it at the required temperature, provision 
should he made for protecting the paraffin from the steam of the heated 
water. 
A very convenient apparatus for this purpose is that of Paul Mayer, which 
will be found, described at p. 146 of Journ. Roy. Mic. Soc., 1883. It may 
be procured from the Zoological Station at Naples (address—“ Direzione 
della Stazione Zoologica, Napoli”), or from M. Paul Rousseau, 24, rue des 
Fosses-St.-Jacques, Paris. See also Amer. Natural., 1886, p. 910; and Journ. 
Roy. Mic. Soc., 1887, p. 167. 
Other similar forms of paraffin-heating apparatus are described in several 
places in the same journal, as also in Zeit.f. wiss. Mik. 
But whenever the worker has gas at his disposition, it will he found pre- 
ferable to employ a regulating stove or thermostat. I recommend the form 
described in Fol’s Lehrbuch, p. 121. Other descriptions of similar apparatus 
will be found also in the above-named journals. 
Amongst apparatus arranged for heating by means of petroleum or similar 
combustibles other than gas may be mentioned the stove manufactured and 
sold by F. Santorius, Gottingen (Zeit. f. wiss. Mik., x, 2, 1893, p. 161), 
and that of Altmann {ibid., p. 221, cf. Centralb. f. Bakteriol., xii, 1892, 
p. 654). 
134. Imbedding in Vacro.—There are objects which, on account of then- 
con sistency or their size, Cannot be penetrated by paraffin in tbe ordinary 
way, even after hours or days in the bath. For such objects, the method of IMBEDDING METHODS PARAFFIN, ETC. 
83 
imbedding in a vacuum renders the greatest service. It not only ensures 
complete penetration in a very short time—a few minutes—but it has the 
further advantage of preventing any falling in of the tissues such as may 
easily happen with objects possessing internal cavities if it be attempted to 
imbed them in the ordinary way. 
The principle of this method is that the objects are put through the 
paraffin-bath in vacuo. In practice this may be realised by means of any 
arrangement that will allow of maintaining paraffin at the necessary tempe- 
rature for keeping it fluid under a vacuum. 
The apparatus of Hoffmann will be found described and figured at p. 230 
of Zool. Anz., 1884. In this arrangement the vacuum is produced by means 
of a pneumatic water aspiration pump, the vessel containing the paraffin 
being placed in a desiccator heated by a water-bath and furnished with a tube 
that brings it into communication with the suction apparatus. This arrange- 
ment is very efficacious and very simple if the laboratory possesses a supply 
of water under sufficient pressure. 
In order to obtain the requisite vacuum without the aid of water under 
pressure, a simple little apparatus has been designed by Fbancotte {Bull. 
Soc. Belg. Micr., 1884, p. 45). In this the vacuum is produced by the con- 
densation of steam. 
Fol (Lehrb., p. 121) employs the vacuum apparatus of Hoffmann, but 
simplifies the arrangement for containing the paraffin. The paraffin is con- 
tained in a stout test-tube furnished with a rubber stopper traversed by a 
tube that puts it into communication with the pump. The lower end of the 
test-tube dips into a water-bath. You pump out the air once or twice, wait 
a few minutes to make sure that no more bubbles rise, then let the air in, 
turn out the object with the paraffin (which by this time will have become 
abnormally hard), and re-imbed in fresh paraffin. 
See also a paper by Pringle, in Journ. Path, and Bacteriol., 1892, p. 117 ; 
or Journ. Roy. Mic. Soc., 1892, pp. 893. 
135. Imbedding and Cooling.—As soon as the objects are 
thoroughly saturated with paraffin they should be imbedded 
by one of the methods given above (§ 129). If the ivatch- 
glass method he followed the paraffin-batlx will naturally have 
been given in the watch-glass used for imbedding, and no 
special imbedding manipulation will be necessary. In any 
case the important point now to be attended to is that the 
paraffin be cooled as rapidly as possible. The object of this is 
to prevent crystallisation of the paraffin, which may happen 
if it be allowed to cool slowly, and to get as homogeneous a 
mass as possible. 
Very small objects may be taken out of the paraffin with a 
needle or small spatula, and put to cool on a block of glass, 
then imbedded in position for cutting on a cone of paraffin 
by means of a heated needle in the manner described above 84 
CHAPTER IX. 
(§ 129). In tlie use of the needle it should be noted that it 
is important to melt as little paraffin as possible at one time, in 
order that that which is melted may cool again as rapidly as 
possible. 
If the watch-glass method be adopted, float the watch-glass 
with the paraffin and objects on to cold water. Do not let it 
sink till all the paraffin has solidified. When cool, cut out 
blocks containing the objects; do this with a slightly warmed 
scalpel. 
If paper trays be taken, cool them on water, holding them 
above the surface with only the bottom immersed until all the 
paraffin has solidified, as if you let them go to the bottom at 
once you will probably get cavities filled with water formed 
in your paraffin. Or you may put them to cool on a block of 
cold metal or stone. 
Selenka recommends cooling the mass by passing a stream 
of cold water through the imbedding tube described above 
<§ 129). 
The objects having been mounted on the carrier of the 
microtome in position for cutting, pare the blocks square to 
the knife, and sufficiently close down to the objects, and go 
round them with a lens. If any bubbles or cavities or opaque 
spots be present, prick with a heated needle till all is smooth 
and homogeneous. Minutes spent in this way are well invested. 
It is said by some workers that it is well to cut within a 
few hours of imbedding if the structure be at all delicate, as 
paraffin may continue to crystallize slowly to a certain extent 
even after rapid cooling. But this danger is very greatly 
diminished if the mass have been properly cooled. And 
according to my experience the damage likely to arise from 
the crystallisation of the paraffin has been greatly exaggerated. 
As stated in § 3, I find no better medium for the preservation 
of tissues than paraffin. 
136. Orientation.—The above-described manipulations of definitive im- 
bedding are in most cases sufficient. But it may be desirable to have the 
object fixed in the cooled block in a more precisely-arranged position, and, 
above all, in a more precisely-marked position, than is possible by these 
simple methods. Here is a method due to Patten {Zeit. f. wiss. Mile., 
xi, 1, 1894, p. 13), which is especially useful when one desires to orient 
accurately large numbers of small and similar objects. You get some writing 
paper of the sort that is made with two sets of raised parallel lines running IMBEDDING METHODS PARAFFIN, ETC. 
85 
at right angles to each other (according to Woodwobth, see below, 
this is known as “linen cloth paper”). Small strips are cut from this, and 
at suitable intervals along them small drops of a mixture of collodion 
and clove oil, of about the consistency of thick honey, are arranged close 
together along one of the ribs that run lengthwise. The objects to be im- 
bedded are cleared in olive oil or oil of bergamot— not turpentine. They 
are taken one by one on the point of a knife, and after the excess of oil has 
been drawn off are transferred each to a drop of the collodion mixture. They 
may be adjusted therein under the dissecting microscope, and will stay in 
any required position. When half a dozen or more objects have been 
oriented in reference to the cross lines (which are to be parallel to the section 
planes) the whole thing is placed in turpentine. This washes out the clove 
oil and fixes the objects very firmly to the paper. The paper with the 
attached objects is now passed through the bath of paraffin and imbedded in 
the usual way. After cooling on water the block is trimmed and the paper 
peeled off, leaving the objects in the paraffin close to the under surface of 
the block. This surface is now seen to be marked by the orienting lines of 
the ribbed paper, and also by any record numbers which may before imbed- 
ding have been written with a soft pencil on the paper. 
A somewhat more complicated form of this process has been described by 
Woodwobth, Bull. Mus. Comp. Zool., xxxviii, 1893, p. 45. 
A similar process has also been described by Field and Maetin in Zeit.f. 
uriss. Mik., xi, i, 1894, p. 11, small strips of gelatin being used instead of 
paper. 
137. Cutting and Section-stretching.—Paraffin sections are 
cut dry—that is, with a knife not moistened with alcohol or 
other liquid. By this means better sections are obtained, but 
a difficulty generally arises owing to the tendency of sections 
so cut to curl up on the blade of the knife. It is sometimes 
difficult by any means to unroll a thin section that has curled. 
To prevent sections from rolling, the following points should 
be attended to. 
First and foremost, the paraffin must not be too hard, but 
must be taken of a melting-point suitable to the temperature 
of the laboratory (for the winter season, the temperature of 
the laboratory being between 15° and 17° C., a paraffin 
melting at about 45° C. should be taken; for hot summer 
weather, laboratory at 22° C., a paraffin of 48° C. melting- 
point ; but see further, § 142). 
The exact degree of hardness necessary must be determined 
by experiment. If, after cutting has begun, the paraffin be 
found to be too hard, it may be softened by the following simple 
expedient (Fol, Lehrbuch. d. vergl. milcr. Anat., p. 123) :— 
A lamp provided with a parabolic reflector is set up near the 86 
CHAPTER IX. 
microtome in such a position that the heat-rays of the flame 
are thrown by the reflector on to the imbedded object. The 
right temperature is obtained by adjusting the distance of the 
lamp. This is not a mere theoretical fancy, but a very practical 
hint. I find that a reflector is not necessary, and that a 
mere spirit-lamp set up near the object will sometimes bring 
the paraffin to the right consistency in a few minutes. 
If, on the contrary, the paraffin be found too soft, it may 
be hardened by exposing it to the cooling influence of a lump 
of ice placed in the focus of a similar reflector. 
It is often sufficient to moderate the temperature of the 
room by opening or closing the window, stirring the fire, 
setting up a screen, or the like. 
Secondly, the knife should be set square, for the oblique 
position produces rolling, and the more the knife is oblique 
the more do the sections roll. 
Thirdly, it is better to cut ribbons than disconnected sec- 
tions ; ribbons of sections will often cut perfectly flat, even 
when the same mass will only give rolled sections if cut dis- 
connectedly. 
Special masses having less tendency to roll than pure 
paraffin have been proposed. Thus a mass composed of four 
parts of hard paraffin and one of vaselin has been recom- 
mended. I recommend, however, that all such mixtures be 
avoided. 
Mechanical means may be employed. The simplest of these 
and perhaps the best is as follows : 
During the cutting the edge of the section that begins to 
curl is caught and held down on the blade of the knife by 
means of a small camel-hair brush with a flat point, or by a 
small spatula made by running a piece of paper on to the 
back of a scalpel. Or the section is held down by means of 
an instrument called a “ section-stretcher.” This consists 
essentially of a little metallic roller suspended over the object 
to be cut in such a way as to rest on its free surface with a 
pressure that can be delicately regulated so as to be sufficient 
to keep the section flat without in any way hindering the 
knife from gliding beneath it. 
According to Ref. Handbook Med. Sci., Supp., p. 441, a 
very simple section-smoother may be made as follows :—The 
head and point are cut off an ordinary pin, which is then fixed IMBEDDING METHODS—PAltAFFIN, ETC. 
87 
parallel to the edge of the knife by pressing- its ends down 
into two small pellets of beeswax to the proper depth. 
Amongst the more complicated devices that have been 
imagined, that of Born (Zeit.f. wiss. Mik., x, 2, 1893, p. 157, 
or Journ. Roy. Mic. Soc., 1894, p. 132) appears to me to be 
the most recommendable. It must be allowed that all these 
instruments are difficult to use, and that if they are not 
perfectly adjusted they may easily injure the sections. 
See the descriptions of various forms of section-stretchers, Zool. Anzeig., 
vol. vi, 1883, p. 100 (Schultze) ; Mitth. Zool. Stat. Neapel, iv, 1883, p. 429 
(Mayer, Andres, and Giesbrecht) ; Arch.f. mik. Anat., xxiii, 1884, ji. 537 
(Decker) ; Bull. Soc. Beige Mic., x, 1883, p. 55 (Francotte) ; The Micro- 
scope, February, 1884 (Gage and Smith) ; Whitman’s Metli. in Mic. Anat., 
1885, p. 91; Zeit. f. wiss. Mik., iv, 2,1887, p. 218 (Strasser) ; as well as 
Journ. Boy. Mic. Soc., iii, pp. 450, 916, and other places. 
Another plan is to allow the sections to roll, but to control 
the rolling. To this end, the block of paraffin is pared to the 
shape of a wedge five or six times as long as broad, the object 
being contained in the broad part, and the edge turned 
towards the knife. The sections are allowed to roll and come 
off as coils, the section of the object lying in the outermost 
coil, which will be found to be a very open one—indeed, very 
nearly flat. Lay the coil on a slide with this end downwards, 
warm gently, and the part containing the object will unroll 
completely and lie quite flat. 
Minute examination of paraffin sections sometimes reveals certain dis- 
tortions and dislocations or even raptures of delicate elements. I have 
often noticed that in certain regions of my sections all the karyokinetie 
figures are drawn up to one side (always the same side) of the nucleus, 
leaving the rest of the nucleus empty and vacuolar in appearance. The 
achromatic fibrils of the division spindle are frequently ruptured, and I have 
not rarely found isolated chromosomes lying far from the nucleus in the 
body of cells, or even outside the cells themselves. These phenomena have 
generally been ascribed to “ shrinkage ” caused by the action of the fixing 
agents or the processes of dehydration or imbedding. Heidenhain (Ueber 
Kern u. Protoplasma, in Festschr. Herrn Geheimr. A. v. Kolliker 
gewidm., 1892) thinks that they are often caused by excessive tilt of the 
under surface of the microtome knife. If this he found to he the case, the 
knife should be readjusted by means of a piece of cardboard placed in the 
jaws of the clamp. I would suggest that another cause of these defects is to 
be found in imperfection of the edge of the knife. If the knife be blunt, or 
have a rounded or curled edged caused by untrue honing or stropping, it will 
of course act in respect to minute structures as a plough rather than a 
cutting instrument, and thus produce the appearances described. 88 
CHAPTER IX. 
Devices for heating or for cooling the knife, with a view to the improve- 
ment of cutting, have been described; see van Walsem in Zeit. f. wiss. 
Mik., xi, 2,1894, p. 218. I have myself sometimes found it advantageous 
to warm the knife. 
138. Chain or Ribbon Section-cutting.—If a series of paraffin 
sections be cut in succession and not removed from the knife 
one by one as cut, but allowed to lie undisturbed on the 
blade, it not unfrequently happens that they adhere to one 
another by the edges so as to form a chain which may be taken 
up and transferred to a slide without breaking up, thus 
greatly lightening the labour of mounting a series. The fol- 
lowing appear to me to be the factors necessary for the pro- 
duction of a chain. 
Firstly, the paraffin must be of a melting-point having a 
certain relation to the temperature of the laboratory. Small 
sections can always be made to chain when cut from a good 
paraffin of 45° C. melting-point in a, room in which the ther- 
mometer stands at 16° to 17° C. (The temperatures quoted 
apply to the case of rooms heated by an open fire, and pro- 
bably would not apply to the case of rooms heated by closed 
stoves, such as are usual in Germany.) At 15° C. the paraffin 
will be found a trifle hard. At 22° C. the proper melting- 
point of the paraffin will probably be found at about 48° C., 
but my observations at these temperatures are less extended. 
Secondly, the knife should he set square. Thirdly, the block 
of paraffin should be pared down very close to the object and 
should be cut so as to present a straight edge parallel to the 
knife edge ; and the opposite edge should also be parallel to 
this. Fourthly, the sections ought to be cut rapidly, with 
the swiftest strokes that can be produced. For it is the 
sharp impact of the knife that slightly heats, and therefore 
slightly softens the near edge of the paraffin, and thus causes 
the sections to cohere. It is by no means necessary for this 
purpose to have recourse to special mechanical contrivances, 
as in the so-called ribbon microtomes. The Thoma microtome 
well flooded with oil is sufficient. But the automatic micro- 
tomes are certainly most advantageous for this purpose, and 
amongst them the Cambridge Rocking Microtome, the 
Reinhold-Giltay, and the Minot, may be quoted as giving 
admirable results. IMBEDDING METHODS PARAFFIN, ETC. 
89 
Various plans, such as coating the edges of the paraffin with softer 
paraffin, or with Canada balsam, or the employment of specially prepared 
paraffin, have been recommended, with the idea that they help the sections 
to stick. None of these devices is necessary. For the prepared paraffin of 
Spee, Brass, and Foettinger, see below, § 143. 
It sometimes, though rarely, happens that the ribbon becomes electrified 
during the cutting, and twists and curls about in the air in a most fantastic 
manner. It may be got flat by warming slightly; but there is no known 
means of preventing the electrification. 
139. Collodionisation of Sections.—Some objects are by 
nature so brittle that, notwithstanding all precautions taken in 
imbedding and previous preparation, they break or crumble 
before the knife, or furnish sections so friable that it is im- 
possible to mount them in the ordinary way without some 
impairment of their integrity. Ova are frequently in this 
case. The remedy for this state of things consists in cover- 
ing the exposed surface of the object just before cutting each 
section with a thin layer of colloidin, which serves to hold 
together the loose parts of even the most fragile sections in 
a wonderfully efficacious way; and the same treatment applied 
to tissues which are not specially fragile will enable the ope- 
rator to cut sections considerably thinner than can be obtained 
in the usual way. Butschli has obtained in this manner 
sections of less than 1 /u in thickness. 
The primitive form of the process was to place a drop of 
collodion on the free surface of each section just before cutting 
it. But this practice has two defects ; the quantity of collo- 
dion employed sensibly softens the paraffin, and the thick 
layer of collodion when dry causes the sections to roll. 
Mark (Amer. Natural., 1885, p. 628; cf. Journ. Roy. Mic. 
Soc., 1885, p. 738) gives the following directions : 
“ Have ready a little very fluid collodion in a small bottle, 
through the cork of which passes a small camel-hair brush, 
which just dips into the collodion with its tip. The collodion 
should be of such a consistency that when applied in a thin 
layer to a surface of paraffin it dries in two or three seconds 
without leaving a shiny surface. Collodion of this consistency 
does not produce a membrane on the paraffin in drying, and 
therefore has no tendency to cause sections to roll. It has 
further the advantage that it penetrates to a certain depth 
below the surface of the preparation, and fixes the deeper 90 
CHAPTER IX. 
layers of it in their places. The collodion must he diluted 
will ether as soon as it begins to show signs of leaving a shiny 
surface on the paraffiu. 
“Take the brush out of the collodion, wipe it against the neck 
of the bottle, so as to have it merely moist with collodion, and 
quickly pass it over the free surface of the preparation. Care 
must be taken not to let the collodion touch the vertical sur- 
faces of the paraffin, especially not the one which as tua-ned 
towards the operator, as that will probably cause the section 
to become stuck to the edge or under sui-face of the knife. 
As soon as the collodion is dry, which ought to be in two or 
three seconds, cut the section, withdraw the knife, and pass 
the collodion brush over the newly-exposed surface of the 
paraffin. Whilst this last layer of collodion is di’ying, take 
up the section from the knife and place it with the collodion- 
ised surface downwards on a slide pi-epared with fixative of 
Schaellibaum. Then cut the second section, and repeat the 
manipulations just described in the same order. A skilful ope- 
rator can cut l’ibbons of sections, collodionising each section.” 
Henking (Zeit. f. wiss. Mik., iii, 4, 1886, p. 478) objects to 
the above process that the ether of the collodion softens the 
paraffin, and proposes a solution of paraffin in absolute alcohol. 
The solution is made by scraping paraffin into absolute alcohol. 
For extremely brittle objects, such as ova of Phalangida, 
the same author recommends a thin (light yellow) solution of 
shellac in absolute alcohol. 
Heidek (Embryonalentic. v. Hydrophilus, 1889, p. 12 ; cf. 
Zeit. f. wiss. Mik., viii, 4, 1892, p. 509) eaaiploys a solution 
made by mixing a solution of gum mastic in ethei*, of a 
syrupy consistency, with an equal volume of collodion, and 
diluting the mixture with ether until quite thin and liquid. 
Babl (ibid., xi, 2, 1894, p. 170) employs superheated 
kept at a temperature of about 100° C. on a water- 
bath. This plan has the advantage of efficiently filling up 
any cavities there may be in the objects, and also of pi-event- 
ing the sections fi’om rolling. 
140. Clearing and Mounting. — Tlie sections having been 
obtained are generally mounted on a slide in serial order by 
one of the methods described in the chapter on “ Serial Section 
Mounting.” If the sections have rolled or become folded on IMBEDDING METHODS PARAFFIN, ETC. 
91 
cutting they must be unrolled and smoothed out before being 
fixed to the slide. 
The most efficacious plan for unrolling sections is perhaps 
the combined treatment with fluid and heat (Gaskell, Quart. 
Journ. Mic. 8r,i., xxxi, 1890, p. 382; M. Duval, Journ. de 
VAnat. et de la Physiol., 1891, p. 26; Henneguy, ibid., 1891, 
p. 398; Gulland, Journ. of Anat. and Physiol., 1891, p. 56 ; 
and others). The rolled sections are either floated on to the 
surface of warm water or warm alcohol contained in a watch- 
glass or suitable dish, which causes them to flatten out, and 
are then transferred to a slide to be mounted in any desired 
manner. Or the slide has a layer of water spread over it, 
the sections are laid on the water, and the slide is heated (to 
about 45° to 50° C.) until the sections flatten out, which 
happens in a few instants. The method can be made avail- 
able for fixing series of sections to the slide; the further 
details necessary for the successful accomplishment of this 
are given in §§ 195 and 199. 
Van Walsem (Zeit. f. wiss. Mik., xi, 2, 1894, p. 228) describes a plan 
according to which the sections are arranged on a strip of parchment-paper 
which is moistened and passed over a warmed cylinder revolving in water on 
the principle of a postage-stamp dampener (see abstract with illustration in 
Journ. Boy. Mic. Soc., 1895, p. 121). 
The sections having” been duly smoothed by one of these 
processes, and duly fixed to the slide (unless it is desired to 
keep them loose) all that now remains is to get rid of the 
paraffin and mount or stain as the case may be. The following 
solvents of paraffin have been recommended for freeing sec- 
tions from the paraffin with which they are infiltrated:—Tur- 
pentine, warm turpentine, a mixture of 4 parts of essence of 
turpentine with 1 of creasote, creasote, a mixture of turpen- 
tine and oil of cloves, benzin, toluol, xylol, thin solution of 
Canada balsam in xylol (only applicable to very thin sections), 
hot absolute alcohol, naphtha, or any other paraffin oil of low 
boiling-point. Any of these may be used, but xylol and toluol 
are probably in most respects the best. 
Naphtha does very well for sections that are to be passed 
through alcohol for staining, but is not safe for those that are 
to be put direct into balsam, as it frequently precipitates 
Canada balsam (with colophonium there is less risk). 
If the slide be warmed to the melting-point of the paraffin, 92 
CHAPTER IX. 
a few seconds will suffice to remove the paraffin if the slide 
be plunged into a tube of xylol or toluol. The sections may 
be mounted direct from the xylol or the slide may be brought 
into a tube of alcohol to remove the solvent for staining. 
The removal of the paraffin is greatly facilitated by the 
following simple expedient (shown me by Professor v. 
Korotneff). Warm the slide over a flame, and whilst the 
paraffin is still melted hold it close before your lips and blow 
down on it vigorously. The paraffin and collodion or albumen 
or other medium used for gumming the sections to the slide 
are scattered right and left over the slide, leaving the sections 
high and dry. The splashes of paraffin are wiped off whilst 
warm with a cloth, and the slide being put at once into a tube 
of xylol, the remainder of the paraffin is dissolved out in a 
few seconds. This process also favours the adhesion of the 
sections. 
141. Recapitulation of the Paraffin Method, as recommended 
to be practised.—Put into a small test-tube enough oil of 
cedar to cover your object. On to the oil pour carefully the 
same quantity of absolute alcohol. Take your (already de- 
hydrated) object and put it carefully into the alcohol. Leave 
it until it has sunk to the bottom of the cedar oil. Wait till 
the refraction lines, § 111, have vanished. Then put it into 
paraffin kept at melting-point in a watch-glass. Let the 
paraffin be of the very lowest melting point that will give suffi- 
ciently thin sections, and to this end work in a cool place, so 
as not to be obliged to go above 45° C. if possible (see also 
next section). After a time change the paraffin (i. e. put the 
object into a fresh watch-glass with clean paraffin) once, or 
twice if the object be at all large. As soon as the object is 
thoroughly soaked with paraffin float the watch-glass on cold 
water. When cool, cut out a block of paraffin containing the 
object, and fix it with a heated needle on a cone of paraffin 
already mounted on the object-carrier of the microtome. 
Pare it square, and as close down to the object as possible on 
all sides except the one turned towards the knife; this had 
better have a wall of a millimetre or two, or more, according: 
to the size of the object, left standing. Set the knife square. 
Set the block square to the knife-edge. Cut sections in 
chains or ribbons, collodionising them if necessary. Fix IMBEDDING METHODS PARAFFIN, ETC. 
93 
them in serial order on a slide according to one of the methods 
given in Chap. XI. Warm, and remove the paraffin with 
xylol. Stain or mount. 
Parcifiin Masses. 
142. Pure Paraffin.—It is now pretty generally admitted 
that pure paraffin forms an imbedding mass greatly superior 
for ordinary work to any of the many fatty mixtures that used 
to be recommended. I have only to repeat here that a’paraf- 
fin melting at 45° C. is that which in my experience gives 
the best results so long as the temperature of the laboratory is 
between 15° and 17° C.; whilst for a temperature of 22° C. a 
paraffin melting at 48° is required. If the temperature of 
your laboi*atory have risen much above 22° C. you had better 
give it up, for good section-cutting with paraffin under such 
conditions is next to impossible. 
Paraffin of the melting-points named is easily found in 
commerce. Intermediate sorts may be made by mixing hard 
and soft paraffin. Two parts of paraffin melting at 50° 
with one of paraffin melting at 36° C. give a mass melting 
at 48° C. 
Many workers of undoubted competence prefer masses 
somewhat harder than those recommended, viz. of melting- 
points varying between 50° and 55° C. for the normal tem- 
perature of the laboratory. Some authors still recommend 
masses melting at 60° C. or higher. I can only repeat 
that I am convinced that, besides being most hurtful to 
tissues, such masses have no raison d’etre whatever in temperate 
climates. 
The figures above given have been repeatedly verified and 
are undoubtedly correct. But an important explanation 
remains to be made. The statements refer to work with the 
Thoma sliding microtome. I have since ascertained that 
microtomes with fixed knives, such as the Cambridge, the 
Minot, or the Reinhold-Giltay, will give good results, so far 
as cutting is concerned, with much harder paraffins. This is 
an advantage, so far as the obtaining of very thin sections is 
concerned; but it remains true that for delicate work it is 
well in the interest of the preservation of the tissues, to use 
a paraffin of as low a melting-point as possible. 94 
CHAPTER IX. 
Paraffin had better be obtained from Griibler, or one of the 
known dealers in microscopic reagents. Gaule recommends 
that the bluish transparent sorts be taken. I should say, 
transparent by all means, but, if possible, rosy rather than 
bluish. New paraffin is bluish; if kept long, which is well, 
it generally becomes rosy. 
143. Prepared Paraffin (Pure).—Graf Spee (Zeit.f. wiss. Mile., 
ii, 1885, p. 8) recommends the following preparation of com- 
mercial paraffin as giving a mass particularly favourable for 
ribbon-section cutting. Paraffin of about 50° C. melting- 
point is taken and heated in a porcelain capsule by means of 
a spirit lamp. After a time disagreeable white vapours are 
given off, and the mass shrinks a little. This result is arrived 
at in from one to six hours, according to the quality of the 
paraffin. The mass then becomes brownish yellow, and after 
cooling shows an unctuous or soapy surface on being cut. 
The melting-point will be found to have risen several degrees. 
This mass may be obtained ready prepared from Griibler. 
Brass [loc. cit., p. 300) recommends the use of paraffin that 
has been kept for some years, as such has less tendency to 
crystallise than new paraffin. In this I concur. 
Foettinger recommends {Arch, de Biol., vi, 1885, p. 124) a somewhat 
complicated treatment with caustic potash, in which I have no faith (it was 
tried by one of the writers of the Traite des Metli. techn., during the pre- 
paration of that work). 
144. Paraffin Mixtures and other Similar Masses.—Of these 
the only ones that I think can be recommended for a moment 
are the following :—Schulgin’s paraffin with a little cerisin 
(this is evidently what Schulgin means by “ ceresin ”). Or, 
instead of cerisin, white wax (see Zool. Anz., 1883, p. 21), 
or the mixture of Brass (Zeit. f. wiss. Mih., ii, 1885, p. 301), 
who recommends four to six parts of white wax to 100 of 
paraffin. Sections may be cleared with benzin. Van Walsem 
[ibid., xi, 2, 1894, p. 216) advises for large objects 5 percent, 
of yellow wax. 
Soap Masses. 
145. Utility of Soap Masses.—Soap masses certainly have 
many good points. The solvent is alcohol; the mass is highly IMBEDDING METHODS PARAFFIN, ETC. 
95 
transparent, very penetrating, and a good mass cuts far better 
that even paraffin. The mass may be cut either dry or with 
alcohol. As to the preservation of tissues, the mass is alka- 
line, which is against it; yet some workers still prefer soap 
to paraffin, and it has been recommended by so experienced a 
worker as Chun, for Siphonophora (certainly as delicate a 
class of objects as any that exist), on the ground of its pro- 
ducing less shrinkage than paraffin. It is evident that soap 
masses may render service for imbedding such structures as 
-\vill not stand complete dehydration without shrinking. Soap 
imbedding is, in short, a semi-wet process. 
146. Transparent Soap (Polzam, Morjpli. Jahrb., iii, 1877, 
3tes Heft, p. 558).—The following account is taken from 
Salensky’s paper on the gemmation of Salpa (loc. cit.). 
Take good white soap (“ gewdhnliche Kernseife ”), cut it 
up into thin slices, and put them to dry in the sun for some 
days until they become white. The slices are then to be 
rubbed up to a fine powder, which is mixed with spirit to the 
consistency of porridge. Now mix the porridge with alcohol 
and glycerin in such proportions that the whole shall contain, 
for every ten parts by weight of the soap, 22 parts of glycerin 
and 35 parts of alcohol (90 per cent.). Let the whole simmer 
until there is obtained a perfectly transparent, syrupy, some- 
what yellow fluid. 
The objects, previously dehydrated in alcohol, are imbedded 
in this mass in the usual manner. 
The mass may be removed from the sections either by means 
of water or of very dilute alcohol. 
147. Transparent Soap (Kadyi, Zool. Anz., 37, 1879, vol. ii, 
p. 477).—Twenty-five grms. of shavings of stearate of soda 
soap (any stearate of soda soap will do, but the most to be 
recommended is the sort known in commerce as “weisse 
Wachskernseife ”) are heated in a retort with 100 c.c. of 96 
per cent, alcohol over a water-batli until the whole is dissolved. 
Filter if necessary. If a drop of the solution be now poured 
into a watch-glass it will be seen that it almost immediately 
solidifies into a white mass. This is not what is wanted, and 
is a sign that the solution does not contain water enough. 96 
CHAPTER IX. 
Small quantities of water are therefore added by degrees to 
the solution, and the effect tested from time to time by pouring 
a drop of the mixture into a watch-glass. The mass will be 
seen to become more and more pellucid until a point is reached 
at which it is almost perfectly transparent, with merely the 
slightest blue opalescence. The preparation of the mass is 
then complete. 
It is not possible to state a 'priori the exact proportion of 
water that should be added, as this naturally depends on the 
amount of water already present in the sample of soap taken. 
In very many cases it will be found that for about 120 grms. 
of soap solution 5 to 10 grms. of water will be required. 
It is necessary to be very cautious in adding the water, as 
if too much be taken the mass solidifies more slowly or not at 
all. The greatest amount of elasticity and consistency is 
possessed by the mass at the moment in which it contains 
exactly the minimum amount of water necessary to make it 
transparent. 
The reasons for this process are explained as follows :—Stearate of soda 
soap is soluble in divers proportions in warm alcohol. On cooling, the solu- 
tion either solidifies into a homogeneous and pellucid mass, or into a white 
granular mass; or, in certain cases, does not solidify at all. The result in 
each case depends on the proportion of water present in the solution. For 
instance, if 5 to 6 parts of a tolerably dry soap he dissolved in 100 parts of 
96 per cent, alcohol, a solution is generally obtained that solidifies into a 
transparent mass. But such a mass is too soft, and its melting-point too 
low ; it melts by the heat of the finger. If now, in order to get a harder 
mass, you add more soap, you will get a solution that solidifies on cooling 
into a white granular mass; and it is only after adding to it a certain 
{small) quantity of water that you will obtain a solution that solidifies on 
cooling into a transparent mass. If you add more water than is just 
absolutely necessary to this end the mass will have too high a melting-point, 
and will solidify more slowly ; and if still more water be added the solution 
will not solidify for hours, or, indeed, not at all. The more soap you have 
in your alcoholic solution the more water must you add in order to get a 
transparent mass, and the more may you add without depriving the solution 
of the faculty of solidifying. Besides the mass prepared in the proportions 
given above, useful masses may be made for certain purposes with 10, 20, 
30, 40 per cent., or more or less of soap in alcohol. Weisker has employed 
a mass composed of about equal parts by weight of soap and alcohol. Such 
a mass is transparent, but yellow and oily, and takes a long time to solidify 
AVhen cool it is very tough. It requires a considerable temperature to 
liquefy it, and has less penetrating power than the more alcoholic masses. 
It is, however, very suitable for hard, and especially for cliitinous structures. IMBEDDING METHODS, PARAFEIN, ETC. 
97 
The mass recommended above boils at about 60° to 70° C. 
Objects should be imbedded in it in a watch-glass or in paper 
cases in the usual way. Whilst cutting, wet the knife and 
the mass with sti’ong alcohol (one advantage of this method 
is that the knife remains perfectly clean). The sections are 
brought into 96 per cent, alcohol, which frees them from 
the mass instantaneously if warmed, and after a time if left 
cold. 
Gelatin Masses 
148. Gelatin Imbedding is a method that has the advantage 
of being applicable to tissues that have not been in the least 
degree dehydrated, and may render great service in the study 
of very watery objects. 
The modus operandi is, on the whole, the same as for other 
fusion masses, with the difference that the objects are prepared 
by penetration with water instead of alcohol or a clearing 
agent. After the cooling of the mass it may sometimes be 
cut at once, but it is generally necessary to harden it. This 
may be done by treatment for a few minutes with absolute 
alcohol (Kaiser), or for a few days with 90 per cent, alcohol 
(Klebs) or chromic acid (Klebs), or it may be frozen 
(SOLLAS). 
The mass is removed from the sections by means of warm 
water. 
149. Klebs’ Gelatin (Glycerin Jelly) (Arch. f. mik. Anat., v, 
1869, p. 165).—A concentrated solution of isinglass is mixed 
with half its volume of glycerin. 
150. Kaiser’s Gelatin (Bot. Centralb., i, 1880, p. 25; Journ. 
Boy. Mic. Soc., iii, 1880, p. 504). — One part by weight of the 
finest French gelatin is left for about two hours in 6 parts by 
weight of water; 7 parts of glycerin are added, and for 
every 100 grms. of the mixture 1 grm. of concentrated car- 
bolic acid. The whole is warmed for ten to fifteen minutes, 
stirring all the while, until the whole of the flakes produced 
by the carbolic acid have disappeared. Filter whilst warm 
through the finest spun glass, which has been previously 
washed in water and laid whilst wet in the filter. 98 
CHAPTER IX. 
151. Gerlach’s Gelatin (Gerlach, Unters. a. d. Anat. Inst. 
Erlangen, 1884; Journ. Roy. Mic. Soc., 1885, p. 541).—Take 
gelatin, 40 grms.; saturated solution of arsenious acid, 200 
c.c.; glycerin, 120 c.c. Clarify with white of egg. The 
mass may be kept for years in a well-stoppered bottle. The 
objects to be prepared for imbedding by a bath of one-third 
glycerin. 
152. Brunotti’s Cold Gelatin Mass (Journ. de. Botan., vi, 
1892, p. 194; Journ. Roy. Mic. Soc., 1892, p. 706).—Twenty 
grins, gelatin dissolved wit-li heat in 200 c.c. distilled water, 
and 30 to 40 c.c. of glacial acetic acid with 1 grm. corrosive 
sublimate added after filtering. At the temperature of 
15° C. the mass has the consistence of a thick syrup. Objects 
are prepared by soaking in some of the mass diluted with two 
to three vols. of water, then imbedded in the undiluted mass. 
The mass is then hardened in spirit or bichromate of potash, 
picric acid, or the like. No heat at all is required in this 
process. CHAPTER X. 
COLLODION (CELLOIDIN) AND OTHER IMBEDDING METHODS. 
153. Advantages of the Collodion or Celloidin Method.—Collo- 
dion (or celloidin) masses do not require the employment of 
heat, which may he an important question in the case of some 
very delicate structures. They do not require that the objects 
should be cleared before imbedding, and that is an advantage 
in the case of very large objects. They are quite transparent, 
a quality which facilitates very greatly the orientation of the 
object. And they are specially indicated for very large 
objects, for the soaking in collodion being quite inoffensive 
to the most delicate elements may be prolonged if necessary 
for weeks, thus ensuring the harmless penetration of objects 
that would be literally cooked if they were submitted to a 
paraffin bath of like duration. Lastly, the mass being quite 
transparent, it is not necessary to remove it from the sections 
before staining and mounting them; it may remain, and 
fulfil the function of an admirable support to the tissues, 
holding in their places brittle or detached elements that with- 
out that help would fall to pieces and be lost. 
There are two disadvantages. One is that the process is a 
very long one; as usually practised, the collodion process 
requires some three days for the imbedding of an object that 
can be imbedded in paraffin in an hour (though the time may 
be greatly abridged by Gilson’s rapid process given below). 
Another is that it is impossible to obtain with celloidin sections 
as thin as those furnished by paraffin; the lowest limit I have 
been able to attain to is 7 fx, which for much work is not 
sufficient. 
These considerations seem to justify the assertion that the 
collodion method is a special method, and not a general 
method. 
As to the choice of a process, I urgently recommend the 
recently introduced practice of clearing before cutting, and 
cutting dry as described in §§ 166 and 168. 100 
(JHAPTER X. 
154. Collodion, Celloidin, and Photoxylin.—The collodion 
method is due to Duval (Journ. de VAnat., 1879, p. 185). 
Celloidin, recommended later on by Merkel and Schieffer- 
decker (Arch. f. Anat. u. Phys., 1882, p. 200), is merely a 
patent collodion. It may be obtained through the post by 
writing to Schering’s “ Griine Apotheke,” Wittick and Ben- 
kendorf, Berlin, N. Chaussee-Strasse, No. 19, or from Grubler, 
or the other dealers in histological reagents. It is sent out in 
the form of tablets of a tough gelatinous consistency and 
slightly milky-white transparency. These tablets may, if de- 
sired, be dissolved at once in ether, or a mixture of ether and 
alcohol, to make a collodion of any desired strength. But it is 
better, as recommended by Apathy, to cut them up into thin 
shavings, which should be allowed to dry in the air until they 
become yellow*, transparent, and of a horny consistency, and 
that these be then dissolved in alcohol and ether (sulphuric, 
free from acid). The solutions thus prepared are free from the 
excess of water that is present in the undried celloidin, and 
give after hardening a mass that is more transparent and of 
a better consistency for cutting (Zeit. f. iviss. Mik., vi, 2, 
1889, p. 164). 
Imbedding masses of excellent quality can be prepared with 
ordinary collodion, but celloidin will be found more conve- 
nient to manipulate and furnishes more readily solutions of 
known concentration. Otherwise there is but little to choose 
between the two, and therefore in this work the terms collodion 
and celloidin are used indifferently. 
Pliotoxylin (Krysinsky, Virchow’s Archiv, cviii, 1887, p. 217 ; Busse, 
Zeit.f. wiss. Mik., ix, 1, 1892, p. 47) is a dry substance, of the aspect of 
cotton wool, and chemically nearly related to celloidin. It can be obtained 
either from Scherlng or Grubber. It gives a clear solution in a mixture 
of equal parts of ether and absolute alcohol, and should be used in exactly 
the same way as celloidin. In has the very great advantage of affording a 
mass which after hardening in 85 per cent, alcohol remains perfectly 
transparent. But celloidin or common collodion also give perfectly trans- 
parent masses if cleared in bulk as I recommend should be done (§§ 166— 
168); so that there is no advantage on this head in having recourse to 
photoxylin, unless it be desired to proceed in the old way. 
155. Preparation of Objects.—The objects must first be 
thoroughly dehydrated with absolute alcohol. They are then COLLODION AND OTHER IMBEDDING METHODS. 
101 
soaked till thoroughly penetrated in ether, or, which is better, 
in a mixture of ether and absolute alcohol. Duval (Z. c.) 
takes for this purpose a mixture of ten parts of ether to one 
of alcohol; Schieeeerdecker (and the majority of workers) a 
mixture of equal parts of ether and alcohol; Tubby (in Nature, 
November 17th, 1892, p. 51) advises a mixture of four parts 
of ether and one of alcohol. But the point is one of no great 
importance. 
This stage maybe omitted if the objects are of a sufficiently 
permeable nature, and they may be brought direct from 
alcohol into the collodion bath. 
156. The Collodion. Bath.—The next step is to get the objects 
penetrated with thick collodion. The secret of success here 
is to penetrate them first with thin solutions, then with the 
definitive thick one. (A thin solution may be taken to mean 
one containing from 4 to 6 per cent, of celloidin (dried as 
described in § 154) ; a thick solution one containing 10 to 12 
per cent.) 
If collodion be taken, the thin solution may be made by 
diluting it with ether. If photoxylin or celloidin be taken, 
the solutions are made in a mixture of ether and absolute 
alcohol in equal parts. 
The dried celloidin shavings dissolve very slowly in the 
mixture. Elschnig (Zeit. f. iviss. Mik., x, 4, 1893, p. 443) 
states that solution is obtained much quicker if the shavings 
be first allowed to swell up for twenty-four hours in the neces- 
sary quantity of absolute alcohol, and the ether be added 
afterwards. On trial it seems to me that this is so. 
Busse (op. cit., ix, 1, 1892, p. 47) gives the following pro- 
portions for the successive baths :—No. 1,10 parts by weight 
of photoxylin or perfectly dried celloidin to 150 parts of the 
ether and alcohol mixture : No. 2, 10 parts of photoxylin or 
celloidin to 105 of the mixture; No. 3, 10 parts to 80 of the 
mixture (already used solution may be employed for the first 
bath). 
I generally use only two solutions : one weak one, and one 
strong one corresponding approximately to Busse’s No. 2. 
His No. 3 is so thick that excessive time is required to obtain 
penetration by it. 
The objects ought to remain in the first bath until very 102 
CHAPTER X. 
thoroughly penetrated ;—days, even for small objects,—weeks 
or months for large ones (human embryos of from six to 
twelve weeks, for instance). If the object contain cavities, 
these should be opened to ensure their being filled by the 
mass. 
When the object is duly penetrated by the thin solution, or 
solutions if more than one have been employed, it should be 
brought into the thickest one. This may be done (as first 
described in this work, 1st. edit., 1885, p. 194) by allowing 
the thin solution to concentrate slowly (the stopper of the con- 
taining vessel being raised, for instance by means of a piece 
of paper placed under it), and making up the loss from 
evaporation with thick solution. 
157. Imbedding.—If the object is such that it can be fixed, by 
gamming or otherwise, to the holder of the microtome without 
the intervention of any specially shaped mass of collodion around 
it, and if the presence of such a mass be not required in the 
interest of the orientation of the object or of the production 
of continuous series of sections, no special imbedding is neces- 
sary, and as soon as the objects are duly penetrated by the thick 
solution you may proceed to the hardening part of the pro- 
cess. In other words, it is waste of time to get the object 
into a special block of collodion if that is not rendered desirable 
for the reasons above mentioned. If, however, it be desirable, 
the objects must at this stage, if it has not been done before, be 
imbedded—that is, arranged in position in the thick collodion in 
the receptacle in which they are to be hardened. For the usual 
manipulations see § 129. I recommend the paper thimbles or 
cylindrical trays, Fig. 2, as being very convenient for collodion 
imbedding. The bottoms, however, should be made of soft 
wood in preference to cork ; cork is elastic, and bends in the 
object-holder of the microtome, deforming the mass and 
object. The box should be prepared for the reception of 
the object by pouring into it a drop of collodion, which is 
allowed to dry. The object of this is to prevent bubbles 
coming up through the wood or cork and lodging in the mass. 
Objects may also be imbedded on a piece of pith or leather, 
which should also be prepared with a layer of dry collo- 
dion. 
Watch-glasses, square porcelain water-colour moulds, and COLLODION AND OTHER IMBEDDING METHODS. 
103 
the like, also make convenient imbedding receptacles. Care 
should be taken to have them perfectly dry. 
It not unfrequently happens that during these manipulations 
bubbles make their appearance in the mass. Before pro- 
ceeding with the hardening these should be got rid of. This 
may be done by exposing the whole for an hour or two to the 
vapour of ether in a desiccator or other well-closed vessel. 
Care should be taken that the ether (which may be poured 
on the bottom of the vessel) does not wet the mass (Busse, 
Zeit.f. wiss. Mik., viii, 4, 1892, p. 467). 
158. Orientation.—If it be desired to mark the position of the object 
in the mass in order to facilitate the subsequent orientation of it on the 
object-holder of the microtome, recourse may be had to the method described 
by Eyclesheimek in Amer. Nat., xxvi, 1892, p. 354 (see also Journ. Boy. 
Mic. Soc., 1892, p. 562). The object is imbedded in one of the metal 
boxes described in § 129. The box has its ends and sides perforated at 
regular intervals by small opposite holes. Silk threads are passed through 
these holes from side to side, stretched, and kept tight by sticking them to 
the sides of the box by means of a drop of celloidin, leaving a length of a 
couple of inches hanging loose. The loose ends are soaked in thin celloidin 
solution with which lamp-black has been mixed. The object is arranged in 
position on the framework formed by the taut threads in the box, the mass 
is poured in, and the whole is hardened. After hardening, the celloidin 
holding the ends of the threads is dissolved by means of a drop of ether, 
and the lampblacked ends are pulled through the box. This leaves 
adhering to the bottom of the mass a series of black lines which form 
orientation points. 
Apathy (Zeit. f. wiss. Mik., v, 1, 1888, p. 47) arranges 
objects on a small rectangular plate of gelatin, placed on the 
bottom of the imbedding-recipient. The gelatin is turned 
out with the mass after hardening, and cut with it. The 
edges of the gelatin form good orientation lines. 
Halle and Born (see Zeit.f. iviss. Mik., xii, 3, 1896, p. 364) 
use plates of hardened white of egg, in which a shallow furrow 
for the reception of the objects has been cut by means of a 
special instrument. 
159. Hardening, Preliminary.—This is logically the next step, 
but as a matter of fact is frequently begun before. For the 
different processes of the collodion method so run into one 
another that it is difficult to assign natural lines of demar- 
cation between them. 104 
CHAPTER X. 
The objects being imbedded, and in the stage at which we 
left them at the end of § 156, the treatment should be as 
follows. The receptacles or supports are set with the mass 
under a glass shade, allowing of just enough communication 
with the air to set up a slow evaporation. Or porcelain 
moulds or small dishes may be covered with a lightly fitting 
cover. 
As soon as the added thick collodion (of which only just 
enough to cover the object should have been taken) has so far 
sunk down that the object begins to lie dry, fresh thick solu- 
tion is added, and the whole is left as before. (If the first 
layer of collodion has become too dry, it should be moistened 
with a drop of ether before adding the fresh collodion.) 
Provision should be again made for slow evaporation, either 
in one of the ways above indicated, or, which is perhaps 
better, by setting the objects under an hermetically fitting 
bell-jar, which is lifted for a few seconds only once or twice 
a day. (I have sometimes found it advantageous to set the 
object under a bell-jar together with a dish containing alcohol, 
so that the evaporation is gone through in an atmosphere of 
alcohol. This is especially indicated for very large objects.) 
The whole process is repeated every few hours for, if need 
be, two or three days. 
When the mass has attained a consistency such that the 
ball of a finger (not the nail) no longer leaves an impress on 
it, it should be scooped out of the dish or mould, or have the 
paper removed if it has been imbedded in paper, and be sub- 
mitted to the next stage of the hardening process. (If the 
mass is found to be not quite hard enough to come away 
safely it should be put for a day or two into weak alcohol, 
30 to 70 per cent.) 
160. Hardening, Definitive.—Several methods are available 
for the definitive hardening process. One of these is the 
chloroform method, due to Viallanes (Uech. sur VRist. et le 
Dev. des Insectes, 1883, p. 129). I recommend this method 
for small objects, because I find it much more rapid than 
the alcohol method, whilst giving at least as good a consis- 
tency to the mass. (Schiefferdecker does not find this, v. 
Zeit. f. wiss. Mik.y v, 4, 1881, p. 506.) For large objects 
the method is said to be inferior to the alcohol method, COLLODION AND OTHER IMBEDDING METHODS. 
105 
because the rapid hardening of the external layers is an 
obstacle to the diffusion necessary to the hardening* of the 
inner layers. 
The method consists in bringing the objects into chloroform. 
“Under the influence of this reagent this collodion coagulates 
into a mass having the consistence of wax, but having also an 
elasticity that renders it unbreakable, and having besides 
the precious quality of being admirably transparent, and 
possessing exactly the index of refraction of glass.” 
In some cases a few hours’ immersion is sufficient to g;ive 
the requisite consistence. In no case have my specimens re- 
quired more than three days. But the length of time required 
varies in a very inexplicable way, so that no rule can be given. 
The collodion frequently becomes opaque on being put into 
the chloroform, but regains its transparency after a time. 
Small objects may be hardened by chloroform without 'pre- 
liminary hardening by evaporation. All that is necessary is 
to expose the mass to the air for a few seconds until a mem- 
brane has formed on it, and then bring it into chloroform. If 
the mass is in a test-tube this may be filled up with chloroform, 
and left for two or three days. By this time the collodion 
mass will be considerably hardened, and also somewhat shrunk, 
so that it can be shaken out of the tube. It is then brought 
into fresh chloroform in a larger vessel, where it remains for 
about six days, after which time.it is generally ready for 
cutting. The process is sometimes much more rapid than this. 
Good chloroform is a necessity, as the reaction cannot be 
obtained with samples of chloroform that are not free from 
water. 
The above processes are excellent, but I regard them as 
primitive forms of the chloroform method. I now almost 
always harden in vapour of chloroform. All that is necessary 
is to put the liquid mass (after having removed bubbles as 
directed in § 157) with its recipient into a desiccator on the 
bottom of which a few drops of chloroform have been poured. 
The action is very rapid, and the final consistency of the mass 
at least equal to that obtained by the best alcohol-hardening. 
We shall revert to this subject, § 168. 
The more commonly employed hardening method is the 
alcohol method. The objects are thrown into alcohol and left 
there until they have attained the right consistency (one day 106 
CHAPTER X. 
to several weeks). The bottle or other vessel containing the 
alcohol ought not to he tightly closed, hut shoidd be left at least 
‘partly open. 
The strength of the alcohol is a point on which the practice 
of different writers differs greatly. The question may now 
be considered to be finally settled by experiments, specially 
directed to the clearing up of this point, made by Busse {Zeit. 
f. wiss. Mik., ix, 1, 1892, p. 49), and which I have repeated 
and confirmed. Busse finds that alcohol of about 85 per cent. 
is the best, both as regards the cutting consistency and the 
transparency of the mass. Care must be taken to keep the 
mass moist whilst cutting, as it dries by evaporation very 
quickly. 
Lastly, the mass may be frozen. After preliminary hardening by alcohol 
it is soaked for a few hours in water in order to get rid of the greater part 
of the alcohol (the alcohol should not be removed entirely, or the mass 
may freeze top hard). It is then dipped for a few moments into gum 
mucilage in order to make it adhere to the freezing plate, and is frozen. 
The sections are brought into warm water. If the mass have frozen too 
hard, cut with a knife warmed with warm water. 
A paper has been written by Flormax [Zeit. f. wiss. Mile., vi, 2, 1889, 
p. 184) to recommend that the definite hardening should be done without 
the aid of alcohol or chloroform, by simply cutting out the blocks, turning 
them over, and carefully continuing the evaporation process in the way 
described above. I described this process myself in the first edition of 
this work. No doubt the author is right in claiming for it a superior 
degree of hardening of the mass ; but I doubt whether it is possible to carry 
the hardening much beyond the point attained by the chloroform or alcohol 
method without incurring a very undesirable degree of shrinkage. 
The hardening processes used in the method of clearing 
before cutting, which 1 prefer to all the foregoing, will be de- 
scribed later on, § 168. 
161. Preservation.—-The hardened blocks of collodion may 
be preserved till wanted in weak alcohol (70 per cent.). They 
may also be preserved dry by dipping them into melted 
paraffin (Apathy, Zeit. f. wiss. Mik., v, 1, 1888, p. 45). 
Reference numbers may be written with a soft lead pencil 
on the bottom of the paper trays, or with a yellow oil pencil 
on the bottom of the watch-glasses in which the objects are 
imbedded. On removal of the paper from the collodion after 
hardening, the numbers will be found impressed, on the 
collodion. 107 
COLLODION AND OTHER IMBEDDING METHODS. 
162. Cutting.—If the object has not been stained before 
imbedding, it will form so transparent a mass with the 
collodion that the arrangement of the object and sections in 
the right position may be rendered very difficult. It is, 
therefore, well to stain the collodion lightly, just enough to 
make its outlines visible in the sections. This may be done 
by adding picric acid or other suitable colouring matter 
dissolved in alcohol to the collodion used for imbedding, or 
to the bergamot oil used for clearing. 
To fix to the microtome, proceed as follows. Take a piece 
of soft wood, or, for very small objects, pith, of a size and 
shape adapted to fit the holder of the microtome. Cover it 
with a layer of collodion, which you allow to dry. Take the 
block of collodion, or the impregnated and hardened but not 
imbedded object, cut a slice off the bottom, so as to get a clean 
surface; wet this surface first with absolute alcohol, then with 
ether (or allow it to dry), place one drop of very thick collodion 
on the prepared wood or pith, and press down tightly on to it 
the wetted or dried surface of the block of collodion. Then 
throw the whole into weak (70 per cent.) alcohol for a few 
hours (or even less), or into chloroform, or vapour of chlo- 
roform, for a few minutes, in order that the joint may harden. 
Dr. Lindsay Johnson informs me that he finds it very 
convenient to take for this purpose the cement used by metal 
turners for fastening metal objects on to boxwood chucks. 
The exact composition of this cement varies somewhat, but 
an average one is—beeswax, 1 part; resin, 2 parts. To use 
it, you must get the block of celloidin perfectly dry at the 
bottom, then warm the object-holder slightly, if possible, 
over a flame; drop on to it a few drops of melted cement, 
and press on to it the block of celloidin, which will be firmly 
fixed as soon as the cement is cool—that is, in a few seconds. 
For objects of any considerable size it is important not to 
use cork for mounting on the microtome, especially if the 
object-holder be a vice; for cork bends under the pressure of 
the holder, and the elastic collodion bends with it, deforming 
the object. I have seen large embryos so deformed in this 
way that the sections obtained were true calottes, segments of 
a sphere. If the object-holder be of the cylinder type, as in 
the later forms of the Thoma microtome, the above-described 
accidents will be less likely to happen, and a good cork may 108 
CHAPTER X. 
be used; but even then, I think, wood is safer. Gage has 
recommended bits of glass cylinders. Jelinek (Zeit. f. wins. 
Mik., xi, 2, 1894, p. 237) recommends a sort of vulcanite 
known as “ Stabilit,” which is manufactured for electrical 
insulation purposes. It is supplied in suitable blocks by 
Hermann Dumler, 4, Schwarzpanierstrasse, Vienna, ix, 3 
(presumably also obtainable through Grubler and Co.). 
Sections are cut with a knife kept abundantly wetted with 
alcohol (of 50 to 85 or even 95 per cent.). Some kind of 
drip arrangement will be found very useful here. Apathy 
recommends that the knife be smeared with yellow vaselin; 
it cuts better, is protected from the alcohol, and the mobility 
of the alcohol on the blade is lessened. 
The knife is set in as oblique a position as possible. 
Very brittle sections may be collodionised as explained 
above (§ 139). 
The sections are either brought into alcohol (of 50 to 85 or 
95 per cent.) as fast as they are made ; or if it be desired to 
mount them in series, they are treated according to one of the 
methods described below, in the chapter on “ Serial Section 
Mounting.” 
163. Staining.—The sections may now be stained as desired, 
either loose, or mounted in series on slides or on paper as 
described in the chapter on “ Serial Section Mounting.” It 
is not in general necessary, nor indeed desirable, to remove 
the mass before staining, as it usually either remains colour- 
less or gives up the stain on treatment with alcohol. But 
some of the anilin dyes and some other colours stain it 
strongly, and are not removed with sufficient completeness 
by the processes of dehydration and clearing. If it be 
desired to employ these, the mass may be removed by 
treating the sections with absolute alcohol or ether. 
164. Clearing and Mounting.—You may mount in glycerin 
without removing the mass, which remains as clear as glass 
in that medium. 
You may mount in balsam, also without removing the mass, 
which does no harm, and serves the useful purpose of holding 
the parts of the sections together during the manipulations. 
Dehydrate in alcohol of 95 or 96 per cent, (not absolute, as 
this attacks the collodion). Nikiforow (Zeit. f. wiss. Mik., COLLODION AND OTHER IMBEDDING METHODS. 
109 
viii, 2, 1891, p. 189) recommends a mixture of equal parts of 
alcohol and chloroform. Clear with a substance that does 
not dissolve collodion. The clearing agents most recom- 
mended are origanum oil {01. Origan. Cretici, it is said, should 
be taken, not 01. Orig. Gallici; but see, as to this reagent, 
the remarks in Chap. VII, § 118), bergamot oil (said to 
make sections shrink somewhat), oil of sandal-wood, lavender 
oil, oil of cedar-wood (safe and gives excellent results, but 
acts rather slowly), chloroform, xylol or benzin (may make 
sections shrink if not well dehydrated), or Dunham’s mixture 
of three or four parts of white oil of thyme with one part of 
oil of cloves. (As to oil of thyme, see also “ Origanum Oil ” 
in Chap. VII, § 118). 
Fish {Proc. Amer. Mic. Soc., 1893) advises a mixture of 
one part of red oil of thyme with three parts of castor oil, 
the latter being added in order to counteract the volatility 
of the thyme oil. But later (June, 1895), writing to me, 
Dr. Fish say he has substituted the white oil of thyme for 
the red, and finds it an advantage in orientating. See also 
§ 119. 
Some specimens of clove oil dissolve collodion very slowly, and may be 
used, but I would not be understood to recommend it. The action of 
origanum oil varies much, acccording to the samples; some sorts do not 
clear the collodion, others dissolve it, others pucker it. Minot (Zeit.f. wiss. 
Mik., iii, 2, 1886, p. 175) says that Dunham’s mixture “clarifies the 
sections very readily and softens the celloidin just enough to prevent the 
puckering, which is so annoying with thyme alone.” 
Carbolic acid has been recommended. Weigert {Zeit.f. wiss. Milt., iii, 
4, 1886, p. 480) finds that a mixture of 3 parts of xylol with 1 part of 
carbolic acid (anhydrous) clears well. But it must not be used with the 
basic anilin stains, as it discolours them. For these, anilin oil may be used 
with the xylol, in the place of carbolic acid. 
Anilin oil clears well (it will clear from 70 per cent, alcohol), but unless 
thoroughly removed the preparation becomes yellowish brown. It may he 
removed by soaking in chloroform for twenty-four hours (see van Gieson, 
Amer. Mon. Mic. Journ., 1887, p. 49, or Journ. Boy. Mic. Soc., 1887, 
p. 519, for a review of these clearing agents ; see also § 125). 
Beechwood creasote has been recommended (by M. Flesch). 
Eyclesheimer {Amer. Nat., xxvi, 1892, p. 354 ; Journ. Boy. Mic. Soc., 
1892, p. 565) advises a mixture of equal parts of bergamot oil, cedar oil, and 
carbolic acid. 
165. Review of the Older Celloidin Method.—The older cel- 
loidin method, described in the foregoing pages, is extremely 110 
CHAPTER X. 
lengthy and cumbrous. The operation of infiltrating the 
tissues with the collodion requires days or weeks. The 
hardening process requires nearly as much time. The 
resulting mass has the disadvantage of being opaque or at 
most only translucent, not transparent. The mass has to be 
cut under the surface of alcohol, or at least with constant 
wetting with alcohol and with a knife kept constantly 
wet with alcohol. By the recent method of clearing the 
mass before cutting, a large part of these defects is done 
away with; the resultant mass is as clear as glass, thus allow- 
the most perfect orientation of the object; and as I have 
shown (Lee et Henneguy, Traite des Mcthodes techniques de 
VAnat. Mic., 1896, p. 230) the mass can with advantage be 
cut dry, thereby greatly simplifying the operation of cutting. 
By Gilson’s ingenious Rapid Method, the time necessary for 
hardening is very greatly abridged, and the whole series of 
operations becomes almost as short and simple as the paraffin 
method. I cannot imagine that anyone who has ever em- 
ployed the new method would willingly go back to the old 
one. The following paragraphs describe the new method. 
166. The New Method, by Clearing before Cutting.—This 
process is due, I believe, in the first instance to Bumpus 
(Amer. Natur., xxvi, 1892, p. 80; see Journ. Roy. Mic. 
Soc., 1892, p. 438). He advises clearing the mass, after 
hardening in chloroform, with white oil of thyme or other 
suitable clearing agent (see above, § 164). After clearing, 
the under surface of the block of mass is washed with 
ether, and cemented with thick celloidin solution to a block 
of wood for cutting, in the manner described above, the 
whole being- thrown into chloroform for a few minutes to 
harden the joint. The knife is wetted with the clearing oil, 
and the same oil is employed for covering the exposed surface 
of the object after each cut. Similar recommendations are 
made by Eyclesheimer (op. cit., pp. 354, 563), carbolic acid, 
or glycerin, or the mixture given § 164, being suggested for 
clearing. Professor Gilson writes me that he has for some 
time past adopted the practice of clearing before cutting- 
with cedar oil, as described below, § 167. 
Fish (loc. cit., § 164) also advocates the practice of clearing 
in the mass, recommending the clearing mixture there given. COLLODION AND OTHER IMBEDDING METHODS. 
All the authors above quoted cut in the wet way, that is to 
say, with a knife wetted with the clearing liquid. I have 
found a great improvement in cutting dry, and in employing 
the combined hardening and clearing process of Gilson, given 
below. 
167. Gilson’s Rapid Process.—The following1 rapid method 
communicated to me by Prof. Gilson (April, 1892) has 
the advantage of being the most expeditious of any. The 
object is dehydrated, soaked in ether, and brought into a 
test-tube with collodion, or thin celloidin solution. The tube 
is dipped into a bath of melted paraffin, and the collodion 
allowed to boil (which it does at a very low temperature) 
until it has become of a syrupy consistence. It should be 
boiled down to about one third of its volume. The mass is 
then turned out, mounted on a block of hardened celloidin, 
and the whole hardened in chloroform or in a mixture of chlo- 
roform and cedar oil for about an hour. It is then cleared in 
cedar oil (if hardened in pure chloroform; special clearing- 
will not be necessary if it has been hardened in the mixture). 
It may now be fixed in the microtome and cut, using cedar oil 
to wet the knife, and cover the exposed surface of the object 
after each cut. 
It will be observed that this process is very much more rapid 
than the old process, in two ways—the celloidin bath, being- 
given warm, is greatly abridged ; small objects can be duly 
infiltrated in an hour, where days would be required by the 
old process. The hardening is also much more rapid than 
hardening by alcohol, which requires at least twenty-four 
hours. As collodion boils at a very low temperature, very 
little heat is required, and there is no risk of the tissues 
suffering on that head. 
168, The Dry Cutting Method.—I recommend the following 
as being a further improvement. Penetrate with collodion or 
celloidin either by Gilson’s process, or by soaking in the cold 
in the usual way, § 156. This is a much slower process, but 
does not take up more of the worker’s time, as the specimens 
require no attention whilst in the bath. Imbed as usual, either 
directly on the holder of the microtome, or in a paper tray or 
a water-colour mould or the like. Harden in vapour of chlo- 112 
CHAPTER X. 
roform for from one hour (generally sufficient for small 
objects) to overnight. This is done by putting the preparation 
into a Steinach’s sieve-dish or into a desiccator, on the bottom 
of which a tea-spoonful of chloroform has been poured. 
(The objects may remain for months in the chloroform vapour 
if desired.) As soon as the mass has attained sufficient super- 
ficial hardness, it is, of course, well to turn it out of its re- 
cipient, and turn it over from time to time, in order that it 
may be equally exposed on all sides to the action of the 
vapour. When fairly hard (it is not necessary to wait till 
the mass has attained all the hardness of which it is sus- 
ceptible), throw it into Gilson’s mixture. This should be at 
first a mixture of one part of chloroform with one or two parts 
of cedar oil. From time to time more cedar oil should be 
added, so as to bring the mixture up gradually to nearly pure 
cedar oil. As soon as the object is cleared throughout, the 
mass may be exposed to the air, and the rest of the chlo- 
roform will evaporate gradually. The block may now be 
mounted on the holder of the microtome with a drop of thick 
collodion, § 162, and may either be cut at once, or may be 
preserved indefinitely without change in a stoppered bottle. 
Cut dry, the cut surface will not dry injuriously under 
several hours. The cutting quality of the mass is often 
improved by allowing it to evaporate in the air for some hours. 
The hardening may be done at once in the chloroform and 
cedar-wood mixture, instead of the chloroform vapour, but I 
find the latter process preferable as giving a better hardening. 
And clearing may be done in pure cedar oil instead of the 
mixture, but then it will be very slow, Avhereas in the mixture 
it is extremely rapid. 
169. Double Imbedding in Collodion and Paraffin.—Tins com- 
plicated process is sometimes, though rarely, indicated for objects of 
which it is desired to have very thin sections, and which are too brittle to 
give good sections by the plain paraffin process. 
Kultschizky’s Method (Zeit. f. wiss. Mik., iv, 1, 1887, p. 48).—After 
the collodion bath, the object is soaked in oil of origanum (Oleum Origani 
vulg.). It is then brought into a mixture or origanum oil and paraffin, 
heated to not more than 40° C., and lastly into a bath of pure paraffin. 
The mass may be preserved in the dry state, and may be cut dry. 
Ryder (Queen’s Micr. Bull., 1887, p. 43 ; Journ. Boy. Mic. Soc., 
1888, p. 512) modified the process by substituting chloroform for the 
origanum oil. COLLODION AND OTHER IMBEDDING METHODS. 
113 
Ide (La Cellule, vii, 1891, p. 347, and viii, 1, 1892, p. 114) employed 
with success the following method:—The object is imbedded in collodion in 
a tube by Gilson’s process (supra, § 167) ; the collodion is boiled for forty 
minutes, then brought for fifteen minutes (this is for small objects) into 
chloroform heated to 30° C. containing one fourth part of paraffin dissolved 
in it, then for ten minutes into pure melted paraffin. 
Field and Maktin (Bull. Soc. Zool. cle France, 1894, p. 48), finding that 
it is difficult to get hardened celloidin masses adequately impregnated with 
the paraffin, have worked out the following process of simultaneous 
imbedding. A solution of dried celloidin in a mixture of equal parts of 
absolute alcohol and toluene, of about the consistency of clove oil, is made. 
This solution is saturated with paraffin, added in shavings at a temperature 
not exceeding 20° to 23° C. The tissues are prepared by soaking in some of 
the mixture of alcohol and toluene, and are then penetrated with the 
celloidin-paraffin solution. The mass is hardened by throwing it into a 
saturated solution of paraffin in chloroform or in toluene, and is finally 
imbedded in pure paraffin in the usual way. 
Other Evaporation Masses. 
170. Joliet’s Gum and Glycerin Method (Arch. Znol. exp. et 
gen., x, 1882, p. xliii; Journ. Roy. Mic. Soc. [N.S.], ii, 1882, 
p. 890).—Pure gum arabic dissolved in water to the consistency 
of a thick syrup. (Solutions of gum sold under the name of 
strong white liquid glue colle forte blanche liquicle a froid ”] 
may also be used; they have the advantage of having a uni- 
form consistency.*) Pour a little of the solution into a watch- 
glass, so as not quite to fill it, add from 6 to 10 drops of pure 
glycerin, stir until thoroughly mixed. 
Between the limits of 6 to 10 drops of glycerin the propor- 
tions most suitable to the nature of the object and to the 
season of the year must be found by experimental trials. In 
the winter or in rainy weather less glycerin should be taken 
than in the summer or dry weather. 
It is often well to soak the object in glycerin before putting 
it into the mass. In this case less glycerin should be added 
to the gum, in proportion to the amount of glycerin contained 
in the object. 
The object is imbedded in the mass in the watch-glass, and 
the whole left to dry for from one to four days. When it has 
assumed a cartilaginous consistency, a block containing the 
object is cut out, turned over, and allowed to dry again until 
* It is highly probable that these commercial preparations contain gelatin, 
and perhaps some other gum besides gum arabic. 114 
CHAPTER X. 
wanted for use. A stove, or the sun, may be employed for 
drying, but it is best to dry slowly at the normal temperature. 
The block may be preserved in good condition almost indefi- 
nitely, the gum, when mixed with a sufficient quantity of 
glycerin, never becoming hard or brittle. It is generally 
better to wait till the blocks have assumed such a consistency 
that they cannot be easily bent. It is after having waited 
almost a week that the author always obtained the best sec- 
tions. The gum is dissolved out from the sections by means 
of a drop of water on the slide. The sections are then covered, 
and a drop of glycerin being added, the preparation is com- 
plete as soon as the water has evaporated. 
This process may render service occasionally in the study 
of extremely watery organisms, such as Salpa, or the Cteno- 
phora. 
171. Strieker’s Gum Method (Hdb. d. Gewebel., p. xxiv).—A concen- 
trated solution of gum arabie. The object may he prepared in alcohol and 
imbedded in the gum in a paper case. The whole is thrown into alcohol, and 
after two or three days may be cut. 
I have seen masses of admirable consistency prepared by this simple 
method. 
172. Robertson’s Grape-sugar Method, see Journ. of Anat. and 
Physiol., xxiv, 1890, p. 230; Zeit.f. wiss. Mih., vii, 1, 1890, p. 33. 
173. Hyatt’s Shellac Method, see Am. M. Micr.Journ., i, 1880, p. 8 ; 
Journ. Roy. Mic. Soc., iii, 1880, p. 320. 
This process is merely intended for the purpose of making sections through 
hard chitinous organs consisting of several pieces, such as stings and ovi- 
positors, retaining all the parts in their natural positions. 
174. Von Koch’s Copal Method {Zool. Anz., 2, vol. i, 1878, 
p. 36).—Small pieces of the object are stained in bulk and 
dehydrated with alcohol. A thin solution of copal in chloro- 
form is prepared by triturating small fragments of copal in a 
mortar with fine sand, pouring on chloroform to the powder 
thus obtained, and filtering. The objects are brought into 
a capsule filled with the copal solution. The solution is now 
slowly evaporated by gently heating the capsule on a tile by 
means of a common night-light placed beneath it. As soon 
as the solution is so far concentrated as to draw out into 
threads that are brittle after cooling, the objects are removed 
from the capsule and placed to dry for a few days on the tile, 
in order that they may more quickly become hard. When COLLODION AND OTHER IMBEDDING METHODS. 
115 
they have attained such a degree of hardness that they cannot 
be indented by a finger-nail, sections are cut from them by 
means of a fine saw. The sections are rubbed down even 
and smooth on one side with a hone, and cemented, with this 
side downwards to a slide, by means either of Canada balsam 
or copal solution. The slide is put away for a few days more 
on the warmed tile. As soon as the cement is perfectly hard 
the sections are rubbed down on a grindstone, and then on a 
hone, to the requisite thinness and polish, washed with water, 
and mounted in balsam. 
The process may be varied by imbedding the objects un- 
stained, removing the copal from the sections by soaking in 
chloroform, decalcifying them if necessary, and then staining. 
It is sometimes a good plan after removing the copal, to 
cement a section to a slide by means of hard Canada balsam, 
then decalcify cautiously the exposed half of the specimen, 
wash, and stain it. In this way von Koch was able to demon- 
strate the most delicate lamellae of connective tissue in Isis 
elongata. 
This method was imagined in order to enable the hard and 
soft parts of corals to be studied in their natural relations. 
It is evidently applicable to the study of any structures in 
which hard and soft parts are intimately combined. For 
purposes such as these it is certainly a method of the greatest 
value. 
175. Ehrenbaum’s Colophonium and Wax Method {Zeit.f. miss. 
Mile., 1884, p. 414).—Ehrenbaum recommends that the objects 
be penetrated by a mass consisting of ten parts of colophonium 
to one of wax. The addition of wax makes the mass less 
brittle. Sections are obtained by grinding in the usual way. 
The mass is removed from them by means of turpentine fol- 
lowed by chloroform. 
176. Weil’s Canada Balsam Method (Zeit.f. wiss. Mib, v, 2, 1888, 
p. 200).—Balsam heated till brittle when cold, then dissolved in chloroform. 
Heat the objects in the mass on a water-bath. For further details see 
Journ. Hoy. Mic. Soc., 1888, p. 1042. 
Congelation Masses. 
177. The Freezing Method. —Fresh tissues may be, and are, 
frequently frozen without being included in any mass, and in 116 
CHAPTER X. 
certain cases very satisfactory sections can be obtained in this 
manner. But the formation of ice ciystals frequently causes 
tearing of delicate elements, and it is better to infiltrate the 
tissues with a mass that does not crystallise in the freezing 
mixture, but becomes hard and tough. Gum arabic affords 
such a mass. Some workers used common gum water, which 
is either poured into the well of the microtome or round the 
object on the object plate, according to the form of microtome 
used. 
178. Syrup and Gum Congelation Mass (Hamilton, Journ. of 
Anat. and Rhys., xii, 1878, p. 254).—The hardening reagent 
having been soaked out by water, the tissues are prepared for 
freezing in the following manner, which it is important to 
observe, otherwise it will be found that the crystals of ice so 
break up delicafe tissue as to render it totally useless for 
minute examination. The tissues are to be well soaked in 
syrup. The sugar somewhat retards the freezing, and besides, 
seems to alter the manner of crystallisation, so that instead of 
the ice being spicular in form it becomes granular, and does 
no injury to the parts. 
The syrup requires to be of a particular strength, viz. double 
refined sugar, 2 ounces ; water, 1 fluid ounce. 
Wash the superfluous syrup from the surface, and put into 
the ordinary mucilage for an hour or so before cutting. Imbed 
in the freezing microtome with mucilage in the usual way. 
Float the sections into water. 
179. Gum and Syrup Congelation Mass (Cole, Methods of 
Microscopical Research, 1884, p. xxxix ; Journ. Roy. Mic. Soc. 
[N.S.], iv, 1884, p. 318).—Gum mucilage (B.P.), 5 parts; 
syrup, 3 parts. (For brain and spinal cord, re tinge, and all 
tissues liable to come in pieces put 4 parts of syrup to five of 
gum.) Add 5 grains of pure carbolic acid to eaclx ounce of 
the medium. 
(Gum mucilage [B.P.] is made by dissolving 4 ounces of 
picked gum acacia in 6 ounces of water.) 
The syrup is made by dissolving 1 pound of loaf sugar in 
1 pint of water and boiling. 
This medium is employed for soaking tissues previous to COLLODION AND OTHER IMBEDDING METHODS. 
117 
freezing. They may remain in it for “ any length of time, 
all the year round ” if desired. 
The freezing is conducted as follows :—the gum and syrup 
is removed from the outside of the object by means of a cloth ; 
the spray is set going and a little gum mucilage painted on 
the freezing plate ; the object is placed on this and surrounded 
with gum mucilage; it is thus saturated with gum and syrup, 
but surrounded when being frozen with mucilage only. This 
combination prevents the sections from curling up on the one 
hand, or splintering from being too hard frozen on the other. 
The mass ought to cut like cheese. Should freezing have 
been carried too far, wait for a few seconds. 
180. Dextrin Congelation Mass (Webb, The Microscope, ix, 
1890, p. 344; Journ. Roy. Mic. Soc., 1890, p. 113).—Thick 
solution of dextrin in solution of carbolic acid in water 
(1 in 40). Use heat for making the solution if desired. This 
medium is much cheaper than the gum and syrup mass, and, 
according to Webb, possesses superior cutting qualities. 
181. Gelatin Congelation Mass (Sollas, Quart. Journ. Mic. 
Soc., xxiv, 1884, pp. 163, 164; Journ. Roy. Mic. Soc. [N.S.], 
iv, 1884, p. 316).—“Instead of gum one uses gelatin jelly. 
This is prepared and clarified in the usual manner. It should 
set into a stiff mass when cold The tissue to be cut is 
transferred from water to the melted jelly, and should remain 
in it till well permeated.” 
The sections are transferred to a slide as soon as cut. On 
touching the glass they adhere to it. When enough sections 
have been thus arranged they are covered with a drop of 
glycerin; a cover is put on, and the mount closed with any 
suitable cement. In process of time the glycerin will per- 
meate the gelatin and convert it into glycerin jelly; this 
may be hastened by placing the slide in an oven kept at about 
20° to 30° C. 
182. Gum-Gelatin Congelation Mass (Jacobs, Amer. Natural., 
1885, p. 734; Journ. Roy. Mic. Soc., 1885, p. 900).—Gum 
arabic, 5 parts; gum tragacanth, 1 part; gelatin, 1 part. 
Dissolve in enough warm water (containing one sixth of 
glycerin) to give a mass of the consistency of thin jelly when 
cold. 118 
CHAPTER X. 
183. White of Egg Congelation Mass (Rollett, Denkschr. math, 
naturw. Kl.k. Acad. Wiss. Wien, 1885 ; Zeit.f. wiss. Mile., 1886, p. 92).— 
Small portions of tissue brought in the white of a freshly laid egg on to the 
freezing stage, frozen, and cut. The knife must he well cooled. 
184. Oil of Aniseed Congelation Mass (Kuhne, Centralb. f. 
Bakteriol., xii, 1892, p. 28 ; Journ. Roy. Mic. Soc., 1892, p. 706).—Soak in 
oil of aniseed for twelve to twenty-four hours, freeze and cut, and remove 
the oil from the sections by means of alcohol. 
Y. A. Moore (Amer. Mon. Mic. Journ., 1894, p. 373 ; Journ. Roy. Mic. 
Soc., 1895, p. 247) says that sections may be transferred direct into Canada 
balsam, which is miscible with anise oil. CHAPTER XI. 
SERIAL SECTION MOUNTING. 
185. Choice of a Method.—All the following methods are 
excellent if properly carried out. I recommend for general 
workj the following':—For paraffin sections that have been 
already stained, Schallibaum’s collodion. For paraffin sections 
that are to be stained on the slide, Mayer’s albumen, unless 
the stain to be employed be one that will stain the albumen 
or if the sections be badly folded, in which case take one of 
the water or alcohol methods given in the next section. For 
collodion sections, Mayer’s albumen. For very large collodion 
sections, Weigert’s process. 
Methods for Paraffin Sections. 
186. The Water or Alcohol Method.—The principle of this method 
is due to Gaule (Arch.f. Anat. u. Pliys. [Phys. Abth.'], 1881, p. 156), who 
practised it as follows :—A slide is moistened with alcohol, the sections are 
arranged on it by means of a camel-hair brush, also moistened with alcohol; 
the slide is slightly warmed so as to cause the sections to stick to the slide; 
a cover is put on ; the excess of paraffin is removed by means of a drop of 
pure xylol, and the mount is completed by means of xylol balsam. 
Both the moistening with alcohol and the heating are necessaiy for the 
attachment of the sections to the slide; the effect is not obtainable by 
means of one of these manoeuvres alone. 
In the primitive form given above, Gaule’s method is only applicable to 
the purpose of mounting a small series of sections that do not require to be 
stained or otherwise further manipulated on the slide. Later workers have 
by improvements in the details of the process brought it to a state of 
considerable perfection, so that it may now be said to be a fairly safe 
process for extensive series of sections, and will allow of staining on 
the slide. 
Suchannek (Zeit. f. wiss. Mile., vii, 4, 1891, p. 464) pointed out that the 
slides must be absolutely free from grease in order that the alcohol (50 per 
cent.) or distilled water, which may be used instead, may spread out in a 
thin and uniform layer. Secondly, that the slides should not be warmed to 120 
CHAPTER XL. 
more than 40° C., it being important both that the alcohol should evaporate 
slowly and that the paraffin shoidd only he softened, not melted, until the 
evaporation is complete. Hoyer had already for this reason advised slow 
evaporation at the temperature of the laboratory. 
Gulland (Journ. of Anat. and Physiol., xxvi, 1891, p, 56; Journ. Boy. 
Mic. Soc., 1892, p. 161) floats sections on to the surface of warm water (not 
warm enough to melt the paraffin), or alcohol if preferred, in a dish, and 
thence floats them into position on the slide. The slide is drained, and the 
water evaporated from it at a low temperature as described above. When 
the water of the sections has evaporated completely they become more 
transparent, and look dry. The fixation is then complete, the paraffin may 
be melted and removed by means of any desired solvent, and the sections 
may be mounted, or be stained in any medium, or otherwise manipulated as 
desired. Thin sections will generally be fixed in about an hour ; thick ones 
will require six hours or more. 
Here it may be pointed out that the degree of adhesion seems to depend 
very much on the nature of the sections. Schiefferdecker (Zeit. f. wiss. 
Mik., ix, 2, 1892, p. 202) finds that the larger and thinner sections are, the 
better do they stick, and vice versa. And Mr. Andrew Pringle writes 
me that he finds that tissues thoroughly fixed in chrome solutions, so that 
their albuminoid substances have become quite insoluble, do not adhere 
sufficiently without the aid of some substance to fix them to the slide. He 
prefers arranging sections on the slide with cold water, and then warming 
until the sections flatten out. 
M. Heidenhain (Kern und Protoplasma, p. 114, in “ Festschr. f. 
Kolliker,” 1892) also proceeds in this way, and advises that the heating be 
not carried to above 35° C., at which temperature several hours at least are 
necessary to ensure fixation. He also finds that water is preferable to 
alcohol, which is too mobile on the slide and evaporates too quickly. 
Durham’s method (Quart. Journ. Mic. Sci., xxxiii, 1891, p. 116 ; Journ. 
Boy. Mic. Soc., 1892, p. 293) is the same as the last described, with the 
exception that he uses 70 per cent, alcohol instead of water. 
Wlassax (Mercier’s Coupes du Systhne Nerveux Central, 1894, p. 118) 
places the sections first of all on a drop of warm water on the slide, then 
exposes them to vapour of boiling distilled water, and then puts them in the 
stove. 
The advantage of the water or alcohol method is that as there is nothing 
on the slide that can stain in plasma stains, it gives very clean preparations. 
The disadvantages are that, besides being excessively lengthy, it is certainly 
not safe for chrome-osmium preparations. 
187. Schallibaum’s Collodion Method {Arch. f. mik. Anat., 
1883, p. 565).—One part of collodion is shaken up with three 
to four volumes (according to the consistency of the collodion) 
of clove oil or lavender oil. This should give a clear solution. 
A little is spread thinly on a slide with a small brush. After 
arranging the sections on the prepared surface, warm over a SEEIAL SECTION MOUNTING. 
121 
water-bath, gently, until the clove oil has evaporated (five to 
ten minutes). The sections are then found to be fixed, and 
can be treated for days with turpentine, chloroform, alcohol, 
and watery fluids, without becoming detached. The advan- 
tage of this method is that it allows of staining on the slide. 
If after staining any cloudiness should appear between the 
sections, dehydrate the slide and treat it several times with 
absolute alcohol and turpentine, warming it gently the while; 
or brush the space between the sections repeatedly with a 
brush moistened with clove oil. This cloudiness only arises 
from the collodion solution having been taken too concentrated, 
or having- been laid too thick on the slide. 
I find it is not necessary to evaporate over a water-bath. 
It is sufficient to hold the slide over a spirit lamp until the 
paraffin has melted and the clove oil has collected in drops 
between the sections. Schallibaum has stated elsewhere that 
long evaporation of the slide is necessary if the sections are 
to be secured firmly enough to allow of staining on the slide. 
That is not so. What is necessary is that the paraffin and 
clove oil be thoroughly removed from contact with the sec- 
tions ; and that can be done in a second by melting and 
blowing the paraffin and oil away as described in § 140. 
Rabl (Zeit. /. wiss. Mik., xi, 2, p. 179) says that new- 
solution of Schallibaum, not more than four or five days old, 
will stick fast enough to resist absolute alcohol. He gives 
the proportions of three parts of clove oil to two of col- 
lodion. 
Personally I do not consider Schallibaum’s method so safe 
as Mayer’s albumen (and some other methods) for objects 
that are to be stained on the slide. I recommend it for 
already stained objects, because it is found to work very 
pleasantly. Its great defect is that it does not readily lend 
itself to any device for the flattening out of folded sections. 
I recommend xylol or naphtha for clearing, in preference to 
turpentine. 
Field and Martin {Bull. Soc. Zool. de France, 1894, p. 48) 
say that treatment with xylol, toluol, or benzin makes col- 
lodion more readily soluble in alcohol, and recommend for 
clearing after Schallibaum’s fixative either petroleum ether 
(a light petroleum, density 0 650) or chloroform. 
Gallemaerts {Bull. Soc. Beige de Micro., xv, 1889, p. 56; 122 
CHAPTER XI. 
Zeit. f. wiss. Mile., vi, p. 4, 1889, p. 493), following Drash, em- 
ploys a saturated solution of gun-cotton in acetone, diluted 
to the requisite thinness with absolute alcohol. 
Gage prefers preparing slides with a layer of pure collodion, 
which is allowed to dry, and is rendered adhesive at the instant 
of using by brushing with clove oil. 
Summers (Amer. Mon. Mic. Journ., 1887, p. 73 ; Zeit.f. wiss. 
Mile., iv, 4, 1887, p. 482) also employs a dry layer of collodion, 
which he renders adhesive after the sections are arranged on 
it, by wetting with a mixture of equal parts of alcohol and 
ether. As soon as the mixture has evaporated, the sections 
are found to be fixed. 
Good collodion is essential in this process. 
Strasser (Zeit.f. wiss. Mik., iv, 1, 1887, p. 45) recommends a mixture 
of 2 parts collodion, 2 parts ether, and 3 parts castor oil; or (ibid., vi, 2, 
1889, p. 153) 2 parts of collodion with one of castor oil, the sections being 
painted over with a thicker solution, viz. collodium concentratum duplex 2 
to 3 parts, castor oil 2 parts, and the slide being plunged at once, without 
warming, into a bath of turpentine, in which it remains till the paraffin is 
dissolved (two to ten hours, somewhat less if the whole be put in a stove). 
The turpentine suffices to harden the collodion (benzin, benzol, and chloro- 
form have the same effect). 
188. Strasser’s Collodion-Paper Method (Zeit.f. wiss. Mile., iii, 8, 
1886, p. 346).—This is an extremely complicated modification of Weigert’s 
method for celloidin sections, and is only adapted for use with Strasser’s 
automatic ribbon-microtome. See the original papers in Zeit.f. wiss. Mik., 
iii, 3, 1886, p. 346 ; vi, 2, 1889, p. 154 ; vii, 3, 1890, p. 290; ib., p. 304; 
and ix, 1, 1892, p. 8 ; see also a very short abstract of the last paper in 
Journ. Roy. Mic. Soc., 1892, p. 703, in which is a figure of the “Schnitt- 
Aufklebe-Mikrotom ;” and Zeit. f. wiss. Mile., xii, 2, 1895, p. 154 (Journ. 
Roy. Mic. Soc., 1895, p. 702). 
189. The Shellac Method (Giesbrecht, Zool. Anz., 1881, p. 484).— 
Prepare a stock of slides covered with a thin and even film of shellac. This 
is done as follows :—Make a not too strong solution of brown shellac in abso- 
lute alcohol, filter it thoroughly; warm the slides, and spread over them a 
layer of shellac by means of a glass rod dipped in the solution and drawn 
once over each slide. Let the slides dry. 
Just before beginning to cut your sections take a prepared slide and brush 
it over very thinly with kreasote applied by means of a brush ; this forms a 
sticky surface on which the sections are now arranged one by one as cut, 
care being taken to bring them on to the slide with as little surrounding 
paraffin as possible. 
When all the sections are arranged the slide is heated on a water-bath for SERIAL SECTION MOUNTING. 
123 
about a quarter of an hour at the melting-point of the paraffin. The slide 
is allowed to cool, and the sections are now found to be firmly fixed in the 
shellac. The paraffin is dissolved away by dropping turpentine on to the 
sections, which are then mounted in Canada balsam. There is no danger of 
the sections being floated away by the turpentine, because turpentine does 
not dissolve shellac. 
In the note in the Zool. Anz. above quoted, the shellac solution is stated 
to be prepared with common brown shellac (choosing, of course, by prefer- 
ence the paler sorts), on account of the insolubility of white shellac in 
alcohol. In the Mitth. d. Zool. Stat. of Naples, of the same year, “ bleached 
white shellac” is recommended to be dissolved as before in absolute alcohol. 
In the Journ. Roy. Mic. Soc. (N.S.), vol. ii, 1882, p. 888, it is stated (on 
whose authority is not clear) that the solution is made by mixing 1 part of 
bleached shellac with 10 parts absolute alcohol, and filtering. In the same 
place it is added that “ Dr. Mark uses the bleached shellac in the form in 
which it is prepared for artists as a ‘ fixative ’ for charcoal pictures.” 
The account given in the Mittli. d. Zool. Stat. further varies in one other 
detail from that given in the Zool. Anz. It directs that the shellac slides 
be brushed before cutting with oil of cloves instead of kreasote, the slide 
being slightly warmed before brushing. 
The white shellac of commerce is sometimes not easily soluble in alcohol. 
Kingsley (see Whitman’s Methods in Microscopical Anat., p. 117) recom- 
mends that brown shellac be taken, and bleached by exposure to the sun. 
Caldwell {Quart. Journ. Mic. Soc. [N.S.], lxxxvii, 1882, p. 336) 
simplifies the method by merely brushing over the side (thinly) at the 
moment of using with a strong solution of shellac in anhydrous kreasote. 
(To make the solution, warm the kreasote.) 
In both the foregoing methods it often happens that the shellac becomes 
granular or cloudy on the slide. P. Mayer attributes this to the kreasote 
or clove oil, and proposes to remedy it by employing carbolic acid instead 
(Amer. Natural., 1882, p. 733; Zeit. f. wiss. Mik., iv, 1, 1887, p. 77; 
Journ. Roy. Mic. Soc., 1885, p. 910). Powdered white shellac is heated 
with crystallised carbolic acid till it dissolves, and the solution filtered warm. 
But more recently (Intern. Monatsclir. f. Anat., &c., 1887, Heft 2; Zeit. 
f. wiss. Mik., iv, 1,1887, p. 77) the same author, on the ground that hot car- 
bolic acid attacks some tissues, recommends another method. Slides are pre- 
pared with alcoholic shellac according to Giesbrecht’s plan. The sections 
are arranged on the dry film and gently pressed down on to it, then exposed 
for half a minute to vapour of ether. 
Chloroform softens shellac ; therefore chloroform balsam is not a safe 
mounting medium for sections fixed by these methods. 
These methods do not allow of staining on the slide. 
I feel bound to say that I am at a loss to understand by what virtue 
it is that the shellac method continues to survive, as it certainly seems to do, 
in the face of far more convenient and efficient processes. 
190. Mayer’s Albumen (Mittli. Zool. Stat. Nerrpel, iv, 1883; 
Journ. Hoy. Mic. Soc. [N.S.], iv, 1884, p. 317; Interned. 124 
CHAPTER XI. 
Monatschr.f. Anat., 1887, Heft 2 ; Journ. Hoy. Mic. Soc., 1888, 
p. 160).—White of egg, 50 c.c.; glycerin, 50 c.c.; salicylate 
of soda, 1 grm. Shake them well together, and filter into a 
clean bottle. The filtering may take days or a week, but the 
preparation does not spoil meanwhile. 
Fol (Lehrb., p. 184) takes whipped white of egg, filters it 
through a Bunsen filter, and adds the glycerin and a little 
camphor or carbolic acid. 
I find it convenient to beat up the egg with a little water 
before adding the glycerin and filtering, the salicylate 
being dissolved in the water in the first instance. But in 
view of the good preservation of the mixture it is perhaps 
better not to add Avater ; the salicylate will dissolve without 
it with sufficient shaking. 
According to my experience carbolic acid is perfectly effi- 
cient as a preservative, but is not to be recommended because 
it precipitates a great deal of the albumen. 
A thin layer of the mixture is spread on a cold slide with a 
fine brush, and the sections laid on it and warmed for some 
minutes on a water-bath. (Press the sections ivell down into 
the albumen with a brush.) As the paraffin in the sections 
melts it carries the albumen away from them, and this is one 
of the advantages of the method. The sections may be 
treated with turpentine, alcohol, and aqueous or other stains 
without any danger of their moving. 
It is not necessary to use a water-batli for warming the 
slide. I prefer to warm for an instant over a flame until the 
paraffin melts, and then blow away the melted paraffin as 
described, § 140. The remaining paraffin is instantly removed 
by means of xylol, toluol, or the like. 
It is not necessary to warm the slide at all; the 'paraffin can 
be removed in the cold if desired by putting the slide into 
toluol, xylol, or the like. But the slide should be very 
thoroughly treated with alcohol after removal of the paraffin, 
in order to get rid of the glycerin, which will cause cloudiness 
if not perfectly removed. 
The function of the glycerin is merely to keep the layer of 
albumen moist. 
Miss A. M. Claypole lias written a paper complaining that the method is 
uncertain, because too much heat may injure the tissues, and if too little be SERIAL SECTION MOUNTING. 
125 
applied the albumen will not coagulate. This is a misapprehension. No 
heat whatever is required to coagulate the albumen ; the alcohol will do that 
sufficiently. The complications proposed by Miss Ciaypole are not only 
unnecessary but undesirable. The only object in using heat is to enable 
one to get rid of the paraffin quicker; and it cannot he supposed that 
tissues that have been for perhaps several hours in a bath of paraffin can 
suffer through being exposed for a single instant more to the temperature 
required to melt away the same paraffin from the sections. 
This method allows of the staining of sections on the slide 
with perfect safety, both with alcoholic and aqueous stains. 
This method can be combined with the water process for 
flattening out sections (§ 186), as described by Henneguy 
[Journ. de I’Auat. et de la Physiol., 1891, p. 398). A drop of 
water is spread by means of a glass rod on a slide prepared 
with white-of-egg mixture, the sections are arranged on it, 
the whole is warmed [not to the melting-point of the paraffin) 
until the sections flatten out; the water is then evaporated off 
at a temperature of about 40° C., and as soon as it has entirely 
disappeared the paraffin is melted, and the slide further 
treated as above described. 
See also the careful and detailed description of this method given by 
Ohlmacheb (Journ. Amer. Med. Ass., April, 1893), who has independently 
worked out the same process. 
According to my experience the albumen method is abso- 
lutely safe, and is the one that should in general be preferred 
for staining on the slide. It lias the defect that certain 
plasma-stains (not chromatin stains) colour the albumen very 
strongly and cannot be removed from it. This produces very 
ugly mounts. 
It sometimes happens that the mixture after it has stood 
for some time becomes turbid, a change which is attributed 
to the development of a microbe. I know of no means of 
preventing the mixture from going bad in this way, though I 
have found that it keeps better when freely exposed to the 
sun. It has been stated (Vosseler, Zeit.f. wiss. Mik., vii, 4, 
1891, p. 457) that as soon as the mixture has become turbid 
it loses its adhesive properties and should be thrown away. 
That is not my experience. I find the liquid first becomes 
milky, theu altogether turbid, and at last coagulates, passing 
into a caseous state. But up to the very last it does not in 
the least degree lose its adhesive properties. As long as 126 
CHAPTER XI. 
there is enough moisture in it to moisten the brush, it will 
stick as well as the first day. 
191. Mann’s Albumen Method {Zeit. f. wiss. Mile., xi, 4, 
1894, p.486).—Shake up white of egg with ten volumes of 
distilled water and filter twice through the same paper. 
Spread this on a stock of slides with a glass rod, let them 
drain and dry. Float the sections on to water warmed to 
40° C., pass a slide beneath them, arrange them in order, lift 
them out, put the slide for five minutes on a stove heated to 
35° C., then treat with xylol and alcohol. 
192. Flogel’s Gum Method (Zool. Anz., 1883, p. 565).—Make 
a solution of one part gum arabic in twenty parts water; filter, 
and add a little alcohol to prevent the formation of mould. 
Slides are prepared by pouring the solution over them, and 
draining. (It is important that the slides be so perfectly 
clean as to be evenly wetted all over by the gum solution.) 
Sections may now be cut and laid on the gum surface before 
it has become dry, and floated into the proper position; this 
is the best plan for sections of mm. thickness, and for 
large sections. For thinner and small sections it is best to 
take slides that have completely dried, arrange the sections 
dry on the gum film, and then breathe on it until the gum has 
become sticky. 
A very neat method for cases in which it is not required to 
treat the slide with watery fluids. 
Waddingtojx («Tourn. Queh. M. Club, vi, 1881, p. 199 ; Journ. Roy. Mic. 
Soc. [N.S.], i, 1881, p. 704) gives the following process for preparing 
“ arabin,” a purified gum arabic which has the advantage of not presenting 
a granular appearance under the microscope as ordinary gum arabic does. 
Dissolve clear and white gum arabic in distilled water to the consistency 
of thin mucilage. Filter. Pour the filtrate into rectified alcohol, and shake 
well; the arabin separates as a white pasty mass. Place it on filter-paper, 
and wash with pure alcohol until the washings are free from water. Dry. 
The white powder thus obtained should be dissolved in distilled water and 
filtered twice. It may then be placed on slides, which are drained, dried, 
and put away till wanted. In this condition it may be preserved indefinitely. 
193. Frenzel’s Gum Method (Arch. f. mik. Anat., Bel. xxv, 
1885, p. 51). — Gum avabic is dissolved in water to the con- 
sistency of a thin mucilage, and to this is added aqueous SERIAL SECTION MOUNTING. 
127 
solution of chrome-alum. An excess of the latter does no 
harm. Finally add a little glycerin and a trace of alcohol 
(1. c., p. 142). The slide is prepared with this in the usual 
way ; the sections (either cut dry or in the wet way) are 
gently pressed on to it with a brush and slightly melted on, 
and heated for at most a quarter of an hour at a temperature 
of 30° to 45° C., which suffices to render the gum insoluble. 
This layer has the advantage of not staining with the 
majority of staining fluids; fuchsin and safranin are the 
only ones that stain it to a harmful degree. In the other 
anilins, and in carmine or haematoxylin, it does not stain. 
Watery stains (it is stated) may be used with it. 
194. Born and Wieger’s Quince-Mucilage (Zeit. f. wiss. Mik., 
1885, p. 346).—To two volumes of the ordinary pharma- 
ceutical quince-mucilage (Mucilac/o c-ydonii) add one volume 
of glycerin and a trace of carbolic acid. Spread in a thin 
layer on a carefully cleaned slide, and arrange the sections 
on the moist surface. Heat for twenty minutes at a tempe- 
rature of 30° to 40° C. After removal of the paraffin by 
turpentine the slide is brought for half an hour into absolute 
alcohol. You may then mount, or pass through successive 
alcohols, and stain. Alkaline staining fluids must be avoided, 
as they soften the mucilage and cause the sections to become 
detached. 
195. Geavis’s Agar-agar {Bull. Soc. Beige de Micr., xv, 1889, p. 72; 
Zeit. f. wiss. Mik., vi. 4, 1889, p. 494).—Solution of agar-agar in 1000 
parts of water, to be used as in last section. 
196. Geay’s Gelatin Process (The Microscope, ix, 1889, p. 325 ; Journ, 
Roy. Mic. Soc., 1890, p. 117).—Solution of gelatin in 100 parts of water. 
Use as gum arabic solution, taking care not to melt the paraffin ; let the 
slide dry spontaneously overnight, and remove the paraffin with a suitable 
solvent; remove the solvent with alcohol, and then treat for five minutes 
with 2 per cent, solution of potassium bichromate to render the gelatin 
insoluble. Stain as desired, or mount. 
Hexnegtjy {Legons sur la Cellule, p. 62) takes a solution of gelatin of 
about 1: 5000, to which a trace of bichromate of potash is added at the 
instant of using. The sections are flattened out by warming, the slide is 
drained, and dried for some hours exposed to the light. 
Alleges (Proc. Amer. Mic. Soc., xv, 104 and 192; quoted from Fish, 
ibid., xvii, 1895, p. 321) uses formalin in much the same way. A few 
drops of formalin are added to each gramme of a O'5 to 1 per cent, gelatin 128 
CHAPTER XI. 
solution. A gentle heat is applied to the slide until the paraffin is softened, 
and the superfluous gelatin allowed to drain from the edge of the slide.” 
Eisen (Proc. Cal. Acad. Sci., v, 1895, p. 4 ; Journ. Roy. Mic. Soc., 1895, 
p. 486) practises the same process, explaining that “ the fixing should be 
allowed to harden in the air for at least four hours, or better during the 
night,” after the sections have been arranged on it. 
197. Van Walsem’s Gelatin Process.—Extremely complicated. See 
tbe six pages of description in the original, Zeit. f. wiss. Mile., xi, 2, 1894, 
pp. 229 to 235, or the abstract in Journ. Roy. Mic. Soc., 1895, p. 122. 
198. Obregia’s process given below, for celloidin sections, 
is also applicable to paraffin sections. 
Methods for Watery Sections. 
199. Fol’s Gelatin (Fol, Lehrb., p. 132).—Four grammes of 
gelatin are dissolved in 20 c.c. of glacial acetic acid by heating- 
on a water-bath and agitation. To 5 c.c. of the solution add 
70 c.c. of 70 per cent, alcohol and 1 to 2 c.c. of 5 per cent, 
aqueous solution of chrome-alum. Pour the mixture on to 
the slide and allow it to dry. In a few hours the gelatin 
passes into the insoluble state. It retains, however, the 
property of swelling and becoming somewhat sticky in pre- 
sence of water. The slide may then be immersed in water 
containing the sections ; these can be slid into their places, 
and the whole lifted out: the sections will be found to be 
fixed in their places. 
This method is especially useful for sections made under 
water, large celloidin sections amongst others. 
200. Poli (Malpighia, ii, 1888, 2, 3 ; Zeit. f. wiss. Alik., v, 3, 
1888, p. 361) arranges sections on a layer of melted Kaiser’s 
gelatin, adds glycerin, and covers. See also supra, § 196. 
201. Frenzel and Threlfall’s Gutta-percha (or Caoutchouc) 
Method (Zool. Anz., 1883, pp. 51, 301, and 423).—This extremely elegant 
method is not perfectly safe, the gutta-percha film being liable to tear; and 
is now, I believe, very generally abandoned. 
Methods for Celloidin Sections. 
202. The Albumen Method.—I find that celloidin sections may 
be mounted on Mayer’s albumen, and have the celloidin SERIAL SECTION MOUNTING. 
129 
removed if desired by putting them into ether-alcohol. Care 
must be taken to press them down very thoroughly on to the 
albumen. 
203. Summer’s Ether Method (Amer. Mon. Mic. Journ., 1887, 
p. 73; Zeit. f. iviss. Mile., iv, 4, 1887, p.482; Journ. Boy. 
Mic. Soc., 1887, p. 523).—Besides the method given above 
(§ 187), which is applicable to celloidin sections, but is 
needlessly complicated, Summers recommends the following 
simpler method :—Place the sections in 95 per cent, alcohol 
for a minute or two, arrange on the slide, and then pour over 
the sections sulphuric ether vapour, from a bottle partly full 
of liquid ether. The celloidin will immediately soften and 
become perfectly transparent. Place the slide in 80 percent, 
alcohol, or even directly in 95 per cent, if desired. The 
sections, it is said, will be found to be firmly fixed, and may 
be stained if desii’ed. I have myself not found this method 
safe. 
Schiefferdecker (Zeit. f. wiss. Mile., v, 4, 1888, p. 507) re- 
commends that the slide be one that has been previously pre- 
pared with a layer of collodion if it is desired to stain on the 
slide; but if not a clean slide is perfectly sufficient. The 
slide may of course be treated with ether vapour in a prepara- 
tion glass or similar arrangement. 
Gage (Proc. Amer. Soc. Mic., 1892, p. 82) advises that the 
slide be one that has been previously coated with a O'5 per 
cent, solution of gelatin and dried; the collodion adheres 
much more strongly to a gelatinised surface. 
204. Eyclesheimer’s Method (Amer. Natural., xxvi, 1892, 
p. 354 ; Journ. Boy. Mic. Soc., 1892, p. 565).—The sections are 
arranged on a slide with enough alcohol to keep them moist, 
hut not enough to float them. They are covered with a strip 
of toilet paper, which is kept down in place by winding thread 
round the slide. Care should be taken to have the turns of 
the thread passing between the sections, not over them. You 
may now stain and clear in any way that may be desired. 
After clearing, you cut the thread, remove the paper by 
rolling it up from one end, and mount. 
205. Apathy’s Oil of Bergamot Method (Mitth. Zool. Stat. 
Neapel, 1887, p. 742 : Zeit. f. wiss. Mik., v, 1, 1888, p. 46, 130 
CHAPTER XI. 
and v, 3, 1888, p. 360; Journ. Roy. Mic. Soc., 1888, p. 670). 
—Cut with a knife smeared with vaselin (§ 162) and wetted 
with 95 per cent, alcohol. Float the sections, as cut, on 
bergamot oil (must be green, must mix perfectly with 90 per 
cent, alcohol, and must not smell of turpentine). The sections 
spread themselves out on the surface of the oil; before they 
sink, each one is pushed by means of a needle into its place 
on a slip of tracing-paper dipped into the oil. (A good size 
for the paper is about as broad as the slide, and three times 
as long as the cover.) When the requisite number of sections 
have been arranged on the paper, you drain the paper, dry the 
under side of it with blotting-paper, turn it over, and gently 
press it down with blotting-paper on to a carefully dried slide. 
Eemove the paper by rolling it up from one end. The sections 
remain adhering to the slide, and may have the remaining 
bergamot oil removed from them by means of a cigarette 
paper. If they are already stained, nothing remains but to 
add balsam and a cover. 
In the case of unstained or very small objects, it is well to add a little 
alcoholic solution of safranin to the bergamot oil. The celloidin of the sec- 
tions becomes coloured in it in a few seconds, and makes them readily visible. 
The colour disappears after mounting in a few days. 
If tlie sections are to be stained, the slide after removal of 
the bergamot oil is exposed for a few minutes to the vapour 
of a mixture of ether and alcohol, then brought into 90 per 
cent, alcohol, and after a quarter of an hour therein may be 
stained in any fluid that contains 70 per cent, alcohol or more. 
If it be desired to stain in a watery fluid, care must have 
been taken when arranging the sections to let the celloidin of 
each section overlap that of its neighbours at the edges, so 
that the ether vapour may fuse them all into one continuous 
plate. This will become detached from the slide in watery 
fluids, and may then be treated as a single section. 
206. Apathy’s Series-on-the-Knife Method (Zeit.f. wiss. MiJc., 
vi, 2, 1888, p. 168).— The following is in some respects more 
convenient than the oil of bergamot method. The knife is 
well smeared with yellow vaselin rubbed evenly on with the 
finger, and is wetted with alcohol of 70 to 90 per cent. As 
fast as the sections are cut they are drawn with a needle or SERIAL SECTION MOUNTING. 
131 
small brush to a dry part of the blade, and there arranged in 
rows, the celloidin of each section overlapping or at least 
touching that of its neighbours. The rows are the length 
of the cover-glass, and are arranged one under the other so 
as to form a square of the size of the cover-glass. When a 
series (or several series, if you like) has been thus completed, 
the sections are dried by laying blotting-paper on them 
(there is no risk of their becoming attached to it, as they are 
held down by the vaselin). The series is then painted over 
with some of the thickest celloidin solution used for imbed- 
ding, is allowed to evaporate for five minutes in the air, and 
is then either wetted with 70 per cent, alcohol, and allowed 
to remain whilst cutting is proceeded with, or (if no more 
sections are to be cut, or if the knife is now full) the knife is 
removed and brought for half an hour into 70 per cent, alcohol. 
This hardens the celloidin around the sections into a continu- 
ous lamella, which can be easily detached by means of a 
scalpel, and stained, or further treated as desired. It is well 
to bring it at once on to a slide, moisten the edges of the 
celloidin plate with ether and alcohol mixture, so that it may 
not become detached, and bring the whole into the staining 
solution. 
207. Weigert’s Collodion Method (Zeit. f. wiss. Mik., 1885, 
p. 490).—Sections are cut wet with alcohol. Care should be 
taken not to have so much alcohol on the knife as to cause 
the sections to float. Prepare a slip of porous but tough 
paper (Weigert recommends “ closet paper ”), of about twice 
the width of the sections. Soak it in alcohol, take it by both 
ends, stretch it slightly, and lower it on to the section that 
is on the knife. The section will adhere to the paper, and is 
taken up by moving the slip horizontally or slightly upwards, 
away from the edge of the knife. Take up the first section 
towards the end of the paper that you hold in your left hand, 
and let the remaining sections follow in order from left to 
right. After each section has been taken up, the slip is 
placed, whilst the next section is being cut, with the sections 
upwards on a moist surface prepared by arranging several 
layers of blotting-paper, covered with one layer of closet 
paper, in a plate, and saturating the whole with alcohol. 
When all the sections have been arranged on the slip, you 132 
CHAPTER XI. 
pass to the next stage of the process, the collodionisation of 
the series. 
This is done in two steps. The first of these consists in 
transporting the series on to a plate of glass prepared with 
collodion. The plate is prepared beforehand by pouring on 
to it collodion and causing it to spread out into a thin layer, 
as photographers do, and allowing it to dry. (A number of 
the plates maybe prepared and kept indefinitely in stock; 
microscope slides will do for series of small sections.) Take 
one of these plates; lay the slip of paper with the sections 
on the plate, the sections downwards; press it down gently 
and evenly, and the sections will adhere to the collodion, then 
carefully remove the paper. (Do not place more than one or 
at most two lines of sections on the same plate, for those first 
placed run the risk of becoming dry whilst you are placing 
the others.) This finishes the first stage of the collodionising 
process. 
Now remove with blotting-paper any excess of alcohol that 
may remain on or around the sections, pour collodion over 
them, and get it to spread in an even layer. As soon as this 
layer is dry at the surface you may write any necessary indi- 
cations on it with a small brush charged with methylen blue 
(the colour will remain fast throughout all subsequent manipu- 
lations) . 
The plate may now either be put away till wanted in 80 per 
cent, alcohol, or may be brought into a staining fluid. Wei- 
gert recommends his hsematoxylin process, but other watery 
stains may be used. The watery fluid causes the double sheet 
of collodion to become detached from the glass, holding the 
sections fast between its folds. It is then easy to stain, wash, 
dehydrate, and mount in the usual way, merely taking care 
not to use alcohol of more than 90 to 96 per cent, for dehy- 
dration. Weigert recommends for clearing the above- 
described mixture of xylol and carbolic acid (§ 164). Both 
the dehydration and the clearing take rather longer with the 
collodionised series than with free sections. 
The series should be cut into the desired lengths for 
mounting whilst in the alcohol. It is perhaps safer to lay 
them out for cutting on a strip of closet paper saturated with 
alcohol. SERIAL SECTION MOUNTING. 
133 
It is hardly necessary to comment on the great value of this 
beautiful method. 
It is suggested by STRASSEBthat gummed paper might be an improvement 
on the glass plates used in this process—especially for very large sections. 
See ante, § 187. 
The modification of Weigert’s method proposed by Wintebsteiner (Zeit. 
f.wiss. Mik., x, 3, 1893, p. 316) consists in suppi’essing the alignment of the 
sections on the strip of paper, and slipping them direct from the knife onto 
the prepared glass. 
208. Obregia’s Method.—This method was originally de- 
scribed in the Neurologisches Centralb. for 1890, and is given 
in the third edition of Woodhead’s Practical Pathology. It 
has been recommended for class purposes as being very safe 
and convenient by Gulland (Journ. of Path., February, 1893). 
Slides, or glass plates of any size, are coated with a solution 
made of— 
Syrupy solution of powdered candy- 
sugar made with boiling distilled 
water ...... 30 c.c. 
Absolute alcohol . . . . 20 ,, 
Transparent syrupy solution of pure 
dextrin made with distilled water . 10 „ 
They are dried slowly for two or three days until the 
surface is just sticky to the moist finger. Sections are 
arranged and heated for a few minutes to a temperature 
slightly above the melting-point of the paraffin. The paraffin 
is removed by some solvent, such as xylol or naphtha, and 
this is in turn removed by alcohol. The alcohol is poured 
off, and the sections are covered with solution of celloidin or 
with a solution of 3 per cent, of photoxylin in a mixture of 
equal parts of ether and absolute alcohol. The plates ai-e 
left to evaporate in a horizontal position, and when the sec- 
tions are required the sheet of collodion is cut into ribbons, 
which are floated off in water, and further treated as desired, 
e.g. as in Weigert’s process, § 207. (It is well to divide the 
sheet of collodion into ribbons by running the point of a knife 
down it as soon as evaporation has produced a very slight 
solidification, and the evaporation must not be artificially 
hastened.) 
This is the process for paraffin sections; for celloidin 134 
CHAPTER XI. 
sections the sections are taken up in order on a strip of paper 
as in Weigert’s method, and laid down on the glass in the 
same way, and then covered with the photoxylin solution and 
evaporated as described. The advantage of Obregia’s 
pi’ocess is that it is equally applicable to paraffin sections, to 
celloidin sections, and to sections of material that has not 
been imbedded at all. 
209. GriACOMiNi’s collodion-gelatin process for large sections, see 
Gazzelta delle Cliniclie, November, 1885, Zeit.f. wiss. Mile., 1885, p. 531, or 
the first edition of the Traite of Lee et Henneguy, p. 392. CHAPTER XII. 
STAINING. 
210. The Kinds of Stains.—Stains are either General or 
Special (otherwise called Specific, or Selective, or Elective). 
A general stain is one that takes effect on all the elements of 
a preparation. A special, specific, selective, or elective stain 
is one that takes effect only on some of them, certain elements 
being made prominent by being coloured, the rest either 
remaining colourless or being coloured with a different 
intensity or in a different tone. To obtain this differentiation 
is the chief object for which colouring reagents are employed 
in microscopic anatomy. 
Two chief kinds of this selection may be distinguished,— 
histological selection and cytological selection. In the 
former an entire tissue or group of tissue-elements is promi- 
nently stained, the elements of other sorts present in the 
preparation remaining colourless or being at all events 
differently stained, as in a successful impregnation of nerve- 
endings by means of gold chloride. This is the kind of 
stain that is generally meant by a specific stain. In the 
latter the stain seizes on one of the constituent elements of 
cells in general, namely, either the nucleus or the extra- 
nuclear parts. 
Stains that thus exhibit a selective affinity for the sub- 
stance of nuclei—nuclear or chromatin stains—form at present 
by far the most important class of stains—in zootomy at any 
rate. What the zootomist wants, and the histologist too, in 
the great majority of cases, is either to differentiate the 
intimate structures of cells by means of a colour reaction, in 
order to study them for their own sakes, or to have the 
nuclei of tissues marked out by- staining in the midst of the 
unstained material in such a way that they may form land- 
marks to catch the eye, which is then able to follow out with 136 
CHAPTER XII. 
ease the contours and relations of the elements to which the 
nuclei belong ; the extra-nuclear parts of these elements being 
expressly left unstained in order that as little light as possible 
may be absorbed in passing through the preparation. Pos- 
sibly this may be an irrational procedure, but it has hitherta 
been found in practice to be the most efficient for general 
work. 
To these two kinds of selective stains must be added a 
third group, the plasmatic stains, consisting of those few stains 
that take effect in a special way on cytoplasm, or formed 
tissue or ground-substance, leaving the chromatic substance 
of nuclei as far as possible unstained. In this book, therefore,, 
stains are looked upon as being (1) General stains; (2) 
Selective stains; the latter group being subdivided into (a) 
Nuclear, (b) Plasmatic, (c) Histologically Selective, or Specific. 
This classification, however, is not followed in the arrange- 
ment of the special paragraphs, it being more practical to- 
follow an order based on the chemical nature of the staining 
agents, and on convenience of exposition. 
Some writers have divided stains into nuclear, general, and selective. 
This arrangement appeal’s to me faulty because every nuclear stain is eo ipso 
selective, and because it ignores the subdivisions of selective stains. 
211. The Methods of Staining.—Colouring matters possessing 
so great an affinity for certain elements of tissues that they 
may be left to produce the desired electivity of stain without 
any special manipulation on the part of the operator, are un- 
fortunately rare. In practice, selective staining is arrived at 
in two ways- In the one, which may be called the progressive 
or direct method, you make use of a colouring reagent 
that stains the element desired to be selected more quickly 
than the elements you wish to have unstained; and you stop 
the process and fix the colour at the moment when the 
former are just sufficiently stained, and the latter not affected 
to an injurious extent, or not affected at all, by the colour. 
This is what happens, for instance, when you stain the nuclei 
of a preparation by treatment with very dilute hsematoxylin : 
you get, at a certain moment, a fairly pure nuclear stain; 
but if you were to prolong the treatment, the extra-nuclear 
elements would take up the colour, and the selectivity of the 
stain would be lost. It may be noted of this method that it STAINTNG. 
137 
is in general the method of fast stains (“ echte Farbung ”), 
and that it renders great services in the colouring of specimens 
in toto,—a procedure which is not possible with the chief 
stains of the other class (the anilins). It is the old method 
of carmine and haematoxylin staining. 
The second, the regressive or indirect method, is the method 
of overstaining followed by partial discoloration. You begin 
by staining all the elements of your preparation indis- 
criminately, and you then wash out the colour from all the 
elements except those which you desire to have stained, 
these retaining the colour more obstinately than the others 
in virtue of a certain not yet satisfactorily explained affinity. 
This is what happens, for instance, when you stain a section 
of one deep red in all its elements with safranin, and then 
treating it for a few seconds with alcohol, extract the colour 
from all but the chromatin and nucleoli of the nuclei. It is 
in this method that the coal-tar colours find their chief 
employment. It is in general applicable only to sections, 
and not to staining objects in toto (the case of borax-carmine 
is probably only a seeming exception to this statement). 
It is a method, however, of very wide applicability, and 
gives, perhaps, the most brilliant results that have hitherto 
been attained. 
In previous editions the expressions "‘direct” and “indirect” staining 
methods were alone used. The expressions “ progressive ” and “ regressive ” 
are due to M. Heidenhaix, and appear to me to be preferable. 
212. The State of the Tissues to be Stained.—It is generally 
found that precise stains can only be obtained with carefully 
fixed (i. e. hardened) tissues. Dead, but not artificially hard- 
ened tissues stain indeed, but not generally in a precise 
manner. Living tissue elements in general do not stain at 
all, but resist the action of colouring reagents till they are 
killed by them (see, however, next section). 
It appears probable, as was first pointed out, I believe, by 
Paul Mayer, that the usual histological stains obtained with 
fixed tissues are brought about in two ways. Either they 
result from the combination of the colouring agent with 
certain organic or inorganic salts,—phosphates, for instance, 
that existed in the tissue elements during life and were thrown 
down in situ by the fixing or hardening agent employed, as 138 
CHAPTER XII. 
seems to happen when such a fixing agent as alcohol is 
employed. Or they result from the combination of the 
colouring agent with certain compounds that did not pre-exist 
in the tissues, but were formed by the combination of the con- 
stituents of the tissues with the chemical elements brought 
to them by the fixing agent, as seems to happen when such a 
fixing agent as chromic acid is employed—the compounds in 
question being probably chiefly metal albuminates. These 
considerations will serve to show to how great an extent the 
quality of a stain is dependent on the nature of the previous 
treatment the tissues have undergone. 
213. Staining “intra vitam.”—Some few substances possess 
the property of staining—or rather, tingeing—living cells 
without greatly impairing their vitality. Such are—in very 
dilute solutions—cyanin (or quinolein), methylen blue, 
Bismarck brown, anilin black, and, under certain conditions, 
dahlia and eosin, gentian violet, with perhaps methyl violet, 
and some others whose action is not yet sufficiently esta- 
blished by experiment. Congo, even in strong solution, is 
not toxic to some organisms, and stains some structures (see 
Scholtz, Centralb. f. d. med. Wiss., 1886, p. 449; also Journ. 
Roy. Mic. Soc., 1886, p. 1092). Living Rotifera are in 
part successfully stained by it during life. (The paper of 
Martinotti, Zeit. f. wiss. Mik., v, 3, 1888, p. 305, may be 
consulted on this subject.) More recently, neutral red 
(Neutralrotli) has been greatly recommended by Ehrlich (see 
Chap. XVI). 
As to the employment of these reagents, it may be noted 
that they must be taken in a state of extreme dilution, and in 
neutral or feebly alkaline solution—acids being of course 
toxic to cells. Thus employed, they will be found to tinge 
with colour the cytoplasm of certain cells during life (never, 
so far as I know, nuclear chromatin during life ;—if this stain, 
it is a sign that death has set in). The stain is sometimes 
diffused throughout the general substance of the cytoplasm, 
sometimes limited to certain granules in it, which have been 
taken, perhaps without sufficient reason, to be identical with the 
granules of Altmann (Altmanu’s Studien iiber die Zelle, 1886). 
Since the publication of the last edition I have made a 
considerable number of observations on this subject, and have STAINING. 
139 
come to the same conclusion as Galleotti (Zeit.f. wiss. Mile., 
xi, 2, 1894, p. 172), namely, that the so-called “ intra vitam ” 
stains are not true stains at all. The diffused coloration 
above mentioned appears always, if the cell that shows it have 
remained in a state of unimpaired vitality, to be due to simple 
absorption or imbibition of the colouring matter by the cell, 
not to a chemical combination of the colouring matter with 
any of the constituents of the cell. If a cell thus coloured 
be transported into a medium free from the colouring matter 
it will give up unchanged the colour it had imbibed, which 
seems to be a sufficient proof that the colouring matter had 
not entered into any chemical combination with the elements 
of the cell, but was simply held in a mechanical way in the 
interstices of its substance. If, on the other hand, there has 
been produced the above-mentioned coloration of certain 
granules or other cell-contents, it is possible that this may 
be a true stain in the sense of being a chemical combination. 
It may be so, but it certainly is not always so, as may 
sometimes be proved with the greatest ease by putting the 
cell into a colourless medium and observing the supposed 
stain disappear. And in cases in which this does not happen, 
in which therefore a more or less fast stain has been obtained, 
it is invariably found that the stain is limited to cell-contents 
that do not form an integral part of the living texture of the 
cell; the cell itself may be living, but they are not. These 
granules or other cell-contents may be granules formed of 
substances that have been absorbed by the cell from without 
—food-granules ; or they may be katabolic products, consist- 
ing of matter that is no longer alive and is destined to be 
shortly expelled from the cell; or they may be elements that 
form indeed an integral part of the living texture of the cell 
but have been injuriously affected by the colouring matter, 
and for that or some other reason are in a state of diminished 
vitality,—they are parts of the cell that are being killed by 
the colouring reagent or that have been totally killed by 
it whilst the rest survives ; in no case do they consist of 
matter that is fully and perfectly alive. I am inclined to 
think that the chief scientific value of the so-called vital or 
intra-vitam stains will be found to lie in the fact that they 
may furnish us with the means of distinguishing the living 
constituents of a cell from the non-living ones, and even of 140 
CHAPTER XII. 
recognising amongst the living ones those that possess only 
a relatively low or impaired degree of vitality. 
Apart, however, from the question whether the elements 
stained by the so-called “ vital ” stains are truly living or 
not, it must be conceded that this mode of treating living 
cells has frequently a measure of practical utility. It often 
enables us to map out physiological or morphological tracts 
that would otherwise be unrecognisable or less readily recog- 
nisable in the living state. 
I have frequently found gentian, dahlia, and methylen 
blue, added to indifferent liquids, extremely useful in the exa- 
mination of tissue-cells. Quinolei'n and Bismarck brown are 
well-known aids to the study of Infusoria. Methylen blue 
has a specific affinity for sensory nerves, and is an extremely 
important reagent (see post, Chap. XVI). According to my 
experience, methylen blue is the most generally useful of 
these stains. It has the valuable point that it is perfectly 
soluble in saline solutions, and may therefore be employed 
with marine oi'ganisms by simply adding it to sea water. The 
others are not thus soluble to a practical extent, but I find 
that gentian and dahlia become so if a trace of chloral hydrate 
—025 per cent, is ample enough—be added to the saline 
solution. Any of these reagents may be rubbed up with 
serum, or other “ indifferent ” liquid. 
Methylen blue may be fixed in the tissues, and permanent 
preparations made, by one or other of the methods described 
in Chap. XVI. Bismarck brown stains may be fixed with 02 
per cent, chromic acid or with sublimate solution (Mayer), 
and the preparations may be stained with safranin, care being 
taken not to expose them too long to the action of alcohol. 
214. Substantive and Adjective Staining; Mordants.—In the 
industry of dyeing, colouring matters are divided into two 
classes, according to their behaviour with respect to the 
material to be dyed. Certain dyes are absorbed directly from 
their solution by the material immersed therein, and combine 
with it directly. In this case the material is said to be sub- 
stantively dyed, and the colouring matter is called a substantive 
colouring matter. 
Other dyes do not combine directly with the material to be 
acted on, but this material must first be charged with some STAINING. 
141 
substance known as a mordant (generally a metallic salt or 
hydrate) before it will combine with the colouring matter. 
These are known as adjective colouring matters.* 
Animal tissues have in general a considerable affinity for 
colouring matters, taking them up directly from their solutions. 
In consequence, the great majority of histological stains are 
obtained by substantive staining of the tissues. Still, as has 
been already pointed out, it seems probable that many of the 
histological stains that are obtained without intentional 
mordanting of the tissues, should yet in strictness be attributed 
to the class of adjective stains. This would be the case 
whenever there is reason to suppose that the stain obtained 
results from a combination of the colouring matter with some 
metallic salt or hydrate that is not a constituent of the living 
tissue, but has been brought into it by the fixing* or hardening 
reagents, these reagents playing the part of mordants though 
■only intentionally employed for another purpose. This would 
appear to be the case with the stains, or some of them, ob- 
tained after fixation with corrosive sublimate, alum, salts of 
iron, of platinum, of palladium, of uranium, and, for certain 
tissue elements and certain colours, chromium. And further, 
the mordanting substance may not only be present unin- 
tentionally in the fixing or hardening agents, it may be 
present unintentionally, or with imperfect realisation of its 
import, in the staining solutions themselves. Such is pre- 
sumably the part played by alum in many of the stains in 
which it figures as an ingredient. Iodine also plays in some 
staining processes a part which seems only explicable on the 
supposition that it acts as a mordant. 
In some staining processes, however, mordants are inten- 
tionally resorted to in order to fix the stain. Mordanting has 
long been employed in some liasmatein staining processes, such 
as that of M. Heidenhaix. More lately it has been resorted 
to for staining with tar colours, as in the curious “ inversion ” 
process of Rawitz. These processes will be explained in their 
respective paragraphs. Here it remains only to note of what 
sort are the advantages secured by this mode of staining. It 
must be admitted that mordants are in some cases of use by 
* For an excellent popular exposition of this subject see Benedikt and 
Ivnecht’s ‘ Chemistry of the Coal-tar Colours ’ (George Bell and Sons). 142 
CHAPTER XI f. 
enabling us to fix colouring matter in tissue elements that 
would otherwise be rebellious to staining. And they have in 
some cases the advantage of affording a very convenient 
means of regressive staining. For it happens that the colour- 
compounds thrown down in mordanted tissues are in many 
cases specially soluble in an excess of the mordant; so that 
the solution of the mordant itself forms a very appropriate 
decolourising agent. 
Recognising these advantages, it must still, I think, be said 
that there seems to be some danger at the present moment 
that the practice of employing mordants may degenerate into 
an abuse. For surely the primary use and intention of an 
histological stain (not of an industrial dye) is, that it should 
select and reveal those elements of tissues that have a natural 
affinity for its colouring matter. That end is attained in the 
manner least open to objection by the use of substantive 
stains, the natural affinities of the tissues and the colouring 
matter here coming spontaneously and unconstrained into 
play. Not so in the case of adjective staining. Here the 
colour is as it were forcibly compelled into an unnatural union 
with all or many of the elements of the tissue, including many 
which have no natural affinity whatever for the colour. In 
such preparations the distinction between chromatic and 
achromatic elements is obliterated; and the interpretation of 
the images afforded by them is open to more serious causes 
of error than in the case of substantive stains. 
Attention may be called here to the theoretically interesting 
<c invert ” stain obtained by Rawitz by the use of mordants 
(see post, Chap. XVI). In this curious process, a colour— 
safranin, for instance—which when used substantively is a 
pure chromatin stain, becomes by the use of mordants a pure 
plasma stain, staining cytoplasm deeply, and leaving the 
chromatin of nuclei absolutely unaffected. This strange effect 
can hardly be satisfactorily explained at present. Are we to 
suppose that the affinities of the nucleic acid of the chromatin 
are so fully satisfied by combination with the mordants, that 
it is unable further to combine with the colouring matter? 
It may be noted in a general way that the following sub- 
stances may be found useful as mordants, for enhancing the 
resistance of many tar colours to the alcohol employed for de- 
colourising, and for producing a stronger stain. STAINING. 
143 
Iodine: sections may be treated for a few minutes before 
staining with tincture of iodine. 
Permanganate of potash : see Henneguy’s process. 
Formaldehyde : see Ohlmacher’s process. 
Aniline : see Safranin. 
Chromic acid : see Bizzozero’s process. 
215. Choice of a Stain.—The following maybe recommended 
with confidence for general work :—For sections, Mayer’s 
hsemalum; or, for chromosmium objects more especially, 
safranin or thionin. If a plasma stain be required at the 
same time as a chromatin stain, I strongly recommend Kern- 
schwarz, followed by safranin (see Kernsclnvarz). 
For staining in toto, Grenadier’s alcoholic borax-carmine 
or Mayer’s carmalum, unless the object be so impermeable as 
to require a more highly alcoholised stain, in which case take 
Mayer’s jparacarmine, or for chromic acid objects Mayer’s 
hsemacalcium. 
For fresh tissues or small entire objects, methyl green, if it 
is not important to have permanent preparations ; if it is, 
take carmalum or alum-carmine. 
Picric acid may be used for double-staining in bulk after 
carmine or htematoxylin. 
Many others of the numerous stains discussed in the follow- 
ing chapters render most valuable services, and will be 
found recommended in the special paragraphs as occasion 
dictates. 
The beginner will probably do well not to use a double stain 
where a single one will do. To do so is too often to go farther 
and fare worse. 
216. Staining Reagents and Chemicals.—You are not likely 
to succeed in staining, especially in the beautiful processes of 
staining with coal-tar colours, unless you see to it that you are 
working with chemicals of the proper quality. You cannot 
ensure this by going to a generally trustworthy house for 
chemical products—at all events, not in the case of coal-tar 
colours. It is not sufficient that these should be what they 
are commercially described to be; they may be pure, and yet 
not give good stains. They must (in the case of anilins, at all 
events) be the identical products used in their work by the 144 
CHAPTER Xll. 
authors who have described and recommended them (see the 
note on the numerous safranins in the market, sub voce 
Safranin). I therefore feel constrained to advise everybody to 
get his reagents—at all events his anilins—from the well- 
known chemists Grubler or Miinder. Grubler has all the 
tried reagents in stock, and supplies only such as have been 
found by experiment with tissues to furnish the desired stain. 
He also makes up fixing and staining solutions, injection and 
imbedding masses, &c., according to the classical formulas, 
and sends them out neatly packed and ready for use. From 
experience I can most highly recommend these preparations, 
which are in nine cases out of ten better than those the 
observer is likely to make for himself. They may be ordered 
from the price list, or by quoting the numbers of the formulae 
in this work. The address is : Herrn Dr. G. Grubler & Co., 
Chemiker, Baiersche Strasse 63, Leipzig. Grubler can cor- 
respond in English. 
His preparations can be obtained in London from Mr. R. 
Kanthack, 21, Golden Square, Regent Street, W., who is also 
the authorised agent for the microscopes and apparatus of 
Zeiss, also for the microtomes of Becker, and the bacterio- 
logical apparatus of F. & M. Lautenschlaeger, &c. 
Miinder’s address is : Herrn Dr. G. Munder, Mikroskopisch- 
chemisches Institut, Gottingen. 
Messrs. Squire & Sons, chemists, 413, Oxford Street, 
London, W., also make a speciality of microscopical reagents, 
and I am told furnish excellent products. CHAPTER XIII. 
CARMINE AND COCHINEAL STAINS. 
217. The Theory of Carmine Staining.—I take the following 
from the important paper of Mayer, “ Ueber das Farben mit 
Carmin, Cochenille, und Hamatein-Thonerde,” in Mitth a. d. 
Zool. Station zu Neapel, Bd. x, Heft 3, 1892, p. 480. The 
rationale of staining with carmine has hitherto been obscured 
by the erroneous notion that carmine is nothing but carminic 
acid with at most certain impurities. This is not the case. 
According to the analysis of Liebermann (Ber. d. Chem. Qes., 
Jahrg. 18, 1886, pp. 1969—1975) carmine is a very 'peculiar 
alumina-lime-protein compound of carminic acid, a true chemi- 
cal compound from which at all events aluminium and calcium 
can no more be absent than sodium from salt. Analysis 
gives about 17 per cent, of wqter, 20 per cent, nitrogenous 
matters, 56 per cent, carminic acid, at least 3 per cent, alumina, 
and 3 per cent, lime, together with a small proportion of 
magnesia, potash, soda, phosphoric acid, and a trace of wax. 
Mayer has come to the conclusion that in the processes of 
histological staining (not of industrial dyeing) the active 
factors of the compound are, besides the carminic acid, only 
the alumina, and in some cases, the lime. The other bases are 
inactive ; the nitrogenous matters, so far as they have any 
influence at all, are an obstacle, as it is they that give rise to 
the well-known putrefaction of the solutions. 
Having arrived at these conclusions, it seemed logical to 
admit that carminic acid, instead of carmine, should he taken 
us the basis of staining solutions. This had already been pro- 
posed by Dimmock, whose paper (Amer. Natural., xviii, 1884, 
pp. 324-7) I quoted at length in the first edition of this work. 
But Diinmock's proposals were not very successful, for the 
reason that he had omitted from his solutions the essential 
•element, the alumina. He stained, for instance, with pure 146 
CHAPTER XIII. 
alcoholic solution of carminic acid, or of carminate of ammonia. 
Such solutions stain, but stain weakly and diffusely. 
Mayer therefore sought for appropriate means of introduc- 
ing the necessary alumina into the solutions ; with the results 
that will be set forth in the next §. 
218. Carminic Acid occurs as a purple-brown mass, easily 
soluble both in water and in alcohol. It is (according to 
Nietzki, Chemie der organischen Farbstoffe, Berlin, 1889, 
pp. 231—234) a weak (Liebermann says a strong) dibasic acid, 
which forms soluble salts with the alkaline metals, insoluble 
violet-coloured ones with the earthy and heavy metals. Very 
little is known concerning the chemical nature of these salts. 
The alumina salt (carminate of alumina) has the remarkable 
property of being soluble not only in acids and acid salts, such 
as alum, but also in alkalies and alkaline salts, such as borax, 
provided that only water or weak alcohol be employed as the 
menstruum. It may be obtained by precipitating a solution 
of carminic acid or of carminate of ammonia by means of 
acetate of alumina. It is also precipitated from the above- 
named solutions by chloride of aluminium, but only in part; 
whilst if alum be taken no precipitate is produced, the car- 
minate of alumina remaining in solution. Hence the com- 
position of the staining fluid given below under the name of 
Carmalum. 
When chloride of aluminium is taken, a precipitate of car- 
minate of alumina is formed, as stated above. But this pre- 
cipitate will redissolve if more chloride of aluminium be 
cautiously added. This gives the staining fluid described in 
§ 224, which may be convenient in cases in which it is not 
desirable to work with a fluid containing alum. 
Both of these solutions stain in a violet tone, something 
like alum-carmine. A redder tone may be obtained by 
adding calcium chloride to the carmalum solution. But this 
is not advisable, for calcium chloride added to carmalum 
precipitates the solution with formation of gypsum. Of 
course, this does not occur with the aluminium chloride 
solution; but for other reasons the addition does not give 
satisfactory results with the chloride of aluminium solution 
mentioned above. But it does give good results when com- 
bined with an alcoholic chloride of aluminium solution, and CARMINE AND COCHINEAL STAINS. 
147 
thus solves at once the problem of obtaining a red stain and 
an alcoholic staining fluid. This is described below under 
the name of Paracarmine. 
If the foregoing explanations of the rationale of carmine 
staining be compared with the remarks on the theory of 
staining with haematoxylin given in the next Chapter, an 
interesting parallelism will be observed. In both processes, 
it is not the colouring- matter alone which is active, but the 
colouring matter combined with alumina. The stain is 
always got with carminic acid + alumina, or with heematein 
+ alumina ; other substances, such as lime, occasionally 
playing a part. 
219. The foregoing considerations deal with the theory of 
staining with Carmine ; we have now to consider the Theory 
of Staining with Cochineal. According to Mayer, whose 
earliest researches are confirmed by his latest (Mitth. Zool. 
Stat. zu Neapel, x, 3, 1892, p. 496), the active principle of 
extract or tincture of cochineal (as used in histology) is not 
free carminic acid, but carminic acid chemically combined 
with a base which is not lime, but some alkali. The pure 
aqueous extract contains only traces of lime, the alcoholic 
none at all. The watery extract made with alum, or cochineal- 
alum carmine (§ 226), owes its staining power to the formation 
of a carminate of alumina, the general properties of which 
have been discussed above when treating of the theory of 
carmine staining (§ 218). The tincture made with pure 
alcohol, on the other hand, contains only the above-mentioned 
carminate of some alkali. This carminate alone stains weakly 
and diffusely (like carminic acid alone). But if in the tissues 
treated with it it meet with lime salts, alumina or magnesia 
salts, or even metallic salts capable of combining with it and 
forming insoluble coloured precipitates in the tissues, then a 
strong and selective stain may result. As a matter of fact, 
the simple cochineal tincture of Mayer given in § 241 does 
give splendid results with certain objects (i. e. such as contain 
the salts in question). But it is unfortunately equally certain 
that such objects are rather rare than otherwise, and that 
with the majority of objects the stain is a very poor one. 
But if the necessary salts be added to the tincture itself, 
then a solution ought to result containing the necessary 148 
CHAPTER XIII. 
elements for affording a strong and selective stain with all 
classes of objects. This proves to be the case; whence 
Mayer’s new formula, § 242. 
220. General Remarks.—What are the carmine stains useful 
for? Is it for staining fresh tissues? With the exception 
of aceto-carmine, no. Is it for staining sections ? Again, 
no; for, in nine cases out of ten, sections are better stained 
by some of the anilin stains and by some hsematem stains 
than they can be in any carmine stain. Is it for staining 
entire objects ?—for staining in the mass ? Yes; for in 
many, if not in most cases, that can be done more satisfac- 
torily by means of carmine than by means of any other known 
agent. 
Haematoxylin (or hasmatem) has a disastrous tendency to 
overstain; and the tar-colours are with hardly an exception 
entirely inapplicable to staining in bulk. 
Overstainsmayin all cases be washed out with weak HC1 (e. g. 
0‘1 per cent.). Henneguy (Journ. de VAnat. etde la Physiol., 
xxvii, 1891, p. 400) states that overstains may be completely 
removed by means of permanganate of potash. All carmine 
stains, with the exception of aceto-carmine, are permanent in 
balsam. None of the acid stains, nor any of Grenacher’s 
fluids, should be used with calcareous structures that it is wished 
to preserve, unless they be taken in a state of extreme 
dilution. 
221. Choice of a Carmine Stain.—Grenadier’s alcoholic borax- 
carmine may be recommended to the beginner as being the 
easiest of these stains to work with. 
In view of the far greater simplicity and precision of the 
methods proposed by Mayer, it is probable that they, or at all 
events methods conforming to the principles above laid down, 
will gradually take the place of the old methods. Not, indeed, 
that some of the old stains will not hold their ground. Alum- 
carmine will remain a superb stain, and borax-carmine remain 
superior to paracarmine in the power and brilliancy of its 
stain; but the superior precision of Mayer’s methods will 
doubtless be acknowledged. And it is perhaps not too much 
to hope that fewer formulas of the old roundabout, happy-go- 
lucky sort, with commercial carmine or hasmatoxylin as the 
chief ingredient, will henceforth be published. CARMINE AND COCHINEAL STAINS. 
149 
In view of my conviction that the doctrines of Mayer concerning carmine 
staining are true, I have suppressed a very large number of formulie that I 
hold to be as practically superfluous as they are theoretically irrational. See 
previous editions. 
222. Pure Carminic Acid may be obtained from E. Merck of 
Darmstadt, or from Dr. Gf. Grubler & Co. (63, Bayersche 
Strasse, Leipzig). The price at present is from 2s. to 4s. per 
10 grms. 
Dr. Mayer writes me that the quality is sometimes not all 
that could be desired. Recent samples have been found to 
contain a perceptible quantity of ammonia or some fixed 
alkali. 
a. Aqueous Carmine Stains. 
223. Mayer’s Carmalum (Mitth. Zool. Stat. zu Neajpel, x, 3, 
1892, p. 489).—Carminic acid, 1 grin.; alum, 10 grins.; distilled 
water, 200 c.c. Dissolve with lieat (if necessary : I have been 
able to make my solutions in the cold). Decant or filter. Add 
some antiseptic, either a few crystals of thymol, or 0-l per cent, 
salicylic acid, or 0'5 per cent, salicylate of soda. The solution 
will then keep. A clearish red tluid with a violet tinge. It 
stains well in bulk even osmium objects. If washed out with 
distilled water only, the plasma will remain somewhat stained. 
If this be not desired, wash out carefully with alum solution, 
or, in difficult cases, with weak acid. The general effect is 
that of an alum-carmine stain. A notable difference between 
the two is that carmalum stains well in bulk, which alum-carmine 
is not very suitable for when used in the ordinary way; but 
see § 227. 
a. Acid. 
A weaker solution may be made by taking from three to five times as 
much alum and five times as much water, and dissolving in the cold, which 
may be convenient. This is a very close equivalent of alum-carmine, 
giving, however, a somewhat redder stain. I find this solution very weak 
for ordinary work. 
It should be noted that with either solution the objects to 
be stained should not have an alkaline reaction. The other 
properties of these solutions are very similar to those of alum- 
carmine. 150 
CHAPTER XIII. 
224. Mayer’s Aqueous Aluminium-Chloride-Solution (Mitth. Zool. 
Stat. zu Neapel, x, 3, 1892, p. 490).—Carminic acid 1 grin., chloride of 
aluminium, 3 grms. ; water, 200 c.c. Add an antiseptic, as for carmalum. 
Use as carmalum. The stain is of a blue-violet colour, very powerful, 
and elective. But it is not so pure a stain as that of carmalum, plasma 
being more strongly coloured. It is recommended only as a substitute for 
carmalum in cases in which the latter is counter-indicated on account of the 
presence of alum or the like. 
225. Alum-carmine (Grenacher’s formula, Arch. mik. Anat., 
xvi, 1879, p. 465).—An aqueous solution (of 1 to 5 per cent, 
strength, or any other strength that may be preferred) of com- 
mon or ammonia alum is boiled for ten or twenty minutes 
with \ to 1 per cent, of powdered carmine. (It is perhaps 
the safer plan to take the alum solution highly concentrated 
in the first instance, and after boiling the carmine in it dilute 
to the desired strength.) When cool filter. 
This stain must be avoided in the case of calcareous struc- 
tures that it is wished to preserve. 
Tizzoxi (Bull. Sc. Med. Bologna, 1884, p. 259), Pisenti (Gazz. degli 
Ospetali, No. 24; Zeit.f. wiss. Mik., ii, 1885, p. 378), and Grieb (Mem. 
Soc. Hal. Sci., t. vi, No. 9, 1887 ; Zeit. f. wiss. Mik., vii, 1, 1890, p. 47) 
have given modifications of Grenadier’s formula which do not appear to me 
rational. 
Alum-carmine is one of the best stains to be found outside 
the coal-tar colours. It is particularly to be recommended to 
the beginner, as it is easy to work with; it is hardly possible 
to overstain with it (except muscle). Its chief defect is that 
it is not very penetrating, and therefore quite unsuitable for 
staining objects of considerable size in the mass. This defect 
may, however, be to some extent overcome by employing the 
acid formula of Henneguy (§ 227), if it be not convenient to 
use Mayer’s carmalum. 
The stain is permanent in balsam, and in aqueous media 
if not acid. 
226. Cochineal Alum-Carmine (Partsch,Arch.f. mik. Anat., 
xiv, 1877, p. 180).—Powdered cochineal is boiled for some 
time in a 5 per cent, solution of alum, the decoction filtered, 
and a little salicylic acid added to preserve it from mould. 
Another method of preparation has been given by Czokor 
{Arch. f. mik. Anat., xviii, 1880, p. 413). Mayer has care- CARMINE AND COCHINEAL STAINS. 
151 
fully examined both, and finds that Partsch’s is the more 
rational, the proportion of alum in it being- exactly right, 
whilst in Czokor’s it is insufficient. Partsch’s fluid has also 
the advantage of keeping better. 
The formula known as Klein’s cochineal fluid (which appears to have 
been first published in the Ann. and Mag. Nat. Hist., viii, 1881, p. 232) is 
identical with that of Czokor. 
The formula recommended by Rabl in Zeit. f. wiss. Mik., xi, 2, 1894, 
p. 168, is only an insignificant modification of that of Czokor. 
Both these solutions are to all intents and purposes “alum- 
carmines.” They give a stain that is practically identical 
with that of alum-carmine made from carmine, with perhaps 
even more delicate differentiations (but that depends so much 
on the quality of the carmine, the quality of the cochineal, 
and the nature of the objects to be stained that no absolute 
rule can be stated). On the whole, it seems to be a mere 
matter of convenience whether the one or the other should 
be preferred. The cochineal fluids should be used in exactly 
the same way as the carmine fluid. 
It has been lately recommended by Herrick (Journ. Comp. 
Neur., Cincinnati, vol. i, 1891, p. 134) as a “vast improve- 
ment ” to make the solution with sulphate of aluminium 
instead of alum (quoted from Mayer’s paper, Mitth. Zool. Stat. 
Neapel, x, 3, 1892, p. 496). 
227. Acetic Acid Alum-Carmine (Henneguy, Traite des Meth. 
techn., Lee et Henneguy, 1887, p. 88).—Excess of carmine is 
boiled in saturated solution of potash alum. After cooling- 
add 10 per cent, of glacial acetic acid, and leave to settle for 
some days. The deposit of carmine and alum that forms 
during that time is removed by filtration. 
For staining, enough of the solution is added to distilled 
water to give it a deep rose tint. In order to ensure rapid 
diffusion, it is well to bring the tissues into the stain direct 
from alcohol. Stain for twenty-four to forty-eight hours, 
and wash for an hour or two in distilled water. (It is 
important that the water should be distilled in order to avoid 
the formation of crystals.) Dehydrate with alcohol and 
mount in balsam. You can mount in glycerin, but the pre- 
parations do not keep so well as in balsam. 152 
CHAPTER XIII. 
The advantage of this carmine is that it has greater power 
of penetration than the non-acidified alum-carmine, and stains 
deep-seated layers of tissue just as well as the superficial 
ones. The colour of the stain is a somewhat inelegant violet, 
but this can be changed to a warmer tone by treating the 
objects with dilute HC1, as for borax-carmine objects. 
228. Alum-Carmine and Picric Acid.—Alum-carmine objects 
may be double-stained with picric acid. Legal (Morph. 
Jahrb., viii, p. 358) combines the two stains by mixing ten 
vols. of alum-carmine with one of saturated picric acid 
solution. I consider this to be a very recoinmendable practice. 
229. Aceto-Carmine (Acetic Acid Carmine) (Schneider’s formula, 
Zool. Anzeig., No. 56, 1880, p. 254).—To boiling acetic acid of 45 per cent, 
strength add carmine until no more will dissolve, and filter. (Forty-five 
per cent, acetic acid is, according to Schneider, the strength that dissolves 
the largest proportion of carmine). 
To use the solution you may either dilute it to 1 per cent, strength, and 
use the dilute solution for slow staining; or a drop of the concentrated 
solution may be added to a fresh preparation under the cover-glass. If you 
use the concentrated solution it fixes and stains at the same time, and 
hence may render service for the study of fresh objects. It is very pene- 
trating, a quality that enables it to be used where ordinary reagents would 
totally fail. The stain is a pure nuclear one. Unfortunately the pre- 
parations cannot be preserved, and for this and other reasons the stain is of 
very restricted applicability. 
Zacharias (seeZeit.f. wiss. Mik., v, 3, 1888, p. 371) adds to this solution 
wood vinegar (acetum pyrolignosum) in the proportion of 1 drop to 10 c.c. 
A similar stain has been prepared with formic acid by Pianese (see 
Zeit.f. wiss. Mile., x, 4,1894, p. 502). Probably for almost all the purposes 
for which aceto-carmine is useful, methyl-green will give better results. 
230. Iron Carmine (Zacharias, Zool. Anz., No. 440, 1894, 
p. 62).—Stain thoroughly for several hours (Zacharias 
stains in an aceto-carmine, of which I suppress the formula, 
for, as pointed out to me by Dr. Mayer, and as I have 
verified, carmalum does just as well). Einse the objects with 
dilute acetic acid, and bring them (taking care not to touch 
them with metallic instruments, if the aceto-carmine have 
been taken) into a 1 per cent, solution of ammoniated citrate 
of iron (the pharmaceutical Ferri et Ammoniee Gitras). Leave 
them till thoroughly penetrated, for as much as two or three 
hours if need be. In this solution they take on a black tint CAEMINE AND COCHINEAL STAINS. 
153 
(with sections this happens in a few minutes). They should 
be removed as soon as the reaction has taken place throughout, 
otherwise there is risk of over-blackening. Wash for several 
hours in distilled water, dehydrate and mount in balsam. 
This is at the same time a chromatin stain and a plasma 
stain. In my preparations chromatin is blue and plasmatic 
elements brown. I consider the method may render service 
in some cases. Amongst other things, spindles and spindle- 
relics are fairly well brought out by it. It can be applied to 
the coloration of entire objects (provided that they be small, 
Mayer). 
/3. So-called “Neutral,” and Alkaline. 
231. As to Piero-carmine.—The term “ picro-carmine ” is commonly 
used to denote a whole tribe of solutions in which carmine, ammonia, and 
picric acid exist uncombined in haphazard proportions. Ranvier, to whom 
we owe the invention of picro-carmine, claims that when prepared by his 
process it results as a definite chemical substance, a double salt of picric and 
carminic acid and ammonia, or picro-carminate of ammonia. But this has 
never been scientifically demonstrated. Picro-carmine gives good differen- 
tiations, and has the great merit of being less hurtful to most tissues than 
other aqueous alkaline carmines. It should be understood that the chief 
value of picrocarmine does not lie in its capacity of affording a double stain. 
The double stain, if that is all that is wanted, can be just as well or better 
obtained by staining first with borax-carmine, or the like, and after-staining 
with picric acid. The essential point about picro-carmine is that it is a 
fairly neutral fluid. Ranvier was, in fact, led to add picric acid to ammo- 
niacal solution of carmine by the desire of neutralising the ammonia, that 
is all. 
For slow staining, dilute solutions may advantageously have 1 or 2 per 
cent, of chloral hydrate added to them. 
Overstains may be washed out with hydrochloric acid, say 0-5 per cent., in 
water, alcohol, or glycerin. 
Preparations should be mounted in balsam, or if in glycerin this should be 
acidulated with 1 per cent, of acetic or, better, formic acid. 
232. Ranvier’s Piero-carmine or “ Piero-carminate of Ammonia.” 
—The method of preparation employed in the Laboratory of Histology of 
the College de France, kindly communicated to myself and Henneguy for 
our Traite des Metli. teclin. (q. v. p. 451) by M. Yignal, one of the assist- 
ants there, is as follows : 
Take- 
Water 1000 parts. 
Picric acid ....... 20 „ 
Carmine ....... 10 ,, 
Ammonia ....... 50 „ 154 
CHAPTER XIII. 
Put them into a stoppered bottle and leave them for two or three months 
in a warm place. Then put them into a large crystallising dish and let 
them putrefy. When the liquid has become reduced by evaporation to four 
fifths of its original volume, remove the crystals that have formed at the 
bottom, dry them, and dissolve them in a little warm water. Filter the solu- 
tion, and examine it with the microscope to see whether the carmine is really 
dissolved. If not, add water and ammonia, and let the solution putrefy 
again ; evaporate and examine as before. When you have got your carmine 
combined, evaporate the solution to dryness in a stove, and reduce the 
picro-carminate to powder. 
For staining, dissolve 1 grm. of the powder in 100 grms. of water, and 
add a crystal of thymol to prevent the development of mould. 
233. Ranvier’s Original Formula (Traite, p. 100) was as follows :— 
To a saturated solution of picric acid add carmine (dissolved in ammonia) to 
saturation. Evaporate down to one fifth the original volume in a drying 
oven ; and separate by filtration the precipitate, pour in carmine, that forms 
in the liquid when cool. Evaporate the mother-liquid to dryness, and you 
will obtain the picro-carminate in the form of a crystalline powder of the 
colour of red ochre. It ought to dissolve completely in distilled water; a 
1 per cent, solution is best for use. 
234. Other Formulae for Piero-carmine.—I have tried most of the 
following, and found no real advantage in any of them:—Gage, Am. M. Mic. 
Journ., i, 1880, p. 22 ; Journ. Roy. Mic. Soc., vol. iii, p. 501 (very elaborate 
and has not afforded me a soluble carmine.). Fol, Lehrb. d. vergl. mile. 
Anat., p. 195. Rutherford, Pract. Hist., p. 173. Paul Mater, Mitth. 
Zool. Stat. Neapel, ii, p. 20. Baber, Mon. Micro. Journ., xii, p. 48. 
Pergens, Carnoy’s Biologie cellulaire, p. 92. Hoyer, Biol. Centralb., ii, 
1882, p. 17. Bizzozero, Zeit.f. wiss. Mik., 1885, p. 539. Ivlemensiewics, 
Sitzb. Akad. Wiss. Wien, lxxviii, 1878, iii, Juni; Zeit.f. wiss. Mile., i, 1884, 
p. 501. Cuccati, Zeit. f. wiss. Mik., vi, 1, 1889, p. 42. 
For Soda-Piero-Carmine see Lowenthal, Anat. Anzeig., ii, 1887, No. 1, 
p. 22, and Zeit. f. wiss. Mik., x, 3, 1893, p. 313; Anon., Journ. Roy. 
Mic. Soc., 1888, p. 518; Squire’s Methods and Formulse, Ac, 1892, 
p. 35. 
Weigert corrects unsatisfactory samples of picro-carmine in the following 
way (Virchow's Archiv, Bd. lxxxiv, pp. 275, 315 ; Zool. Jahrb., 1881, 
p. 40):—Small quantities of acetic acid are added “ until the first slight 
precipitate appears even after stirring.” The whole is again put away for 
twenty-four hours more, when it will be found that there has formed a pre- 
cipitate that can only partially be removed by filtration ; ammonia is then 
added drop by drop at intervals of twenty-four hours, until the solution 
becomes clear. If the solution stains too yellow, acetic acid is added ; if it 
overstains red, a little ammonia is again added. All badly staining samples 
of picro-carmine may, according to Weigert, be improved in the same way by 
addition of acetic acid or ammonia. I consider it to be a very happy-go- 
lucliy process. CARMINE AND COCHINEAL STAINS. 
155 
235. Lithium-Carmine (Orth, Berlin, klin. Woclienschr., xxviii, 1883, 
p. 421).—Superfluous, and macerates considerably (Mayer). 
236. Ammonia-Carmine.—In my opinion there is no valid excuse for 
using ammonia-carmine at all at the present day. 
If, however, such a stain he used, care should he taken to get rid of the 
free ammonia as completely as possible. This may he done by boiling until 
the excess of ammonia has evaporated. (So long as free ammonia is present 
large bubbles are formed in the fluid, and the latter shows a dark purple 
colour. When the free ammonia has evaporated small bubbles appear, and 
the solution takes a brighter red tint.) 
One per cent, each of carmine and ammonia in distilled water is a good 
proportion. 
But a safer mode of preparation is that of Ranvier, as follows (kindly 
communicated by Dr. Malassez, see Traite des Alethodes techniques, 
&c., of Lee and Henneguy, 1st. edit., p. 82).—Make a simple solution of 
carmine in water with a slight excess of ammonia, and expose it to the 
air in a deep crystallising dish until it is entirely dried up. It should 
be allowed to putrefy if possible. Dissolve the dry deposit in pure water, 
and filter. 
y. Other Aqueous Carmines (Acid and Alkaline). 
237. Schweigger-Seidel’s Acid Carmine (Ranvieb, Traite, p. 99). 
Hamann’s Acid Carmine (Intern. Alon. f. Anat. u. Hist., i, 5, 1884; 
Zeit. f. iciss. Mile., ii, 1885, p. 87). Neutral Borax-Carmine (Niki- 
forow, Zeit. f. wiss. Alik., v, 3, 1888, p. 337). Neutral Borax-Carmine 
(Grenacher, Arch. f. mik. Anat., xvi, 1879, p. 466). Haug’s modifications 
of this, Zeit. f. wiss. Mile., vii, 2, 1890, p. 151, and viii, 1, 1891, p. 52. 
AY oodward’s Borax-Carmine (see Monthly Mic. Journ., vii, 1872, p. 38; 
Am. Quart. Mic. Journ., i, 1879, p. 220; Journ. Roy. Mic. Soc., ii, p. 613). 
H. Gibbes’ Borax Carmine (see Journ. Roy. Mic. Soc., iii, 1883, p. 390). 
Delage's Osmium-Carmine (Arch, cle Zool. exp. et gen., iv, ser. 2, 
1886; Zeit. f. wiss. Alik., iii, 2, 1886, p. 240). Rollett’s Carminroth 
(see Zeit. f. wiss. Alik., i, p. 91). Perl’s Soluble Carmine (see Frey, 
Das Alikroskop., 7 Auf., and Zeit.f. wiss. Mik., i, p. 91). Carminic Acid 
(see Dihmock, Amer. Natural., xviii, 1884, pp. 324-7; and Journ. Roy. 
Alic. Soc., 1884, pp. 471-4). Boric Acid Carmine (Arcangeli, see 
Proc.-verb. Soc. Toscana Sc. Nat., 1885, p. 283; and Zeit.f. wiss. Mile., 
1885, p. 377). Boric Acid Alum-Carmine, Arcangeli, ibid. Sali- 
cylic Acid Alum-Carmine, Arcangeli, ibid. Salicylic Acid Car- 
mine, Arcangeli, ibid. Picric Acid Carmine, Arcangeli, ibid. 
Picric Acid Carmine, Minot (see Whitman’s Methods in Mic. Anat., 
p. 42). Uranium-Carmine, Gierke, Zeit.f. wiss. Alik., i, 1884, p. 92; 
Schmaus, ibid., viii, 2,1891; cf. Aliinchener med. Woclienschr., 1891, No. 8, 
p. 147. Carbonate of Soda Carmine, Cuccati, Zeit.f. wiss. Alik., iv, 1, 
1887, p. 50. 156 
CHAPTER XIII. 
b. Alcoholic Carmine Stains. 
238. Alcoholic Borax-Carmine (Grenacher, Arch. f. mik. 
Anat., xvi, 1879, p. 466, et seq.).—Take a concentrated solution 
of carmine in borax solution (2 to 3 per cent, carmine to 4 per 
cent, borax); boil it for half an hour or more; dilute it with 
about an equal volume of 70 per cent, alcohol, allow it to 
stand some time (twenty-four hours—Mayer), and filter. Or 
the mixture of carmine and borax solution is allowed to stand 
for two or three days and occasionally stirred; the greater part 
of the carmine will dissolve. To the solution is added an 
equal bulk of 70 per cent, alcohol; the mixture is allowed to 
stand for a week, and then is filtered. If on keeping more 
carmine is deposited, it must be refiltered. 
Preparations should remain in the stain until they are 
thoroughly penetrated (for days if necessary), and then be 
brought (without first washing out) into alcohol (of 70 per 
cent.; this is absolutely necessary—Mayer) acidulated with 
4 to 6 drops of hydrochloric acid to each 100 c.c. of alcohol. 
They are left in this until the stain is differentiated, and may 
then be washed or hardened in neutral alcohol. Fopr drops 
of HC1 is generally enough. Three drops I find not quite 
sufficient. The stained objects should remain in the acidu- 
lated alcohol till they acquire a bright transparent look. 
This may require days (Mayer). 
For delicate objects, and for very impermeable objects, it 
may be well to increase the proportion of alcohol in the stain ; 
it may conveniently be raised to about 50 per cent. It 
should not exceed 60 per cent, in any case (Mayer). 
This stain is probably by far the most popular of any for 
staining in the mass. It is easy to use, and gives a most 
splendid coloration. But it is not so penetrating as is 
commonly supposed, and has the defect of sometimes forming 
precipitates in the cavities of bulky objects which cannot be 
removed by washing out. And it must be remembered that 
the fluid is alkaline, and therefore not suitable for delicate 
cytological work. I believe that some otherwise excellent 
cytological work has been vitiated by over-confidence in this 
reagent, and will have to be done over again. 
239. Mayer’s Paracarmine (MitthZool. Stat. zu Neapel, x, 
3, 1892, p. 491).—Carminic acid; 1 grin.; chloride of alu- CARMINE AND COCHINEAL STAINS. 
157 
minium, 0*5 grm.; chloride of calcium, 4 grms.; 70 per cent, 
alcohol, 100 c.c. Dissolve cold or warm, allow to settle, and 
filter. A light red liquid, specially adapted for staining in 
bulk, and the nearest approach to a substitute for Grenadier’s 
alcoholic borax-carmine that has yet been discovered. 
Objects to be stained should not have an alkaline reaction, 
nor contain any considerable amount of carbonate of lime 
(spicules or skeletal parts of corals, &c.), which would give 
rise to precipitates (Mayer). Wash out sections, or objects 
intended to be sectioned, with pure 70 per cent, alcohol. 
Objects intended to be mounted whole may be washed out 
with a weak solution of aluminium chloride in alcohol, or if 
this be not sufficient, with 5 per cent, common acetic acid (or 
2’5 per cent, glacial acetic acid) in alcohol. 
For staining bulky objects with large cavities, such as 
Salpa, the solution should be diluted (with alcohol); and as 
this may cause precipitates to form during the staining, 
especially if the objects are not very clean, it is advisable to 
slightly acidify the dilute solutions. 
Paracarmine gives a nuclear stain of a red colour, though 
not so fiery red as that of borax-carmine. Its points of 
superiority over borax-carmine are that it is not alkaline, 
therefore less hurtful to tissues; that it is more highly 
alcoholic, therefore more penetrating; that it has less tendency 
to form granular precipitates in the interior of objects, and 
that it generally keeps perfectly without precipitating (mine 
has precipitated somewhat, though not to an injurious extent). 
240. Alcoholic Hydrochloric-Acid Carmine.—In view of the above- 
mentioned defects of borax-carmine—defective penetration, and a tendency 
to form insoluble granular precipitates in the interior of objects—it is 
desirable to possess a powerful staining medium more highly alcoholised and 
of acid reaction. Hydrochloric acid carmine possesses these qualities, and 
may be useful in the case of objects for which Mayer’s paracarmine may 
not suffice. It may, for instance, be frequently useful in work on Arthropoda, 
especially the marine forms. 
Grenachee’s receipt {Arcli. f. mih. Anat., xvi, 1879, p. 468) will he 
found extremely troublesome by those who are not expert at neutralising. 
The following method, due to Paul Mayer (quoted from Garbini’s 
Manuale per La Technica moclerna del Microscopic), first ed., p. 46), is easy 
and gives excellent results. Take 100 grms. of alcohol (either absolute or of 
any weaker grade), 1 or 2 drops of HC1, and an excess of carmine, and boil 
until you get a clear solution, taking care that there remains an excess of 
carmine. This ought to give a nuclear stain, without the aid of HC1 for 158 
CHAPTER XIII. 
washing out. If overstaining or diffusion should occur, wash out with 
alcohol, very slightly acidulated with HC1. 
Brass (Zeit.f. wiss. Mile., ii, 1885, p. 303) takes 100 c.c. of 70 per cent, 
alcohol, 15 drops of HCl, and an excess of carmine. An old formula of 
Paul Mayer’s (Mittli. Zool. Slat. Neapel, iv, 1883, p. 521; Journ. Boy. 
Mic. Soc. [N.S.], iv, 1884, p. 317), which gives a more powerful stain than 
the preceding, is somewhat inconvenient to carry out. A more recent formula 
of Mayer’s (Intern. Monatsschr. f. Anat., &c., 1887, p. 43) is as follows : 
Carmine, 4 grms.; water, 15 c.c.; hydrochloric acid, 30 drops. Boil till the 
carmine is dissolved, add 95 c.c. of 85 per cent, alcohol, and neutralise by 
adding ammonia until the carmine begins to precipitate. 
If it be desired to dilute any of these solutions, it should be done with 
alcohol, not water, and alcohol should he taken for washing out. 
241. Alcoholic Cochineal (Mayer’s Old Formula, Mitth. Zool. 
Stat. Neapel, ii, 1881, p. 14).—Cochineal in coarse powder is 
macerated for several days in alcohol of 70 per cent. For 
each gramme of the cochineal there is required 8 to 10 c.c. of 
the alcohol. Stir frequently. Filter, and the resulting clear, 
deep red solution is fit for staining. 
The objects to be stained must previously be imbibed with 
alcohol of 70 per cent, and alcohol of the same strength must 
be used for washing out or for diluting the staining solution. 
The washing out must be repeated with fresh alcohol until the 
latter takes up no more colour. Warm alcohol acts more 
rapidly than cold. Overstaining seldom happens; it may be 
corrected by means of 70 per cent, alcohol, containing per 
cent, hydrochloric or 1 per cent, acetic acid. 
Small objects and thin sections may be stained in a few 
minutes, larger animals require hours or days. 
A nuclear stain, slightly affecting protoplasm. The colour 
varies with the reaction of the tissues, and the presence or 
absence of certain salts in them. Crustacea with thick chi- 
tinous integuments are generally stained red, most other 
organisms blue. The stain is also often of different colours 
in different tissue elements of the same preparation. Glands 
or their secretion often stain grey-green. In embryos of 
Lumbricus Kleinenberg found the vessels to stain red, their 
contents of an intense blue. 
Acids lighten the stain and make it yellowish red. Caustic 
alkalies turn it to a deep purple. 
The best stains are obtained in the case of objects that have 
been prepared with chromic or picric acid combinations, or CARMINE AND COCHINEAL STAINS. 
159 
with absolute alcohol. Osmic acid preparations stain very 
weakly unless they have been previously bleached. The acid 
must be carefully washed out before staining, or a diffuse 
stain will result. The stain is permanent in oil of cloves and 
balsam. 
The object for which this stain was imagined is to obtain 
an alcoholic staining fluid whose high penetrating power 
allows it to be employed in the case of organisms, such as 
Arthropoda, whose chitinous investments are but very slightly 
permeable by aqueous solutions of carmine. 
I have treated this stain at considerable length because I 
am convinced that it ought to be better known. It is very 
useful in many cases (Annelids, for instance), and indispen- 
sable for Arthropoda. On the other hand, it gives a very 
poor stain with unsuitable objects, and the majority of the 
objects with which the histologist has to do are in this case. 
For this reason Mayer has been led to devise the fluid de- 
scribed in the following paragraph, which, containing in 
itself the salts necessary for producing a rich and energetic 
selectivity of stain, gives good results with all classes of ob- 
jects. He almost appears to intend it entirely to take the 
place of the old fluid. But I think the old fluid cannot yet 
be discarded. It has over the new fluid the (for some cases 
considerable) advantage of being more highly alcoholic; and 
it does not contain free acid, so that it can be used with cal- 
careous structures which it is wished to preserve—which the 
new fluid cannot. For specimens of Pluteus, for instance, I 
find it excellent. 
242. Mayer’s Alcoholic Cochineal, New Formula (Mitth. Zool. 
Stat. Neap el, x, 3, 1892, p. 498).—Cochineal, 5 grins.; chloride 
of calcium, 5 grins.; chloride of aluminium, 05 grm.; nitric 
acid of F20 sp. gr., 8 drops; 50 per cent, alcohol, 100 c.c. 
Powder the cochineal finely and rub up in a mortar with the 
salts, add the alcohol and acid, heat to boiling-point, leave to 
cool, leave for some days standing with frequent agitation, 
filter. 
Use as the old tincture, the objects being prepared and 
washed out with 50 per cent, alcohol. The stain is like that 
of paraearmine, but not quite so strong and not so sharp. 
Mayer only recommends it as a succedaneum of paraearmine. 160 
CHAPTER. XIII. 
The object for which it was devised has been explained in 
the last paragraph, where also some limitations to its useful- 
ness have been indicated. 
For the chemical theory involved in the composition of 
these tinctures see also the last paragraph, and supra, § 219. CHAPTER XIV. 
HiEMATEIN (H2EMATOXYLIN) STAINS. 
243. Theory of Haematoxylin Staining.—It appears to be now 
thoroughly well established (see Nietzki, Chemie der organ- 
isclien Fdrbstojfe, Berlin, Springer, 1889, pp. 215—217) that 
the active colouring principle of hematoxylin dyes is heema- 
tein ; and further, that the haematein of the usual histological 
staining solutions is a product of the oxidation of their con- 
tained hematoxylin by means of the air to which they are 
exposed (see Mayer, “ TJeber das Farben mit Hdmatoxylin,” 
in Mitth. a. d. Zool. Station zn Neapel, Bd. x, Heft 1, 1891, 
pp. 170—186; Unna, “ TJeber die Reifung unserer Fdrbstojfe,” 
in Zeit. f. wiss. Mile., viii, 4, 1892, p. 483). This change is 
known as “ ripening,” and until it has taken place the solu- 
tions are not fit to use for staining. 
Hitherto it has been the practice to rely (quite uncon- 
sciously so far as the chemical theory is concerned) on the 
spontaneous absorption by the solutions of oxygen from the 
air to effect this “ ripening,” but it has now been discovered 
(by both Mayer and Unna independently) that nothing is 
easier than to bring about the reaction artificially; all that is 
necessary being, for instance, to add to a solution of hema- 
toxylin containing alum a little neutralised solution of per- 
oxide of hydrogen. The solution becomes almost instanta- 
neously dark blue, “ ripe,” and fit for staining, thus definitely 
confirming the truth of the hypothesis. 
Mayer goes further. A solution of pure uncombined 
haematein would not afford a selective stain such as we require 
in histology ; it would be at most a dye. The usual solutions 
(I am not here speaking of Weigert’s or similar processes) all 
contain alum, and Mayer holds that the active agent in them 
is a compound of haematein with alumina (“ Haemate'in- 
Thonerde ”—haemateate of alumina). He holds that this salt 
is precipitated in the tissues (chiefly in the nuclei) by certain 162 
CHAPTER XIV. 
organic and inorganic salts there present (for instance, phos- 
phates) ; perhaps also by other organic bodies belonging to 
the tissues. Such salts are known to exist in living tissues. 
By the process of “ fixing ” they are fixed there,—either in 
the same chemical state, as when such an agent as alcohol is 
used for fixing; or as fresh compounds formed by the action 
of the fixing agent itself, as when such a fixing medium as 
sublimate is used. 
The chief factor in the composition of these stains is, 
therefore—after the colouring substance itself,—alumina, to 
which, however, may be added some other bodies, such as 
lime, which may play a secondary role (as, for instance, chlo- 
ride of calcium does in Kleinenberg’s solution). 
It follows that the first difficulty with which the worker 
with hagmatoxylin stains has to contend is that of getting his 
hsematoxylin duly oxidised into hsematein, in order to the for- 
mation of the desired hsematein-alumina compound, or lake. If 
this be done by the hitherto customary process of leaving the 
solutions to “ ripen ” by the action of the air, it is necessary to 
wait for a long time before the reaction is obtained. During 
all this time, it may be weeks or months, there is no means, 
except repeated trial, of ascertaining whether the solution at 
any moment contains sufficient haematein to afford a good 
stain. And here a second difficulty arises; the oxidising pro- 
cess continuing, the solutions become “ over-ripe ; ” the haema- 
tein, through further oxidation, passes over into colourless 
compounds, and the solutions begin to precipitate. They are 
therefore, in reality, a mixture in constantly varying propor- 
tions of “ unripe,” “ ripe,” and “ over-ripe ” constituents (the 
first and last being useless for staining purposes), and, in con- 
sequence, their staining power is very inconstant. 
These difficulties are only very imperfectly got over by 
any of the very various methods that have been proposed 
until lately. Now, however, thanks to the important dis- 
coveries of Mayer and Unna, a great step in advance has been 
taken. 
The great point in Mayer’s work is that not hsematoxylin, 
but hseviatein, should be taken in the first instance for making 
the staining solutions. This at once relieves us from the 
tedious and uncertain process of “ ripening” in the old way. 
We have a ripe solution to begin with, and we know that it HJ1MATEIN (HEMATOXYLIN) STAINS. 
163 
must be ripe. A discovery of Unna’s, to be mentioned below, 
affords a means of preventing the “ over-ripening ” brought 
about by excessive oxidation. 
244. Unna’s Half-ripe Constant Stock Solution (Zeit. f. wiss. Mik., 
viii, 4, 1892, p. 483). 
This result may be attained by simply adding a reducing agent to the 
•solution. Various reducing agents are available for this purpose (see the 
original paper); the most convenient method is the simple addition of a little 
sulphur. The following formula is recommended : 
Hmmatoxylin ........ 1 
Alum ......... 10 
Alcohol ......... 100 
Water ......... 200 
Sublimed sulphur ....... 2 
If the sulphur be added to the hsematoxylin solution only when the latter 
has become somewhat strongly blue, i. e. after two or three days’ time, the 
«tage of oxidation attained by the solution will be fixed by the sulphur. The 
■solution in this state may be used for staining. It will not give so energetic 
a stain as the solutions totally ripened with peroxide of hydrogen (see the 
paper quoted), and the stain will be somewhat more diffuse, but washing out 
with acids will not be necessary. If an energetic and more purely nuclear 
stain be desired, the solution should be treated with neutralised peroxide of 
hydrogen, as described in the paper quoted; but this process is cumbrous 
and unnecessary, and I suppress it. Mayer (Mitth. Zool. Stat. Neapel, xii, 
'2,1896, p. 309) finds that the sulphur process does not preserve the solutions 
for long, whilst for some unexplained reason the simple addition of glycerin 
does ; see below, “ Glych-emalum.” 
245. Concerning Hsematein.—The following is taken from the 
two papers of Mayer quoted above, §§ 217, 243. Hsematein 
occurs as a dark green mass, red by transmitted light, which 
may be rubbed up into a violet powder. It is entirely, though 
with difficulty, soluble in distilled water and in alcohol, giving 
a yellowish-brown solution, which remains clear on addition 
of acetic acid. Alkalies dissolve it with a blue-violet tint. 
It is now found in commerce; but Mayer has hitherto only 
been able to procure it in a pure state from Geigy and Co., 
in Bale. But there is also found in commerce an ammonia- 
compound of hsematein—Haemate'in-Ammoniak, also known 
in commerce as Hasmateinum crystallisation; this may be 
obtained in a sufficiently pure state from E. Merck, of 
Darmstadt. 
This is somewhat more easily soluble in both water and 
alcohol than hgematein is, and does quite as well for staining 164 
CHAPTER XLV. 
purposes. The histologist can easily prepare it for himself as 
follows: 
246. Haemateate of Ammonia \(Mayer, ibid.).—Dissolve 1 
grm. of haematoxylin with the aid of heat in 20 c.c. of distilled 
water, filter if necessary, add 1 c.c. of caustic ammonia (of 
0875 sp. gr.), and bring the purple liquid into a capsule of 
such dimensions that its bottom be not covered to a depth of 
more than half a centimetre. Let the liquid evaporate at the 
ordinary temperature and protect it from dust. The dry 
product will consist of haemateate of ammonia, about equal in 
weight to the haematoxylin taken in the first instance. The 
evaporation should not be hastened by heat, as this may give 
rise to the formation of substances that are insoluble in 
alcohol. The preparation should not be touched, until it is 
dry, with any other instruments than such as are made of 
glass, porcelain, or platinum. 
247. Characters of Hsematein Stains (or of Haematoxylin 
Stains).—It is evident, from what has gone before, that all so- 
called haematoxylin stains are really produced by means of 
haematein. The alum-haematein lakes stain in different tones 
of blue or of red, according to the composition of the staining 
solution. Neutral or alkaline solutions give a blue stain ■ 
acid solutions give a red one. In order to get a blue stain in 
preparations that have come out red through the acidity of 
the staining bath, it is a common practice to treat them with 
weak ammonia, in the belief that the blue colour is restored 
by neutralisation of the acid that is the cause of the redness. 
According to Mayer, the ammonia acts not by neutralising 
the acid, but by precipitating the alumina, which carries down 
the hsematein with it (if no alumina were present the colour 
would be purple, not blue). The same result can generally be 
obtained by merely washing out with common tap-water, 
which is usually sufficiently alkaline (Squire has obtained 
the reaction with distilled water, free from even a trace of 
ammonia), and can be obtained with certainty by treatment 
with bicarbonate of soda or acetate of soda or potash. And 
this is the preferable course, as ammonia is certainly a danger- 
ous thing to treat delicate tissues with. Of course this is a 
different question from that of neutralising with an alkali TLEMATEIN (HdSMATOXYLIN) STAINS. 
165 
tissues that have been treated with an acid to correct over- 
staining. Here the neutralisation may be indicated in the 
interest of the preservation of the stain, or of the tissues them- 
selves. 
Squire {Methods, p. 22) finds that sections can be blued in 
a few seconds by treatment with a 1 ; 1000 solution of bicar- 
bonate of soda in distilled water. Mayer holds that acetate 
of potash is the most inoffensive reagent to take; a strength 
of 0’5 to 1 per cent, may be taken. 
According to Watney (see Phil. Trans., 1812, p. 1085 ; Krause, Intern. 
Zeit.f. Anat. u. Hist., i, p. 154; and M. Flesch, Zeit.f. iviss. Mik., 1885, 
p. 358, from whom I quote) the colour is an intense blue if the solution has 
been made with freshly prepared alum, whilst a red tone is obtained if the 
solution has been made with old alum. The reason of this is that alum 
that has been long kept almost always contains free acid. The red solutions 
are said to exhibit a great affinity for connective tissue, and for the granules 
of “plasma-cells;” whilst the blue solutions show a special affinity for 
mucin and chromatin. But see on this point the remarks of Mayer quoted 
under “ Mucin.” 
It has been discovered by Langhans (see Max Flesch, loc. cit.) that it is 
possible to obtain these two elective reactions with one and the same solution. 
All that is necessary is to stain with the solution of Delafield, mount the 
preparations in balsam, and expose them for some time to the light. The 
reaction is not obtained with glycerin mounts. 
None of the purely aqueous solutions are perfectly stable; 
only one or two are fairly so. They can, however, be made to 
keep for a sufficient time, many months, by the addition of 
glycerin (Ehrlich) ; but the addition of the glycerin, as is 
well known, slows the stain greatly, and makes it somewhat 
less precise. (See below, Glychsemalum). 
It has been explained above that the old hsematoxylin 
solutions when freshly prepared stain badly and diffusely; 
they ought either to be “ ripened” by Unna’s method (§ 243) 
or to be allowed to “ ripen ” spontaneously before use. This 
takes, according to the nature of the solution, a few hours, or 
days, or months. In my opinion there is no legitimate excuse 
for not making up stains with haematein, and the old formulas 
for haematoxylin solutions ought to be discarded. 
All of the solutions, when in good staining order, have a 
great tendency to over-stain. Over-stains may be corrected 
by washing out with weak acids (e. g. OT to O’2 or even 0*5 
per cent, of hydrochloric acid), but this is not favorable to 166 
CHAPTER XIY. 
the permanence of the stain. If acids be used, it is well ta 
re-neutralise afterwards with ammonia or bicarbonate of 
soda (01 per cent.). 
Bicarbonate of soda may be used for neutralisation with 
alcohol as the vehicle. It should be dissolved by the aid of 
heat in 70 per cent, alcohol (very little will dissolve), and the 
solution filtered after cooling. For neutralising tissues, three 
to five drops of the solution are added to a watch-glass full of 
70 per cent, alcohol in which the tissues are soaked (von 
Wistinghausen, Mittli. Zool. Stat. Neapel, x, 1891, p. 41; Zeit. 
f. wiss. Mik., x, 4, 1893, p. 480). 
The best plan of all is to avoid over-staining by staining 
very carefully in extremely dilute solutions. Kawitz (Anat. 
Anz., xi, 10, 1895, p. 301) gives the proportion of one to three 
drops of a strong alum-hsematein solution to 25 to 50 c.c. of 
distilled water. Sections of picric or sublimate material may 
remain in this without over-staining for twenty-four to forty- 
eight hours. Chrom-osmium material is of course much less 
likely to over-stain, but even in this case the best results are 
obtained with very dilute solutions. It should be noted that 
the purest chromatin stains are obtained by staining for a 
short time (sublimate sections half an hour, say) in solutions 
of medium strength, such as hgemalum diluted ten to twenty- 
fold with water. The stain obtained either with very strong 
solutions, or with the slow stain of the dilute solutions, is at 
the same time a plasma-stain, which of course may or may not 
be desired. Chrom-osmium material will not yield a pure 
chromatin stain unless it is very fresh; it is consequently 
next to impossible to obtain the reaction with paraffin sections 
of such material; they constantly give a plasma-stain in 
addition to the chromatin stain, which is not the case with 
sublimate material. Chrom-osmium material, if not quite 
fresh, will require at least as many hours’ staining as sublimate 
material does minutes. 
The stain is fairly permanent in balsam, but is sure to fade 
a little, and may fade a great deal. If acids have been used 
after staining, great care should be taken to wash them out 
thoroughly before mounting. In aqueous media the stain 
cannot be relied on to keep (this refers to the old solutions : 
Mayer finds that his hsematein preparations have kept well 
both in aqueous media and, with certain precautions, in HJ5MATKIN (HiEMATOXYLTN) STAINS. 
167 
balsam). Turpentine-balsam should not be used (Mayer, 
in litt.), nor, I find, should alcohol-balsam. 
248. General Remarks.—We have the coal-tar colours for 
staining sections, and we have carmine and cochineal for 
staining in the mass. What, then, do we want heematein 
for ? The answer is that we sometimes want it for staining, 
either sections or in the mass, on account of the faculty it has 
of staining tissues that have been treated with chromic and 
osmic mixtures. This it does in general better than any 
carmine or cochineal. We want it also for some special pur- 
poses, such as staining the Nebenkern and achromatic figures 
of nuclei, and for nerve reseai'ches and other special histo- 
logical objects. It is also a more powerful stain than carmine; 
and according to the mode of employment affords either a 
chromatin stain or a sometimes valuable plasma-stain. The 
chief haematein plasma-stains are found not amongst the 
alum-haematein lakes, but amongst the other lakes, iron or 
chrome lakes, &c. The alum lakes will be treated of first; 
then the other lakes, partly in this chapter and partly in 
Part II (Neurological Methods, Methods of Weigert, Pal, &c.). 
In view of the very great advantages offered by the mode 
of preparation worked out by Mayer I have no hesitation in 
saying that the old methods for preparing alumina-haematein 
lakes ought to be entirely discarded, and Mayer’s formulae 
only, or at least only such formulae as conform to the 
principles laid down by him, used instead. For not only are 
his modes of preparation the most scientific, but, according to 
my experience, his solutions are found in practice to be greatly 
superior to the old ones. 
a. Alumina-hsematein Lakes. 
249. Mayer’s Haemalum (Alum-haematein Solution, Haema- 
laun; Mayer, Mitth. a. d. Zool. Stat. zu Neapel, x, 1, 1891, 
p. 172; see also Zeit. f. iviss. Mik., viii, 3, 1891, p. 338, a 
very exact report, but somewhat short considering the im- 
portance of Mayer’s paper).—One grm. of the colouring matter 
(either hasmatein or the ammonia salt, §§ 245, 246) dissolved 
with heat in 50 c.c. of 90 per cent, alcohol, and added to a 
solution of 50 gr. of alum in a litre of distilled water. Allow 168 
CHAPTER XIV. 
the mixtui’e to cool and settle, filter if necessary, and add a 
crystal of thymol to preserve from mould. 
A dark liquid of about the tint of Grenadier's borax-car- 
mine. It answers to Bohmer’s hasmatoxylin,* stains at least 
as well, either at first, for it is ripe from the beginning, or later. 
It may be used either in the concentrated form or diluted. 
Concentrated, it stains almost instantaneously (sections have 
been stained by merely pouring the liquid over them). 
Diluted twentyfold with distilled water it will still stain 
through the tentacles of a Tubularia in an hour. (Spring 
water or tap water containing lime must not be used for 
diluting; perhaps weak solution of alum in distilled water is 
the best means of all.) After staining, sections may be 
washed out either with distilled or common water. The 
solution is admirable for staining in bulk. Large objects will, 
however, require twenty-four hours’ staining, and should be 
washed out for the same time (this should be done with 1 per 
cent, alum solution if a sharp nuclear stain be desired). The 
solution unfortunately does not keep perfectly, but becomes 
weak with age (see § 251). Mayer’s preparations have kept 
well in glycerin (care being taken not to have it acid), also in 
balsam. It is to be noted that if oil of bergamot be used for 
clearing, it must be thoroughly removed by means of oil of 
turpentine before mounting; and that oil of cloves is danger- 
ous. It is best (Mayer, in litt.) to use only xylol, benzol, or 
chloroform, and to mount in xylol-balsam or chloroform- 
balsam. 
Heemalum may be mixed with alum-carmine, Saurefuchsin, 
or the like, to make a double-staining mixture; but it seems 
preferable to use the solutions in succession. 
250. Mayer's Acid Hsemalum {ibid., p. 174, note).—This 
is heeraalum with 2 per cent, glacial acetic acid (or 4 per 
cent, common acetic acid). To be used as the last, wash- 
ing out with ordinary water in order to obtain a blue- 
violet tint of stain. It has the advantage of being a perhaps 
even more precise nuclear stain than the last formula; and 
further, the solution does not precipitate through decompo- 
* As with Bohmer’s formula, it is not necessary to conform exactly to 
the proportions given, and a rough and ready hsemalum solution may he at 
any time extemporised by adding a few drops of alcoholic solution of 
haematein to an alum solution of any desired strength. HiEMATEIN (HAEMATOXYLIN) STAINS. 
169 
sition caused by the ammonia of the air and the alkali of the 
glass of bottles, which the simple haemalum solution does to a 
certain extent (though this does not affect its staining power). 
251. Mayer’s Glychaemalum (Miffli. Zool. Slat. Neapel, xii, 
2, 1896, p. 310).—Mayer has found that haemalum does not 
keep perfectly (I have also found that my solution has not 
kept in a sufficiently satisfactory manner). By adding 
glycerin it may be made to keep perfectly clear for over a 
year; but as this slows the stain, more haematein should be 
added. The following formula is recommended :—Haematein 
0-4 grm. (to be rubbed up in a few drops of glycerin in a 
mortar till it dissolves); alum, 5 grms.; glycerin, 30; dis- 
tilled water, 70. The stain is not quite so purely nuclear as 
that of the simple haemalum,—ground substance of cartilage, 
for instance, being stained. 
252. Hansen’s Solution (Zool. Anz., 1895, p. 158).—Hansen 
having found that commercial haematein is not of constant 
quality, sought for a convenient means of oxidising haema- 
toxylin into haematein,and recommends the following process. 
One grm. of haematoxylin crystals is dissolved in 10 
grms. of absolute alcohol. Twenty grammes of potash alum 
are dissolved with the aid of heat in 200 grms. of distilled 
watei*, and the solution filtered after cooling. After twenty- 
four hours the two solutions are mixed. 
Three c.c. of concentrated aqueous solution of perman- 
ganate of potash are put into a porcelain capsule (one part of 
permanganate dissolves in 10 of water at 15° C.). The 
haematoxylin solution is added, and the whole is gradually 
heated up to boiling-point with constant stirring. The solu- 
tion turns to a dark reddish violet. The boiling is continued 
for half a minute or a minute. The solution is then cooled, 
which is best done by floating the capsule on to cold water. 
It may be filtered after cooling if desired, but this is seldom 
necessary. The solution is now ripe and ready for use. 
The object of the permaganate in this process is of course 
to effect the instantaneous oxidation of the haematoxylin. 
Hansen fancies that it has another point of utility : that by 
destroying all germs that may be present in the ingredients 
it should make the solution keep better than the usual solu- 170 
CHAPTER XIV. 
tions. As a matter of fact it does not; mine formed a 
pellicle and strong precipitate in a few days, and tlie same 
was found at the Naples station (Mayer, in litt.). 
With this exception I find the solution excellent; the stain 
is most beautiful, and cannot be distinguished from that of 
haem alum. 
Mayer (Mitth. Zool. Stcit. Neapel, xii, 2, 1896, p. 309) 
thinks the presence of the manganese in the solution favours 
the formation of the precipitate. He suggests that the 
oxidation might be brought about by taking an alcoholic 
solution of hsematoxylin, leaving it for a short time in contact 
with the permanganate, and then adding it to 5 per cent, 
alum solution. The alcohol would then not bring over 
manganese into the stain. 
253. Bohmer’s Hsematoxylin (Arch. f. mik. Anat., iv, 1868, p. 345; 
Aerzt. Intelligenzbl., Baiern, 1865, p. 382).—Make (a) a solution of 
luematox. cryst. 1 part, alcohol (absolute) 12 parts, and (b) alum 1 part, 
water 240. For staining, add two or three drops of a to a watcli-glassful 
of B. 
The staining solution ought not to be made up at the moment of using, 
but should be made up beforehand and allowed to ripen for some days 
(Mayer says at least two months). The alcoholic solution of hsematoxylin 
may be kept in stock; it becomes brown, but does not lose its properties. 
Washing out may be done with a 0'5 per cent, solution of alum in water, 
or with acids if desired. 
I consider this to be of merely historical interest. 
254. Delafield’s Hsematoxylin (Zeit.f. wiss. Mile., ii, 1885, p. 288).— 
The history of this formula is as follows :—It had long been in use in the 
Institute of Pathology at Heidelberg, when it was communicated by Pfitzner 
to Flemming, who published it and particularly recommended it in his 
Zellstz., &c., p. 388, 1882. Flemming then attributed the formula to 
Grenadier, and in consequence the stain went for years by the name of 
“ Grenadier's hsematoxylin.” Later on Flemming discovered that this 
attribution was erroneous, and attributed the formula to Prudden; and in 
cconsequence it was thenceforth known for some time as “ Prudden’s htema- 
toxylin.” In 1885 matters were set right by Prudden’s explaining that the 
stain was the invention of Delafield, and publishing the correct formula here 
quoted (in the formula as published by Flemming the proportions are some- 
what different). 
To 400 c.c. of saturated solution of ammonia-alum* add 4 grms. of 
luematox. cryst. dissolved in 25 c.c. of strong alcohol. Leave it exposed to 
* Ammonia-alum dissolves in about 11 parts of water. hematein (hematoxylin) stains. 
171 
the light and air in an unstoppered bottle for three or four days. Filter,, 
and add 100 c.c. of glycerin and 100 c.c. of methylio alcohol (CH40). Allow 
the solution to stand until the colour is sufficiently dark, then filter and 
keep in a tightly stoppered bottle. 
This solution keeps well,—it may be said to keep for years. It is well to 
allow it to ripen for at least two months before using it. 
For staining, enough of the solution should be added to pure water to 
make a very dilute stain ; and even then care should be taken not to leave 
objects too long in the fluid. It is an extremely powerful stain. 
Butschli (Unters. iib. mikroscopisclie Sehdume u. das Protoplasma, 
&c., 1892; Zeit. f. wiss. Mik., ix, 2, 1892, p. 197) recommends, under the 
name of “acid hsematoxylin,” solution of Delafield very strongly diluted, 
and with enough acetic acid added to it to give it a decidedly red tint. 
This gives a sharper and more differentiated nuclear stain than the usual 
solution. 
I used Delafield’s solution a good deal a few years ago, in view of its 
faculty for affording a plasma-stain at the same time as a nuclear stain. It 
sometimes gives good images of spindles, &c., but the stain is very opaque. 
There are several better plasma-stains known now, and I should suppress it 
were it not that I see that it is still used by some good workers, Flemming 
amongst the number. It may perhaps still be useful for staining tissues 
that have been overcharged with metallic salts during fixation or hardening, 
and are therefore unusually difficult to stain. 
255. Ehrlich’s Acid Hsematoxylin (Zeit. f. wiss. Mik., 1886, 
p. 150).— 
Water . . . . . . . 100 c.c. 
Absolute alcohol ..... 100 „ 
Glycerin ....... 100 „ 
Glacial acetic acid . . . . 10 „ 
Hsematoxylin ...... 2 grms. 
Alum in excess. 
Let the mixture ripen in the light (with occasional admission of air) 
until it acquires a dark red colour. It will then keep, with constant 
staining power, for years, if kept in a well-stoppered bottle. Sections are 
stained in a few minutes. It is stated that the solution is also very 
appropriate for staining in bulk, as over-staining does not occur ; but this I 
take leave to doubt. 
Of all the old formulae I have tried, this is the one that has given me the 
sharpest chromatin stain. 
Mann (Zeit.j. wiss. Mik., xi, 4, 1895, p. 487) makes up this stain with 
hsematein instead of hsematoxylin. 
250. Ammoniated Hsematoxylin (Squibe, Methods and Formulae, 
&c., p. 24).—“Hsematoxylin, 2 grins.; ammonium carbonate, 0'4 grm.; 
proof spirit, 40 c.c. Dissolve the ammonium carbonate and the hsematoxylin 
in the proof spirit, and expose to the air in a shallow dish for twenty-four 
hours; make up the volume to 40 c.c. with proof spirit (warming if 172 
CHAPTER XIV. 
necessary to dissolve any separated crystals), and add ammonia alum, 2 grms., 
dissolved in distilled water, 80 e.c.; glycerin, 100 c.c.; rectified spirit, 
80 c.c. ; glacial acetic acid, 10 c.c. This solution requires no ‘ ripening,’ and 
is ready to be diluted for use (1—10).” 
I apprehend that the statement that the solution requires no ripening is 
erroneous. I find the solution .precipitates on dilution, and although it 
gives a fine stain, is therefore disagreeable to work with. It has this good 
quality, that it keeps extremely well. 
257. Mayer’s Hsemacalcium (.Mitth. Zool. Stat. Neapel, x, 1, 
1891, p. 182).—Hsematein (or haemateate of ammonia, §§ 245, 
246), 1 grin.; chloride of aluminium, 1 grm.; chloride of 
calcium, 50 grms.; glacial acetic acid. 10 c.c. (or common 
acetic acid, 20 c.c.) ; 70 per cent, alcohol, 600 c.c. Rub up 
finely together the first two ingredients, add the acid and 
alcohol, dissolve either cold or with heat; lastly add the 
chloride of calcium. 
A reddish-violet liquid, imagined as a substitute for Klein- 
enberg’s solution, in view of the many disadvantages of the 
latter (see below). To be used in the main as Kleinenberg’s. 
If the objects stain in too red a tone they should be treated 
with a solution (of about 2 per cent.) of chloride of aluminium 
in alcohol, or with a 0'5 to 1 per cent, solution of acetate of 
soda or potash in absolute alcohol, or with bicarbonate of soda 
(see above, § 247). 
The solution is not perfectly stable, but in course of time 
{Mittli. a. d. Zool. Stat. Neapel, x, 3, 1892, p. 499) turns blue 
and precipitates. To avoid this the mixture should be made 
up in two separate bottles, each containing half of the alcohol 
and of the acid, and one containing besides all the calcium 
chloride, the other all the hsematein and all the aluminium 
chloride, equal quantities being taken from each when re- 
quired for staining. 
With certain objects this solution does not penetrate well, 
the stain being confined to their superficial parts. This may 
be remedied by acidifying the solution, or, which is better, 
by leaving the objects for some time before staining in acid 
alcohol. Anyway objects ought not to have an alkaline re- 
action. If these precautions be taken, it will not be necessary 
to use acid for washing out. For some objects also (e. g. 
Hydroida) the penetrating effect is enhanced by diluting the 
solution with one third volume of glycerin, or by increasing the HAEMATEIN (HAEMATOXYLIN) STAINS. 
173 
proportion of aluminium chloride up to about eight times that 
of the hmmatein. 
The solution is not recommended as giving as good results 
as haemalum,—as a stain it is distinctly inferior; and Mayer 
is of opinion that no alcoholic haematein solution can be made 
to give as precise a stain as the aqueous solutions, for he 
holds that the watery nature of the menstruum is of itself of 
a beneficial effect so far as regards the quality of the stain. 
He recommends it merely as a substitute for Kleinenberg’s 
(in cases in which an alcoholic limmatein stain seems indi- 
cated), as being convenient, easy to prepare, and constant in 
its effects, none of which qualities belong to Kleinenberg’s 
formula. 
258. Kleinenberg’s Haematoxylin (Quart. Journ. Micr. Sci., lxxiv, 
1879, p. 208).—Highly irrational and veiy inconstant in its composition and 
its effects. For a detailed account of the reasons for which I hold that this 
solution should be discarded, see last edition ; also the elaborate criticism of 
Mayer (Mitth. a. cl. Zool. Stat. zu Neapel, 10, 1, 1891, p. 174), and that of 
Squire, in his Methods and Formulae, p. 25. 
The alternative formulae of Squire (l. c.) and of yon Wistinghausen 
(Mitth. Zool. Stat. Neapel, x, 1891, p. 41; Zeit. f. wiss. Mik., x, 4, 1893, 
p. 479) do not appear to get over the difficulty of realising the production 
of the required proportion of aluminium chloride by the double decom- 
position of alum and calcium chloride. 
259. Other Alumina-haematein Lakes.—Ranvier’s Haematoxylin 
(Comptes rend. Ac. Sc., 2 sera., t. xev, p. 1375). Contejean (Bicll. Soc. 
Philomath. Paris, 8, 3, 1891, p. 117). (Cf. Mayer, in Mitth. a. cl. Zool. 
Stcit. zu Neapel, 10, 3, 1892, p. 500.) Critically examined, these methods 
appear to be only roundabout methods of getting a solution of haematein in 
alum. Eenaut’s Glycerin Haematoxylin (“ Glyce'rine Hematoxy- 
lique,” Arch. de. Physiol., 1881, p. 640). Iodine Haematoxylin 
(Cuccati, Zeit. f. wiss. Mik., v, 1, 1888, p. 55). Iodine Haematoxylin 
(Sanfelice, Journ. de Microgr. xiii, 1889, p. 335 ; Journ. Roy. Mic. Soc., 
1889, p. 837). Mayer (Mitth. Zool. Stat. Neapel, 10, 1, 1891, pp. 178,182) 
remarks on this that it is essentially a Bohmer’s solution with somewhat, 
more alcohol. Gage’s chloral solution (Proc. Amer. Mic. Soc., 1893, p. 125 
Journ. Boy. Mic. Soc., 1893, p. 564) ; the addition of chloral is supposed to 
make the solution keep, but does not (Mater, in litt.). Hacke’s, Compt. 
rend. Soc. Biol. Paris, x, 1,1894, pp. 253—369 (complicated and superfluous, 
Mayer). And for some other superfluous formulae see previous editions. 
260. R. Heidenhain’s Haematoxylin (Arch. f. mik. Anat., 1884, 
p. 468, and 1886, p. 383).—Stain for twelve to twenty-four hours in a | 
b. Other Hseviatein Lakes. 174 
CHAPTER XTV. 
per cent, solution of haematoxylin in pure water (distilled water only should 
he used). Soak the objects for the same length, of time in a 0'5 per cent, 
•solution of neutral chromate of potash, which should be changed, if necessary, 
•several times. Wash out the excess of chromate with water, and treat 
further as desired. 
The above is a slightly modified form of the original process, in which 
•staining was done in a stronger haematoxylin solution (0‘5 to 1 per cent.), and 
bichromate was used for washing out instead of neutral chromate. The more 
•recent process gives a sharper chromatin stain. 
The stain succeeds best with alcohol or picric acid objects, but it will 
succeed with chromic objects if they have been very well washed, or with 
material fixed in Flemming’s mixture. 
Objects that have been fixed in corrosive sublimate ought to be very care- 
fully washed out with water (many hours in running water), as neutral 
haematoxylin forms a black precipitate with the excess of sublimate that 
remains after washing out with alcohol (see Toeniee, in Arch. f. mik. Anat., 
1886, p. 181). To avoid this precipitate it is well to stain in the dark. 
The stain is black to grey (haematoxylin forming with chromic salts a 
‘black compound). It is a sharp stain, remarkably rich in detail. 
The process is adapted to staining in hulk. Perhaps its greatest advan- 
tage lies in the fact that you can decolour the objects to any extent by pro- 
longing the washing in the chromate. 
The method may be varied by washing out after staining with alum 
•solution (1 per cent.) instead of a chromate. In this case the stain will 
be blue. 
This is not, so far as I can see, as fine a stain as M. Heidenhain’s iron- 
hsematoxylin, but it has the advantage of being applicable to staining in bulk, 
which the latter is not. It is a plasma-stain as well as a chromatin stain. 
261. Apathy’s Modification of Heidenhain’s Process (Zeit. f. wiss. 
Mik., v, 1,1888, p. 47).—This is an alcoholic method. Stain in a 1 per cent, 
solution of hsematoxylin in 70 or 80 per cent, alcohol. Wash out (for 
“ thin ” sections, i. e. sections of 10 to 15 g, half the time of staining—for 
thicker ” sections of 25 to 40 /t twice the time of staining) in 1 per cent, 
solution of bichromate of potash in 70 to 80 per cent, alcohol. 
The bichromate solution is conveniently prepared by mixing one part of a 
5 per cent, aqueous solution with about four parts of 80 to 90 per cent, 
alcohol. The mixture should he made immediately before using, and should 
he kept from the light (light precipitates it) during the process of decolora- 
tion, and should also he changed for fresh several times during the process. 
After the differentiation of the colour has been accomplished, the objects 
should be thoroughly washed (still in the dark) in several changes of 70 per 
cent, alcohol. 
Preparations made in this manner are said to he more transparent and 
better preserved than those made by Heidenhain’s process. 
For staining celloidin series of sections, Apathy also (Zeit. f. wiss. Mik., 
vi, 2, 1889, p. 170) recommends the following procedure :—Stain in the 
haematoxj'lin solution as above for ten minutes; then remove the excess of HiEMATEIN (HEMATOXYLIN) STAINS. 
175 
haematoxylin fluid from the sections by means of blotting-paper, and bring 
the series for five to ten minutes into 70 per cent, alcohol containing only a 
few drops of a strong (5 per cent.) solution of bichromate. This must be 
done in the dark. If the haematoxylin be not removed with blotting-paper 
as described, the celloidin will take the stain. The sections should appear 
steel-blue to steel-grey. 
262. Iron Haematoxylin (M. Heidenhain, “Uber Kern nnd 
Protoplasma,” in Festschr. Herrn Geheimr. A. v. Kolliker, fyc., 
yewidm., 1892, p. 118 ; Zeit.f. wiss.Mik., ix, 2, 1892, p. 204).— 
This process is based on an old method, now given up, of Benda 
(Verh. Phys. Ges. Berlin, 1885-6, Nos. 12, 13, 14; Arch. f. 
Anat. u. Phys., 1886, p. 562). Benda mordanted as here in 
ferric alum, but differentiated in chromic acid. Heidenhain’s 
process is as follows :—Sections are treated from half an hour 
to at most two or three hours with a 1*5 to 4 percent, solution 
of ferric alum. By this is meant the double salt of the sesqui- 
oxide of iron (NH4)2Fe3(S04)4, in clear violet crystals; the 
double salt of the protoxide, or salt of Mohr in green crystals, 
will not serve. If the crystals have become yellow and 
opaque, they have gone bad, and should be rejected. They 
ought to be kept in a stoppered bottle, the solution should be 
made in the cold {Arch. f. mik. Anat., xliii, 3, 1894, pp. 431, 
435). The sections are then washed with water and stained 
for half an hour in an aqueous solution (of about 05 per 
oent.) of hsematoxylin. (Hasmatoxylin is stated by Heiden- 
hain to give better results than haematein.) They are then 
rinsed with water, and again treated with the iron solution, 
which slowly washes out the stain. The progress of the 
differentiation ought to be controlled under the microscope. 
The sections should to this end be removed from time to time 
from the alum solution, and put into tap water whilst they 
are being examined. This is favorable to the stain. I find 
my sections require from twenty minutes to an hour or more 
for satisfactory differentiation. As soon as a satisfactory 
differentiation has been obtained, the preparations are washed 
for at least a quarter of an hour in running water, but 
not more than an hour, and mounted. The results differ, in 
Heidenhain’s view, according to the duration of the treatment 
with the iron and the stain. If the baths have been of short 
duration, viz. not more than half an hour in the iron and as 
much in the stain, blue preparations will be obtained. These 176 
CHAPTER XIV. 
show a very intense and highly differentiated stain of all 
nuclear structures, achromatic as well as chromatic, cytoplasmic 
structures being pale. If the baths in the iron and in the stain 
have been prolonged (twelve to eighteen hours), and the sub- 
sequent differentiation in the second iron bath also duly pro- 
longed, black preparations will result. These show chromo- 
somes stained, central and polar bodies stained intensely black, 
cytoplasm sometimes colourless, sometimes grey, in which 
case achromatic spindle-fibres and cell-plates are stained, 
connective-tissue fibres black, red blood-corpuscles black, 
micro-organisms sharply stained, striated muscle very finely 
shown. 
Dr. Mayer, writing to me, doubts that the blue or black 
tone is conditioned by the duration of the mordanting and 
staining baths; and my observations confirm this view. 
I most highly recommend this stain, which is one of the 
very finest I am acquainted with. It may be used either with 
sublimate or alcohol material, or after liquid of Flemming. 
The process is easy to manage. It is only appli- 
cable to sections, which should be thin, not more than 8 /u in 
thickness. The preparations are said to be perfectly perma- 
nent. It has been said that this process frequently gives 
rise to amorphous precipitates in the tissues. I find that it 
does sometimes, but not to any very injurious extent. 
A modification of this process, consisting in a foregoing 
stain with Bordeaux, which is said to enhance its power of 
demonstrating centrosomes, will be given under “ Cytological 
Methods.” 
263. Benda’s later Sulphate of Iron Hsematoxylin (Verb,. d. 
Anat. Ges., vii, 1, 1893, p. 161 ; Zeit.f. wiss. Mik., xi, 1,1894, 
p. 69 ; Rawitz, Leitfaden, 1895, p. 73).—This is a later form 
of the old method of Benda mentioned in the last section. 
Benda found that the ferric alum of that process gave rise to 
amorphous precipitates in the tissues. To avoid them he now 
proceeds as follows :—Tissues fixed in any way may be em- 
ployed. Sections are mordanted for twenty-four hours in 
Liquor ferri sulfurici oxidati, P. Gr., diluted with one or two 
volumes of water (this preparation consists of sulphate of 
iron 80 parts, water 40, sulphuric acid 15, and nitric acid 18, 
and contains 10 per cent, of Fe). They are then well washed, HiEMATEIN (HEMATOXYLIN) STAINS. 
177 
first with distilled water, then with tap water, and are brought 
into a 1 per cent, solution of haematoxylin in water, in which 
they remain till they have become thoroughly black. They are 
then washed and differentiated. The differentiation may be 
done either in 30 per cent, acetic acid, in which case the pro- 
gress of the decoloration must be watched; or in a weaker acid, 
which will not require watching ; or in the sulphate solution 
strongly diluted with water. 
This process is said to be applicable to all sorts of organs, 
and to give in particular excellent images of axis-cylinders, 
and of the achromatic figure of cell-division. For this latter 
object it may be useful to add a second stain with Saure- 
fuchsin. Chromatin, centrosomes and spindle relics (Zwischen- 
harper) should then appear black; linin fibrils and spindle- 
fibres red. I find it to be a powerful and sharp stain, but the 
strong iron solution is a disagreeable reagent to handle. If 
the iron solution be taken for the differentiation, it should be 
taken extremely diluted (of a very jpale straw-colour), and the 
progress of the differentiation watched; as if it be only diluted 
about tenfold, for instance, the decoloration is extremely 
rapid. 
264. Iron Hsematoxylin (Butschli, Unters. iiher mikrosleopische 
Schiiume u. clas Protoplasma, &c., 1892; Zeit.f. wiss. Mile., ix, 2, 1892, 
p. 197).—Sections treated with a weak brown aqueous solution of ferric 
acetate, washed with water, and stained in 0'5 per cent, aqueous solution of 
hsematoxylin. This treatment gives a blue-black or brown-black stain of 
extraordinary intensity. The process was used by Butschli for staining 
sections, 1 y. in thickness, of Protozoa. It does not appear to be of 
general applicability. 
265. Benda’s Copper Hsematoxylin {Arch. f. mile. Anat., xxx, 
p. 49).—Fix in Flemming. Make paraffin sections. Stain them as follows :— 
Put them for twenty-four hours into concentrated solution of neutral acetate 
of copper, kept at a temperature of about 40° C., or for forty-eight hours at the 
normal temperature. Wash them out well with water, and stain for a few 
minutes, or to a dark grey or black tint, in 1 per cent, aqueous hsematoxylin 
solution. Decolourise in dilute hydrochloric acid (0'2 per cent.), until the 
sections appear of a fairly light yellow. You decolourise more or less 
according as you wish to have a more or less plasmatic stain or a purely 
nuclear one. The acid must now he neutralised, which may be done by 
putting the sections back into the copper solution until they turn of a 
bluish grey. Then wash, dehydrate, and mount in balsam. See also the 
remarks of Pieesol, in Amer. Mon. Mic. Journ., 1887, p. 155; or in 
Journ. Boy. Mic. Soc., 1888, p. 158; or Zeit. f. wiss. Mile., v, 4, 1888, 
p. 499. This process gives an energetic differentiation of karyokinetic 178 
CHAPTER XIV. 
figures, and is said to be particularly applicable to the study of spermato- 
genesis. It seems to me to be not very easy to manage, as the proper degree 
of differentiation ought to be hit off to a nicety, which is rather a difficult 
matter. 
266. Mallory’s Phospho-molybdic Acid Haematoxylin (Anat. 
Anzeig., 1891, p. 375 ; see also Zeit.f. wiss. Mile., viii, 3, 1891, p. 341). 
267. Minot’s Haematoxylin Methods (Zeit. f. wiss. MiTc., iii, 2, 1886> 
p. 177). CHAPTER XY. 
ON STAINING WITH COAL-TAR COLOURS. 
268. Basic, Acid and Neutral Coal-tar Colours.—Histologists 
generally conceive of the coal-tar colours as divided into 
three groups, according to a principle of classification founded 
on chemical considerations, and introduced into histological 
literature by Ehrlich [Verb. d. Berl. Phys. Ges., May 16th, 
1879 ; in Reichert and Du-Bois Reymond’s Arch. f. Anat. u. 
Phys., phys. Abth., 1879, p. 571). These three groups are 
those of the basic colours, the acid colours, and the neutral 
colours. By a “ basic ” colour is meant a compound in which 
the colouring principle or molecular group to which the com- 
pound owes its colouring properties exists as or chemically 
plays the part of a base combined with a colourless acid. 
For instance, fuchsin or magenta is a basic colour. It is the 
hydrochloride of rosanilin, and its colouring properties are 
due to the rosanilin which exists as a base in the compound, 
and not to the hydrochloric acid of the compound. By an 
“ acid ” colour is meant a compound in which the colouring 
principle exists as or plays the part of an acid. The dye 
known as acid fuchsin or acid magenta (Saurefuchsin) is an 
acid colour. It is the soda salt of di- or tri-sulphoconjugated 
rosanilin, that is of rosanilin di- or tri-sulplionic acid, and its 
colouring properties are due to the rosanilin which exists as 
an acid in the compound, and not to the soda. Or to take a 
simpler case, picrate of ammonia is an acid colour in Ehrlich’s 
sense, and its colouring properties are evidently due to the 
picric acid in it, and not to the ammonia. The neutral 
colouring matters form a very small group ; the only example 
that I can find mentioned in Benedjkt and Knecht’s 
Chemistry of the Coal-tar Colours being artificial indigo, 
obtained from propiolic acid. Ehrlich holds that neutral 
colours are, however, frequently formed by the mixture of the 180 
CHAPTER XV. 
solutions of an acid colour and a basic colour. They are 
generally insoluble in pure water, and hence precipitate when 
the mixture is made, but may be got to re-dissolve by adding 
an excess of the acid colour. 
Now, according to Ehrlich, the basic colours are in general 
chromatin stains,—that is, they have a special affinity for the 
element of nuclei known as chromatin, so that they are 
mostly sharp nuclear stains. The acid colours, on the other 
hand, are, according to him, in general plasma stains,—that is, 
they have a special affinity for cytoplasm and intercellular 
substances. And lastly, the neutral colours exhibit special 
affinities for certain cell-contents; amongst them are found 
some important granule stains. 
I would not for a moment impugn the accuracy or thorough- 
ness of the observations on which this generalisation is based, 
or its utility from a theoretical point of view; but I must say 
that as a general histological classification of tar colours it 
requires to be supplemented by a good deal of explanation 
and restriction. In practical histology we have to take 
account not only of the affinities of a dye for this or that 
cellular element, as they are manifested in progressive stain- 
ing under narrowly limited conditions: we have also to take 
account of the resistance of the stain to the liquids employed 
for washing, for dehydration, for clearing; in short, we have 
to take into account the way in which the dye behaves when 
employed as a regressive stain. This is of peculiar importance 
in the case of the coal-tar colours, for their principal use is 
for the regressive staining of sections destined to be dehy- 
drated by alcohol and mounted in balsam. Now Ehrlich’s 
experiments take no account of these conditions. He worked 
with “ cover-glass preparations ” of isolated cells, such as 
blood and lymph cells, and was thus able to avoid the pro- 
longed washing necessary for most sections, and to suppress 
altogether the dehydration by alcohol, his cover-glass prepara- 
tions being simply dried, after staining, in a stove. In con- 
sequence, his chemical categories of basic colours and acid 
colours fail to correspond precisely to the technical categories 
of chromatin stains and plasma stains. 
For instance, orange is an acid colour; but used as a 
regressive stain I find it will give a very sharp stain of 
chromatin and plasmatic nucleoli: it cannot, therefore, be ON STAINING WITH COAL-TAR COLOURS. 
181 
classed as a mere plasma stain, though it is also a very good 
plasma stain. Saurefuchsin is also an acid colour. It behaves 
in general as a decided plasma stain. But used as a re- 
gressive stain it sometimes, under conditions which I am not 
able to specify, gives a very vigorous stain of chromatin. 
Safranin is a basic colour, but by the use of appropriate 
mordants it can be made to behave as a plasma stain. 
Methylen blue is a basic colour. But, as is well known, when 
employed according to the method worked out by Ehrlich 
for the so-called intra-vitam staining of nerves, it affords a 
stain that is essentially plasmatic, such staining of nuclei as 
may occur in this process being an accidental epiphenomenon. 
Nigrosin is, according to Ehrlich, an acid colour, and should 
therefore be essentially a plasma stain. Yet I find that, used 
as a regressive stain in the same way as safranin, it gives a 
vigorous chromatin stain, cytoplasm being only faintly 
coloured. Bordeaux is an acid colour, but it stains chromatin 
as well as cytoplasm. 
It would seem, therefore, that Ehrlich’s generalisation, how- 
ever important it may be from a theoretical point of view, 
does not hold good as a statement of the behaviour of tar 
colours when employed for staining sections in the usual 
way. It is roughly true that the basic colours are in general 
chromatin stains, and the acid colours in general plasma 
stains; but the rule is subject to many exceptions. 
269. Progressive and Regressive Coal-tar Stains.—Very few 
anilins give a precise nuclear or chromatin stain by the 'pro- 
gressive or direct method (§ 211). Two of them—methyl 
green and Bismarck brown—are pre-eminently chromatin 
stains. Many of the others—for instance, safranin, gentian, 
and especially dahlia—may be made to give a nuclear stain 
with fresh tissues by combining them with acetic acid; but in 
ninety-nine cases out of a hundred are not so suitable for this 
kind of work as the two colours first named, which practically 
form a class apart. 
Again, very few anilins give a pure plasmatic stain (one 
leaving nuclei unaffected). The majority give a diffuse stain, 
which in some few cases becomes, by the application of the 
regressive or indirect method (§ 211), the most precise and 
splendid chromatin stain as yet obtainable by any means. 182 
CHAPTER XY. 
The regressive staining method, or Flemming’s method, 
will form the subject of the present chapter, and the anilin 
chromatin stains will be treated of in the next chapter, the 
anilin plasma stains being reserved for treatment in a later 
chapter. 
General Directions for the Regressive Staining Method, as 
applied to Coal-tar Colours.* 
270. Staining.—Sections only, or material that is thin 
enough to behave like sections, such as some membranes, 
can be stained by this method. 
The solutions employed are made with alcohol, water, or 
anilin, according to the solubility of the colour. There seems 
to be no special object in making them with alcohol if water 
will suffice, the great object being to get as strong a solution 
as ‘possible. Alcohol of 50 per cent, strength, however, may 
be said to constitute a very generally desirable medium. The 
sections must be very thoroughly stained in the solution. As 
a general rule they cannot be left too long in the staining 
fluid. With the powerful solutions obtained with anilin a 
few minutes or half an hour will frequently suffice, but to be 
on the safe side it is frequently well to leave the sections 
twelve to twenty-four hours in the fluid. Up to a certain 
point the more the tissues are stained the better do they 
resist the washing-out process, which is an advantage. For 
researches on nuclei the solutions made with anilin had better 
be employed only with preparations well fixed in chromo- 
aceto-osmic acid, as the basic anilin oil may easily attack 
chromatin if not specially well fixed. 
Material fixed in chromo-osmic mixtures gives a sharper 
and more selective stain than material fixed in sublimate or 
the like. During the staining the tissues become overstained, 
that is charged with colour in an excessive and diffuse 
manner. The stain must therefore now be differentiated by 
removal of the excess of colour. 
* Historically the principle of this method is due to Hermann and 
Boettcher ; hut it is universally known by the name of Flemming, to 
whom is due the credit of having greatly improved the method in its prac- 
tical details. ON STAINING- WITH COAL-TAR COLOURS. 
183 
271. Differentiation.—This is generally done with alcohol, 
sometimes pure, sometimes acidulated (with HC1). The 
stained sections, if loose (celloidin sections), are brought into 
a watch-glassful of alcohol; if mounted in series on a slide 
they are brought into a tube of alcohol (differentiation can 
be done by simply pouring alcohol on to the slide, but it is 
better to use a tube or other bath). It is in either case well 
to just rinse the sections in water, or even to wash them well 
in it, before bringing them into alcohol. 
The sections in the watch-glass are seen to give up their 
colour to the alcohol in clouds, which are at first very rapidly 
formed, afterwards moi’e slowly. The sections on the slide 
are seen, if the slide be gently lifted above the surface of the 
alcohol, to be giving off their colour in the shape of rivers 
running down the glass. In a short time the formation of 
the clouds or of the rivers is seen to be on the point of ceasing; 
the sections have become pale and somewhat transparent, and 
(in the case of chrom-osmium objects) have changed colour, 
owing to the coming into view of the general ground colour 
of the tissues, from which the stain has now been removed. 
(Thus chrom-osmium-safranin sections turn from an opaque 
red to a delicate purple.) At this point the differentiation is 
complete, and the extraction of the colour must he stopped 
instantly (see § 273). 
It is generally directed that absolute alcohol be taken for 
differentiation. This may be well in some cases, but in 
general strong (95 per cent.) spirit is found to answer 
perfectly well. 
The hydrochloric acid alcohol process had better only be 
employed with tissues well fixed with “ Flemming,” as with 
tissues imperfectly fixed it may cause swellings. Further, 
the acid extracts the colour much more quickly from resting 
nuclei than from kinetic nuclei, which is an advantage or a 
disadvantage according to the end in view. 
The proportion of HC1 with which the alcohol should be 
acidified for the acid process should be about 1 : 1000, or 
less; seldom more. 
As a rough and ready guide to the beginner, it may be 
stated that washing out should be done with pure alcohol 
whenever it is desired to have resting nuclei stained as well 184 
CHAPTER XV. 
as dividing nuclei; the other processes serving chiefly to- 
differentiate mitoses. 
Differentiation with pure alcohol is known as “ neutral 
differentiation/’ or “ neutral extraction; ” and differentiation 
with hydrochloric acid is known as “ acid differentiation/’ or 
“ acid extraction.” 
The length of time necessary for differentiating to the 
precise degree required varies considerably with the nature 
of the tissues and the details of the process employed; all 
that can be said is that it generally lies between thirty 
seconds and two minutes. The acid process is vastly more 
rapid than the neutral process, and therefore of course more 
risky. 
In more than one of the methods presently to be described treatment 
with chromic acid or with iodine forms pai't of the differentiating process. 
The rationale of this is somewhat obscure; the most probable point of view 
appears to be that the chromic acid acts as a mordant on the chromatin, and 
helps it to retain the stain. It is known on the one hand that chromic acid 
precipitates safranin from its solutions, so that by admitting a special 
affinity of chromic acid on the other hand for chromatin, and especially for 
chromatin in the kinetic state, the explanation is hypothetically complete. 
The iodine in Bizzozero’s (Gram’s) process also appears to act as a fixative 
of the colour. See in the next chapter the special processes of differentia- 
tion described under Safranin and Gentian Violet. 
272. Substitution.—It was stated above that differentiation is gene- 
rally done with alcohol. There exists another mode of differentiation that is 
both of practical importance and of theoretical interest—one anilin stain may 
be made to wash out another. Thus methylen blue and gentian violet are 
discharged from tissues by aqueous solution of vesuvin or of eosin : fuchsin 
is discharged from tissues by aqueous solution of methylen blue. The 
second stain “ substitutes ” itself for the first in the general “ ground ” of 
the tissues, leaving, if the operation have been successfully carried out, the 
nuclei stained with the first stain, the second forming a “ contrast ” stain. 
Flemming obtains a highly important stain by differentiating in a 
solution of Orange G sections that have been previously stained with 
gentian violet (see his Orange method in the chapter on Plasma Stains). 
Flemming attributes the differentiation in this case to the “ acid ” qualities 
of the Orange. But it should be borne in mind that Orange G is an acid 
colour in two senses of the word; not only is it an acid colouring matter in 
Ehrlich’s sense, but its solution in water has an acid reaction; and it would 
seem possible that it is rather to this than to its “ acid” composition in the 
sense of molecular structure that its power of extracting gentian violet is 
due. I am not able to say how far the “ acid ” nature of dyes in Ehrlich's 
sense confers on them the power of extracting the stains of basic colours, ON STAINING WITH COAL-TAR COLOURS. 
185 
or of less acid colours. It is certain at any rate that this property is also 
possessed by some basic colours, as is testified by two of the examples given 
above, both vesuvin and methylen blue being basic colours. 
In the interesting paper of Resegotti in Zeit.f. wiss. Mile., v, 3, 1888, 
p. 320, it is stated as a very general rule that colours that do not give a 
nuclear stain by the regressive method will wash out those that do. Thus he 
found that— 
Safranin, Dahlia, Methyl Violet, Gentian Violet, Rubin, Victoria Blue, 
Magenta, Basic Fuchsin, are washed out by the following: 
Congo, Methyl Green, Iodine Green, Nigrosin, Methylen Blue, Orange, 
Ponceau, Acid Fuchsin, Aurantia, Cyanin, Eosin, Methylic Eosin, Magdala 
Red, Bordeaux, Vesuvin. But Resegotti’s experiments do not seem to me 
to constitute a case in point. For he used the second colour, if I undei'stand 
him rightly, in alcoholic solution ; so that it remains uncertain how far the 
differentiation should be attributed to the chemical nature of the second 
colour, and how far to the alcohol used as a vehicle. The same remark 
applies to Benda’s Safranin-and-Lichtgriin process. 
273. Clearing.—The differentiation having been carried to 
a satisfactory point, as described in § 271, the extraction of 
the colour may be stopped by putting the sections into water; 
but the general practice is to clear and mount them at once. 
You may clear with clove oil, which will extract some more 
colour from the tissues. Or you may clear with an agent 
that does not attack the stain (cedar oil, bergamot oil, xylol, 
toluol, naphtha, &c.; see the chapter on Clearing Agents). 
If you have used pure alcohol for washing out, you had 
perhaps better clear with clove oil, as pure alcohol does not 
always, if the staining have been very prolonged, extract the 
colour perfectly from extra-nuclear parts. But if you have 
not stained very long, and if you have used acidulated alcohol 
for washing out, clove oil is not necessary, and it may be 
better not to use it, as it somewhat impairs the brilliancy 
of the stain. A special property of clove oil is that it helps 
to differentiate karyokinetic figures, as it decolours resting 
nuclei more rapidly than those in division. 
Some colours are much more sensible to the action of clove 
oil than others; and much depends on the quality of this 
much-adulterated essence. New clove oil extracts the colour 
more quickly than old. 
Series of sections on slides are conveniently cleared by 
pouring the clearing agent over them. 
When the clearing is accomplished to your satisfaction, 186 
CHAPTER XV. 
mount in damar or balsam, or stop the extraction of the 
colour if clove oil have been used by putting the sections 
into some medium that does not affect the stain (xylol, cedar 
oil, &c.). Chloroform should be avoided, either as a clearer 
or as the menstruum for the mounting medium. 
274. General Results.—The results depend in great measure 
on the previous treatment of the tissues. If you have given 
them a prolonged fixation in Flemming’s strong chromo-aceto- 
osmic mixture, and have differentiated after staining with 
acid alcohol and cleared with clove oil, you will get, with 
some special exceptions, nothing stained but nucleoli and the 
chromatin of dividing nuclei, that of resting nuclei remain- 
ing unstained. If you have given a lighter fixation, with 
Flemming’s weak mixture or some other fixing agent not 
specially inimical to staining, and have differentiated after 
staining with pure alcohol, you will get the chromatin of 
resting nuclei stained as well. 
275. Henneguy’s Permanganate Method (Journ. de VAnat. et de la 
Physiol., xxvii, 1891, p. 397).—This method is based on the fact, discovered 
by Henneguy, that permanganate of potassium is a mordant for many 
anilin dyes, and will enable a good stain to be procured in cases in which 
the usual methods fail. 
Sections (from tissues fixed either in the strong solution of Flemming for 
from two to six hours, or in other reagents such as sublimate, liquid of Perenyi, 
liquid of Kleinenberg, alcohol, &c.) are treated for five minutes with 1 per 
cent, solution of permanganate of potassium. They are then washed with 
water and stained (for about half the time that would have been taken if 
they had not been mordanted with the permanganate) in safranin, rubin, 
gentian violet, vesuvin, or the like. The stain that succeeds the best is a 
safi'anin solution prepared with anilin water and absolute alcohol (see below, 
§ 278). After staining they are differentiated with alcohol, followed by clove 
oil in the usual way. This is the delicate part of the process. The progress 
of the decoloration must he watched under the microscope, in order that it 
may be stopped at the proper moment. It goes on in general slowly, and 
the slower it proceeds the more selective will be the resultant stain. The 
decoloration sometimes continues even after the sections have been mounted 
in balsam, especially if all traces of clove oil have not been removed before 
mounting. It may thus happen that preparations which are insufficiently 
washed out at the moment of mounting show a perfectly differentiated stain 
twenty-four or forty-eight hours afterwards. 
If safranin have been taken as the stain the preparations show protoplasm 
of an orange-grey tint, which shows up the most delicate structures, in 
particular the achromatic figures of Cytodieresis. Chromosomes and nuclear ON STAINING WITH COAL-TAR COLOURS. 
187 
membranes are of a brilliant red, attractive spheres and centrosomes less 
strongly stained, but still sharply brought out against the rest of the cyto- 
plasm. 
If it be desired to have the details of protoplasmic structures still more 
markedly brought out, the safranin stain may be preceded by hsematoxylin 
staining (0‘5 per cent, solution of hsematoxylin in 90 per cent, alcohol ten 
minutes, wash with water, treat for ten minutes with 2 per cent, solution of 
bichromate of potash, and wash with distilled water before treating with 
the permanganate). But with preparations that have been fixed in 
Flemming this treatment will hardly be necessary. 
The mordanting action of permanganate of potassium on anilin stains is 
so energetic that if it have been overmuch prolonged before staining with 
safranin, or, still more, with rubin, it becomes almost impossible to wash 
out the sections properly; it may be necessary to leave them for nearly a 
month in clove oil. 
Henneguy’s preparations are certainly most successful, but I do not 
myself think that the chief value of the process lies in the plasma stain 
obtained by means of it, though this may be useful for some purposes. I 
think rather that it may be found useful as a means of slowing the some- 
times inconveniently rapid decoloration of sections treated by the regressive 
method. 
276. Ohlmacher’s Formaldehyde Process (Medical News, February 
16th, 1895).—Ohlmacher has found that formaldehyde is a powerful mordant 
for tar colours. Tissues may either be mordanted separately by treatment for 
a short time (one minute is enough for cover-glass preparations) with a 2 
per cent, to 4 per cent, formalin solution ; or the formalin may be combined 
with the stain. One gramme of fuchsin dissolved in 10 c.c. of absolute 
alcohol may be added to 100 c.c. of 4 per cent, formalin solution. Or 
saturated alcoholic solution of gentian violet may be added to 4 per cent, 
formalin solution in the proportion of 1 : 10. And a like solution may be 
made by adding to the formalin the like proportion of saturated alcoholic 
solution of methyl violet 5 B. Or formalin-methylen blue may be made by 
dissolving 1 grm. of methylen blue in 100 c.c. of the formalin solution. 
Sections are said to stain in half a minute, and to resist alcohol much more 
than is the case with those treated by the usual solutions. The formalin 
solution of safranin (Safranin 0, from Grtibler) is said to give a plasma 
stain comporting itself in all particulars like eosin. 
For the process of Rawitz for adjective staining with tar colours, by 
which they are made to behave as plasma stains, see the chapter on Plasma 
Stains. 
277. Choice of a Stain.—The tar-colour chromatin stains 
are sufficiently numerous, so that it almost seems as though 
we were in presence of an embarras de richesses. One might 
think that it would be quite sufficient for all practical pur- 
poses to possess one good red stain and one good blue one, 
so that, for instance, safranin and thionin or gentian violet 188 
CHAPTER XV. 
should be sufficient for the most exacting of laboratories. 
But I think it must be admitted that for delicate work, at 
any rate, it is desirable to possess one or two more. We 
have to take account of the manner in which these colours 
behave when used in combination with the plasma stains 
that it may be desired to employ. And there is another point 
that is not undeserving of attention. Some of the dyes 
discussed in the following chapter give a stain of a somewhat 
dead or dull quality, so much so that chromosomes and 
nucleoli frequently come out quite opaque. Gentian violet is 
in this case; whilst dahlia, which is otherwise near to it in hue, 
is not. Safranin and anilin green, on the other hand, leave 
the structures beautifully transparent. This is an advantage 
with thick sections, and sometimes for other reasons; but 
this transparency of the elements is unfortunately favorable 
to the production of diffraction lines, which may be a 
hindrance to good definition in delicate work. So that the 
dead colours, such as gentian, have a certain advantage for 
work with very thin sections and where very fine definition 
of chromatin is required; whilst the transparent or semi- 
transparent colours, such as safranin, should be preferred for 
thick sections. I would also add that it always seems to me 
that the blue stains, such as gentian, are less favoi’able for 
work with artificial light. They give more or less dichroic 
images, which are not favorable to good definition. 
The blues seem to have an advantage where it is desired 
to use them in combination with a plasma stain. For there 
are many fair plasma stains, such as Saurefuclisin, eosin, 
orange, in the red or yellow series, but not in the blue series. 
It is difficult, for instance, to find any plasma-stain except 
Kernschwarz that will work in a thoroughly satisfactory way 
with safranin. 
To sum up, I would recommend safranin for a red 
chromatin stain, and thionin or gentian for a blue one, except 
where special conditions, such as are mentioned above, 
suggest another choice. CHAPTER XVI. 
THE COAL-TAR CHROMATIN STAINS. 
a. Regressive Stains. 
278. Safranin.—One of the most important of these stains, 
•on account of its great power, brilliancy, and supei’ior per- 
manence in balsam, and also on account of the divers degrees 
of electivity that it displays for the nuclei and other consti- 
tuent elements of different tissues. 
The great secret of staining with safranin is to get a good 
safranin. It is needful here to insist most urgently on what 
was said above (§216, sub. finem). Before thinking of working 
with this important reagent you should go to Griibler or to 
Miinder and order the safranin you want, specifying whether 
you want it for staining nuclei or for staining elastic fibres, 
or for what other purpose you may require it. 
There are presumably at least a score of sorts of safranin in the market, 
differing to a considerable extent in colour, weight, solubility, and histological 
action. Some are easily soluble in water and not so in alcohol, some the re- 
verse, and some freely soluble in both. Fourteen brands, supplied by 
Griibler and by Miinder, have been studied by Resegotti (Zeit.f. wiss. Mile., 
v, 3, 1888, p. 320). They all gave positive results with the chromic acid 
method, to be detailed below; although Griibler had explained that the 
brands XX, XXBN, TB, had not given positive results (with the usual 
methods). Resegotti obtained his best results with the brands “ Safranin 
wasserloslich,” “Safranin spiritusloslich,” “XX,” “XXBN,” “ TB,” fur- 
nished by Griibler, and with the brands “ Rein,” “ 0,” “ FII,” and “ Cone.,” 
supplied by Miinder. 
The brand I have been using for a long time, and which 
gives good results, is the “ Safranin O ” of Griibler and Co. 
It should be remembered that as the processes of manu- 
facture are constantly changing, the properties of the pro- 
ducts are sure to vary somewhat from time to time. 
Staining.—The majority of safranins are not sufficiently 190 
CHAPTER XVI. 
soluble in water, so that solutions in other menstrua must 
be employed. 
A solution much used some time ago is that of Pfitzner 
(Morph. Jahrb., vi, p. 478, and vii, p. 291), composed of saf- 
ranin 1 part, absolute alcohol 100 parts, and water 200 parts, 
the last to be added only after a few days. 
The solution of Flemming (Arch. f. mik. Anat., xix, 1881, 
p. 317) is a concentrated solution in absolute alcohol, diluted 
with about one half of water. 
The solutions of Babes (Arch. f. mik. Anat., 1883, p. 356) 
are (a) a mixture of equal parts of concentrated alcoholic 
solution and concentrated aqueous solution (this is very much 
to be recommended), and (b) a concentrated or supersaturated 
aqueous solution made with the aid of heat. 
Some people still employ simple aqueous solutions. 
Lastly, there is the anilin solution of Babes (Zeit. f. tviss. 
Mik., iv, 4, 1887, p. 470). It consists of water 100 parts, 
anilin oil 2 parts, and an excess of safranin. The mixture 
should be warmed to from 60° to 80° 0., and filtered through 
a wet filter. This solution will keep for a month or two. 
Zwaardemaker (Zeit.f. wiss. Mik., iv, 2, 1887, p. 212) makes 
a mixture of about equal parts of alcoholic safranin solution 
and anilin water (saturated solution of anilin oil in water;— 
to make it, shake up “ anilin oil,” which is nothing but pure 
anilin, with water, and filter). This, I find, will keep for 
many months, perhaps indefinitely. 
Any of these stains may be used with any of the following 
differentiation processes. Of course you will have to stain 
longer in the weaker solutions. As to the anilin solutions 
see ante, § 270. 
Differentiation.—For general directions for differentiation 
and clearing see above, §§ 271 and 273. 
Flemming’s first method (or neutral differentiation) (1. c. in 
last section).—Differentiate with pure alcohol, followed by 
clove oil. This method stains resting chromatin as well as 
kinetic chromatin. 
Flemming’s second method (or acid differentiation) (Zeit.f. 
wiss. Mik., i, 3, 1884, p. 350).—Differentiate, until hardly any 
more colour comes away, in alcohol acidulated with about 0*5 
per cent, of hydrochloric acid, followed by pure alcohol and 
clove oil. (You may use the HC1 in watery solution if you COAL-TAR CHROMATIN STAINS. 
191 
prefer it.) The strength here given appears unnecessarily 
high. In Flemming’s latest work (on the achromatic struc- 
tures of the cell) he has been using a lower strength, viz. 01 
per cent, at most (see Arch. f. mik. Anat., xxxvii, 1891, 
p. 249) ; and this I find is generally preferable. 
Objects are supposed to have been well fixed—twelve 
hours at least—in the strong chromo-aceto-osmic mixture, 
and stained for some hours. Podwyssozki (Be.itr. z. path. 
Anat. v. Ziegler u. Neuwerk, i, 1886; Zeit. f. wiss. Mik., iii, 3, 
1886, p. 405) prefers to stain for half an hour only, and 
wash out with OT per cent, of HC1 in alcohol. In each of 
these ways you get kinetic chromatin and nucleoli alone 
stained (if the fixation have been performed as above directed). 
Podwyssozki (1. c.) gives another method, which consists in 
differentiating (for from a few seconds to two minutes) in a 
strongly alcoholic solution of picric acid, followed by pure 
alcohol. Same results (except that the stain will be brownish 
instead of pure red). 
Babes employed for differentiating, after staining in the 
aqueous or alcoholic solutions above mentioned, pure alcohol 
followed by oil of turpentine. For sections stained in the 
anilin solution he recommends treatment with iodine, accord- 
ing to the method of Gram (see what is said as to the process, 
of Gram in the paragraph on gentian violet, next section). This 
process has also been recommended by Prenant (Int. Monats- 
schr. f. Anat., &c., iv, 1887, p. 368), who notes that the 
treatment with the iodine solution should be somewhat longer, 
and the treatment with alcohol somewhat shorter than with 
gentian violet sections. 
Mabtinotti and Eesegotti (Zeit.f ’. wiss. Mik., iv, 3,1887, p. 328) recom- 
mend differentiating with a freshly prepared mixture of one part of 0-1 per 
cent, aqueous solution of chromic acid with nine parts of absolute alcohol 
followed by pure alcohol and bergamot oil. In my experience this method 
does not give better results (I think less good) than that of differentiating 
by the simple aqueous solution of chromic acid of Bizzozero followed by 
alcohol (see next section). The latter is certainly a most useful method. It 
should be mentioned that Martinotti and Eesegotti’s results refer to lightly 
stained alcohol-fixed objects, and not to chromo-aceto-osmic objects, which 
may make a great difference. 
GUbbini {Zeit.f. wiss. Mik., v, 2, 1888, p. 170) has recommended that 
sections be dehydrated, after staining, in methylic alcohol (wood spirit), in 
which safranin is only very slightly soluble, and decoloured in a mixture of 192 
CHAPTER XVI. 
two parts of clove oil with one part of cedar oil. I have not been able to 
obtain good results bj this method. 
It has been shown by Ohlmachee (Journ. Amer. Med. Ass., vol. xx, 
iNo. 5, Feb. 4,1893, p. Ill) that if tissues be treated with solutions containing 
iodine or picric acid after staining with safranin, there may he produced in 
'the tissue elements a precipitate of a dark red substance of a crystalline 
nature, but of lanceolate, semilunar, falciform, or navicellar forms. This pre- 
cipitate is formed both in normal and pathological tissue, and occurs either 
-in the nuclei or in the cytoplasm. It is formed readily in carcinomatous 
tissues ; and Ohlmacher makes out a strong case in favour of the conclusion 
to which he has come that many of the bodies that have been described as 
“ coccidia,” “sporozoa,” or other “parasites” of carcinoma are nothing hut 
particles of this precipitate. This refers, amongst much other work, to 
that of Podwyssozki and Sawtschenko. Forewarned is forearmed; but 
if the formation of Ohlmacher’s precipitate should prove to be a very general 
phenomenon, it will be necessary to conclude with him that to follow 
safranin staining with treatment by solutions containing iodine or picric 
acid is not only unscientific but positively dangerous. Of course this is not 
intended to discredit the use of safranin when washed, out with hydrochloric 
acid, alcohol, or the like. 
The reader will remember that safranin may be washed 
out by substitution (see ante, § 272). 
In preparations made with chromo-aceto-osmic acid, 
safranin stains, besides nuclei, elastic fibres, the cell-bodies of 
certain horny epithelia, and the contents of certain gland- 
cells (mucin, under certain imperfectly ascertained condi- 
tions). 
279. Gentian Violet.—One of the most important of these 
stains. It may be used in aqueous solution, or in alcoholic solu- 
tion diluted with about one half of water (Flemming, Zells. 
Kern. u. Zellth., 1882, p. 384), and the stain may be differ- 
entiated with pure alcohol, or (Flemming, Zeit.f. wiss. Mik., 1, 
1884, p. 350) with acidulated alcohol, as directed for safranin. 
Another good way of using it is that due to Bizzozeeo (Zeit. 
f. wiss. Mik., iii, 1, 1886, p. 24). The tissues may be hard- 
ened either in alcohol or in a chromic mixture, but must in 
the latter case have been well washed out with water. The 
staining solution is borrowed from that of Ehrlich for bac- 
teria, and consists of— 
Gentian violet ..... 1 part. 
Alcohol . . . . . .15 parts. 
Anilin oil . . . . . . 3 ,, 
Water . . . . . . 80 „ COAL-TAR CHROMATIN STAINS. 
193 
The sections are stained in it for five or ten minutes or 
longer (for objects from Flemming’s solution it will frequently 
be advisable to stain for as many hours). After staining, 
rinse the sections with alcohol, and bring them into a O'l per 
cent, aqueous solution of chromic acid. After from thirty to 
forty seconds bring them into alcohol, which begins the wash- 
ing out of the colour. After thirty or forty seconds in the 
alcohol put them back for thirty seconds into the chromic acid 
(this is done in order to fix the colour more completely in the 
nuclei). Then bring them back into alcohol for thirty to forty 
seconds, in order to wash out more colour and dehydrate them 
at the same time. Then treat with clove oil, which will extract 
more colour, and after a short time must be changed for fresh, 
in which the sections remain until they are seen to give up 
no more colour, when they are removed and mounted in 
damar. 
You might give a longer treatment with alcohol, and a shorter treatment 
with clove oil, but you would get a slightly different result. Alcohol washes 
out colour freely from kinetic nuclei as well as from resting nuclei, whereas 
clove oil acts much more energetically on the latter than on the former, and 
thus serves to differentiate dividing nuclei. 
In some cases, especially those of tissues whose nuclei have 
a tendency to give up the colour too freely, better results are 
obtained by combining the foregoing method with that of 
Gram for the staining of bacteria (Fortschr. d. Medicin, ii, 1884, 
No. 6; British Med. Journ., Sept. 6th, 1884, p. 486; Journ. 
Boy. Mic. Soc. [N.S.], iv, 1884, p. 817). 
In Gram’s method the sections are treated, after staining, 
with a solution composed of— 
Iodine 1 gramme. 
Iodide of potassium ... 2 grammes. 
Water 300 
In Bizzozero’s adaptation of this process the series of opera- 
tions is as follows:—Stain in the gentian, wash for five 
seconds in alcohol; two minutes in the iodine solution; 
twenty seconds in alcohol; thirty. seconds in the chromic 
acid solution ; fifteen seconds in alcohol; thirty seconds in 
the chromic acid again ; thirty seconds in alcohol; and treat- 
ment with changes of clove oil until final decoloration. 
Nissen (Arch. f. mik. Anat., 1886, p. 338) employs this pro- 
cess with omission of the treatment with chromic acid. 194 
CHAPTER XVI. 
In resting nuclei the nucleoli alone are stained, or the 
chromatin if stained is pale ; in dividing nuclei the chromatin 
is stained with great intensity, being nearly black in the 
equatorial stage. 
This exceedingly powerful stain is quite as precise as that 
of safranin, to which it is perhaps even preferable for much 
work with very thin sections (thick sections with closely 
packed nuclei may easily come out too dark). It lends itself 
well to double-staining with red or yellow plasma stains. 
The stain keeps fairly well in damar, though not so well as 
that of safi-anin. Flemming found that after a year it had 
faded a little, though not so much as liaematoxylin stains (v. 
Zells. Kern. u. Zellth., p. 384). My preparations in turpen- 
tine-colophonium have kept perfectly for many years, if not 
unduly exposed to light. 
Gentian violet in acid solution stains the nuclei of fresh tissues, and dis- 
solved in indifferent media is sometimes very useful for staining intra vitam 
(see above, § 213). 
280. Thionin.—The hydrochloride of thionin, or violet of 
Lauth, is a colour chemically nearly allied to methylen blue. 
It is, I believe, no longer manufactured for industrial purposes, 
but may be obtained from Griibler and Co. It was first intro- 
duced into histological technique by Hoyek, who found it the 
best of all the special stains of mucin. Later on it was warmly 
recommended as a chromatin stain by M. Heidenhain. lean 
thoroughly endorse Heidenhain’s recommendation : thionin 
is about the finest chromatin stain I have seen. I have 
classed it here as a regressive stain, but its action is so 
selective from the first that it may almost be considered to 
be a progressive stain. If you stain for only a short time (a 
few minutes) in a concentrated aqueous solution, hardly any- 
thing but the chromatin will be found to be stained. If the 
staining be prolonged, plasmatic elements will begin to take 
up the colour. After a short stain no special differentia- 
tion is required ; all that is necessary is to rinse with water, 
dehydrate, and mount. After a strong stain you differentiate 
with alcohol in the usual way, with this advantage, that the 
stain is so highly resistent to alcohol that there is no risk 
whatever of overshooting the mark ; the stain will not be 
more extracted in an hour than that of gentian or dahlia is in COAL-TAR CHROMATIN STAINS. 
195 
a minute, so that the process may be controlled under the 
microscope if desired. For this reason I think this stain 
should be specially recommended to beginners. It is a very 
powerful stain, works well either after sublimate or mixture 
of Flemming, gives good definition, and seems to keep well. 
Henneguy (in litt.) says that very good results are got by 
staining strongly and dehydrating with acetone ; but as the 
differentiation with alcohol is so easy I suppose that this 
process is only desirable for certain special purposes. 
281. Other Regressive Stains.—The foregoing, I think, may 
suffice for most practical purposes, but the following may be 
mentioned. 
Dahlia (Flemming, Arch. f. mik. Anat., xix, 1881, p. 
317).—The stain is paler in the nuclei than with gentian or 
safranin. The cytoplasmic granulations of certain cells are 
sharply stained. 
Dahlia is also a useful nuclear stain for fresh tissues (v. Ehrlich, Arch, 
f. mik. Anat., xiii, 1876, p. 263). For these the aqueous solution must be 
acidulated with (five per cent.) acetic acid; or you may stain in a neutral 
solution, and wash out with acidulated water. Dehydrate with alcohol and 
mount in turpentine-colophonium. It is also useful for staining intra vitam 
(see above, § 213). 
For the staining of Ehrlich’s “ plasma cells ” see post, Part II. 
Victoria Blue (Victoriablau) (Lustgarten, Med. Jahrb. k. 
Ges. d. Aerzte zu Wien, 1886, pp. 285—291).—I find this to be 
a very good stain, especially if the sections be previously 
treated for a few minutes with tincture of iodine. 
Victoria has a special affinity for elastic fibres. For this 
object Lustgarten recommends an alcoholic solution of the 
dye diluted with two to four parts of water. Fixation in 
chrom-osmium, or at least in a chromic mixture, is, I believe, 
a necessary condition to this reaction. And you must stain 
for a long1 time. 
Victoria has also a special affinity for mucus-cells, from 
which it is not washed out by alcohol. 
Flemming {Arcli. f. mile. Anat., xix, 1881, pp. 317 and 742) mentions 
also the following: 
Magdala Red (Naphthalin Red, Rose de Naphthaline).—Nearly if 
not quite as good a stain as any of the foregoing, and superior to all except 
safranin in respect of permanency. 196 
CHAPTER XVI. 
Mauvein and Rouge Fluorescent. 
Solid Green. 
Fuchsin (meaning the basic fuchsins, a series of Rosanilin salts having 
very similar reactions, and found in commerce under the names of Fuchsin, 
Anilin Red, Rubin, Rosein, Magenta, Solferino, Corallin).—A good 
but somewhat weak stain. Good results are said to be obtained by substi- 
tution in the following manner (Graser, Deutsche Zeit.f. Chirurgie, xxvii, 
1888, pp. 538—584; Zeit. f. wiss. Mik., v, 3, 1888, p. 378). You either 
employ the colour as directed for methyl violet (post, § 285), or you stain 
for twelve to twenty-four hours in a dilute aqueous solution, wash out for a 
short time in alcohol, stain for a few minutes in aqueous solution of 
methylen blue, and dehydrate with alcohol. A double stain. Chromatin 
and nucleoli red, all the rest blue. 
Ziehi/s Carbolic Fuchsin has been recommended as supe- 
rior to safranin by Schenk ( Ueber Conservirung von Kerntheil- 
ungsjiguren, Bonn, 1890). I do not know where the original 
formula was published, and take this from Zeit. f. iviss. Mik. 
(vii, 1, 1890, p. 39). 
The stain is made either by taking— 
Fuchsin .... 1 gramme, 
Acid, carbol. crist. . . 5 grammes, 
Alcohol . . . 10 ,, 
Aq. dest. .... 100 ,, 
or by saturating a 5 per cent, aqueous solution of carbolic 
acid with concentrated alcoholic solution of fuchsin (the 
saturation of the carbolic solution with fuchsin is made mani- 
fest by the formation of a metallic-looking pellicle on the 
surface of the liquid). The stain is washed out with alcohol 
followed by clove oil. 
Orange, precise but weak. 
Bismarck Brown is not very satisfactory with chromic objects. With 
alcohol objects it gives a good chromatin stain, but cannot be thoroughly 
removed from cytoplasm by any means yet discovered. It has this advan- 
tage, that being sufficiently resistent to alcohol it may be utilised for stain- 
ing entire objects. 
Kaiser (Biblioth. Zool., H. 7, 1 Halfte, 1891; Zeit. f. 
wiss. Mik., viii, 3, 1891, p. 363) has obtained good results 
with Bismarck brown in the following way :—Stain for forty- 
eight hours, and at a temperature of 60° C., in saturated 
solution of Bismarck brown in 60 per cent, alcohol (the solu- 
tion to be made in boiling alcohol), and wash out (until all is COAL-TAR CHROMATIN STAINS. 
197 
decoloured except the karyokinetic figures) in 60 per cent, 
alcohol, containing* 2 per cent, hydrochloric acid or 3 per 
cent, acetic acid. 
To these may be added— 
Methyl Yiolet, perhaps best used according to the method of Resegotti 
given in § 278. 
Benzoazurin has been lately recommended by Martin (see Zeit.f. wiss. 
Mik., vi, 3, 1889, p. 193). Stain for an hour or so in dilute aqueous solu- 
tion, and wash out with HC1 alcohol. 
Methylen blue may be used in the regressive way, and made to afford a 
chromatin stain. 
Nigrosin has been recommended by Eerera (Proc.-Verb. Soc. 
Beige de Mic., 1881, p. 134). I have obtained some fine 
and vigorous chromatin stains with it in the regressive way. 
The stain resists alcohol well. 
With Toluidin Blue I have had some superb stains of 
chromatin, unfortunately accompanied by a diffuse staining 
of cytoplasm. 
Mann (Zeit.f. iviss. Mik., xi, 4, 1894, p. 489) states that 
he has had good results by staining with it after eosin, thus 
obtaining a double stain, the eosin figuring as a plasma stain 
in the combination. 
b. Progressive Stains. 
282. As regards the progressive nuclear stains, the reader is 
reminded that many if not most of the anilins give a nuclear stain of 
greater or less purity if they are used in solutions acidified with acetic 
acid. Under the present heading, only those are mentioned which give in 
all respects, alike as regards precision and permanence, simplicity of 
manipulation and other qualities, a really valuable stain. The very exist- 
ence of methyl green and Bismarck brown is a sufficient reason for being 
silent, in this connection, with regard to the rest. 
283. Methyl Green.—This is the most common in commerce 
of the <c anilin ” greens. It appears to go by the synonyms 
of Methylanilin green, Griinpulver, Vert Lumiere, Lichtgriin ; 
these two last are in reality the names of another colour. 
When first studied by Calberla, in 1874 (Morphol. Jahrb., iii, 
1887, p. 625), it went by the name of Vert en cristaux. It is 
commonly met with in commerce under the name of more 
costly greens, especially under that of Iodine green. It is im- 198 
CHAPTER XYT. 
portant not to confuse it with the latter, nor with Aldehyde 
green (Vert d’Eusebe), nor with the phenylated rosanilins, 
Paris green, and Vert d’Alcali, or Veridine. 
Methyl green is the chloromethylate of zinc and penta- 
methyl-rosalin-violet. It is obtained by the action of methyl 
chloride on methyl violet. The commercial dye frequently 
contains unconverted methyl violet as a consequence of 
defective puidfication. It is sometimes adulterated with 
anilin blue (soluble blue). It is also sometimes adulterated 
with a green bye-product of the manufacture,—the chloride 
of nona-methyl-para-leukanilin (see Benedikt and Knecht’s 
Chemistry of the Coal-tar Colours). 
Methyl green is extremely sensitive to the action of alkalies. 
It is therefore important to use it only in acidified solutions, 
and to use only acid, or at least perfectly neutral fluids for 
washing and mounting. 
This is an extremely important histological reagent. Its chief 
use is as a chromatin stain for fresh or recently fixed, tissues. 
For this purpose it should be used in the form of a strong 
aqueous solution containing a little acetic acid (about 1 per 
cent, in general). The solutions must always be acid. You 
may wash out with water (best acidulated) and mount in some 
acid aqueous medium containing a little of the methyl green in 
solution. The mounting medium, if aqueous, must be acidu- 
lated. 
Employed in this way, methyl green is a pure chromatin 
stain, in the sense of being a precise colour reagent for 
chromatin. For in the nucleus it stains nothing but the 
chromosomes, or chromatin elements : it does not stain plas- 
matic nucleoli, nor caryoplasm, nor achromatic filaments. 
Outside the nucleus it stains some kinds of cytoplasm and 
some kinds of foimied material, especially glandular secretions 
(silk, for instance, and mucin). But the chromatin elements 
are invariably stained of a bright green (with the exception 
of the nuclein of the head of some spermatozoa), whilst 
extra-nuclear structures are in general stained in tones of 
blue or violet. 
Besides being a perfectly precise test for chromatin in the 
nucleus, methyl green has other advantages. Staining is 
instantaneous; over-staining never occurs. The solution 
is very penetrating, kills cells instantly without swelling COAL-TAR CHROMATIN STAINS. 
199 
or other change of form, and preserves their forms for at least 
some hours, so that it may be considered as a delicate fixative. 
It may be combined without 'precipitating with divers fixing* 
or preserving agents. Osmic acid (0*1 to 1 per cent.) may 
be added to it, or it may be combined with solution of Kipart 
and Petit (this, by the way, is an excellent medium for 
washing out in and mounting in). 
Alcoholic solutions may also be used for staining. They 
also should be acidulated icith acetic acid. 
The stain does not keep easily. It is difficult to mount it 
satisfactorily in balsam, because the colour does not resist 
alcohol (unless this be sufficiently charged Avith the colour). 
The resistance of the colour to alcohol is, however (at all 
events if it be used in the Ehrlich-Biondi combination), con- 
siderably increased by treating the sections for a few minutes 
with tincture of iodine before staining (M. Heidenhain). 
And Squire declares that thorough washing with water 
before passing into the alcohol has the shine effect. 
Of preparations mounted with excess of colour in the usual 
aqueous media the most fortunate only survive for a few 
months. Dr. Henneguy, however, writes to me that it keeps 
well in Brun’s glucose medium. 
Undoubtedly methyl green is one of the most valuable 
stains yet known. It is the classical chromatin stain for 
fresh tissues. 
It was first pointed out, I believe, bj Hescbl (Wiener med. Wochenschr., 
2, 1879), that methyl green is a reagent for amyloid degeneration. His 
observations were confirmed by Curschmann (Virchow’s Arch.,vol. lxxix, 1880, 
p. 556), who showed that it colours amyloid substance of an intense violet; 
but this (as pointed out by Squire, Methods and Formulae, &c., Churchill, 
1892, p. 37) may be due to its containing methyl violet as an impurity. 
The following paragraph, translated from the paper by Calberla above 
quoted, appeared inadvertently without comment in the first edition, and has 
since been repeated without comment in several places : 
“ He then found that * the nuclei of subcutaneous connective tissue and 
those of vessels and nerve-sheaths stained rose-red, cells of the corium reddish 
white, and the cells of epidermis greenish blue to pure blue.’ ” 
It is impossible at the present day to make out what can have been the 
origin of this astonishing statement. Methyl green certainly never under 
any circumstances stains nuclei red. I suggested in the last two editions 
that Calberla’s observation should be taken to refer to the results of a double 
stain with methyl green and eosin, which is mentioned in the somewhat ob- 
scurely expressed passage from which the quotation is taken. Paul Mayer 200 
CHAPTER XVI. 
however (in litt., and see Mitth. Zool. Stat. Neapel, xii, 2, 1896, p. 311), has 
examined the passage in question, and is of opinion that it can hardly bear 
the interpretation I put upon it. He prefers to suppose that Calberla’s 
colour contained methyl violet. That is quite a plausible supposition; but 
even then the nuclei could hardly have stained red, unless they were in a state 
of amyloid degeneration. 
Calberla’s misstatement is repeated, and made one of the grounds of an 
important theoretical deduction by Geiesbach, in Zeit. f. wiss. Mik., iii, 3, 
1886, p. 365—an instructive example of the way in which history is written 
in some quarters. 
284. Bismarck Brown (Manchester Brown, Phenylen Brown, 
Vesuvin, La Phenicienne).—A faii'ly pure nuclear stain that 
will work either with fresh tissues or with such as have been 
hardened in chromic acid. 
The colour is not very easily soluble in water. You may 
boil it in water, and filter after a day or two (Weigert, in 
Arch. f. mik. Anat., xv, 1878, p. 258). You may add a little 
acetic or osmic acid to the solution. Maysel (ibid., xviii, 
1880, pp. 237, 250) dissolves the colour in acetic acid (this 
solution does not give a permanent stain). Alcoholic solu- 
tions may also be used; or Calberla’s glycerin-and-alcohol 
mixture, or dilute glycerin (say of 40 per cent, to 50 per 
cent.) may very advantageously be employed. 
The watery solutions must be frequently filtered. The 
addition to them of carbolic acid has lately been recommeuded 
(vide Journ. Roy. Mic. Soc., 1886, p. 908). Bismarck brown 
stains rapidly, but never overstains. The stain is permanent 
both in balsam and in glycerin. 
The chief use of this colour is for staining objects in toto ; 
but it may also be employed for staining sections by the 
regressive method. 
As has been noted above (§ 213), Bismarck brown has the 
property of imparting a quasi-stain to certain cellular elements 
during life (for this purpose it is necessary to see that the 
colour employed be pure and neutral). 
285. Methyl Violet (Methylanilin = anilin-violet = Paris violet = in- 
chiostro di Leonardi).—The following process has been recommended by 
Orth (Amer. Mon. Micr. Journ., i, 1880, p. 143; Journ. Roy. Mic. Soc., 
N.S., i, 1881, p. 137):—Sections are to be soaked in water, and then brought 
into the following solution: 
Anilin violet ...... 1 part. 
Acetic acid 300 parts. COAL-TAR CHROMATIN STAINS. 
201 
Mount, without washing out, but simply draining, in acetate of potash 
(acetate 2 parts, water 1 part). 
The stain will probably fade within a year or two. 
This process does not appear to be of more than very limited applicability. 
The following, however, due to Graser (Deutsche Zeit. f. Chirurgie, xxvii, 
1888, pp. 538—584; Zeit. f. wiss. Mik., v, 3, 1888, p. 378), may be very 
generally useful. 
Sections are stained for from twelve to twenty-four hours in a (presum- 
ably aqueous) solution so dilute that at the end of that time the sections 
will have taken up all the colour from the liquid. They are then washed 
out for a short time in acidulated alcohol, and then in pure alcohol (followed 
presumably by clearing and mounting in balsam). Schiefferdecker, whose 
account is here quoted, says that the results, as regards nuclear figures, are 
even finer than with safranin. The method is applicable to objects fixed in 
“ Flemming.” 
A useful stain for fresh tissues is also obtained by using dilute acetic acid 
in the manner recommended above (by Ehrlich) for Dahlia, (§ 281). 
Amyloid matter appears red in preparations stained with methyl violet. 
This appears to be an optical effect (see the curious experiments of Cap- 
parelli, in Archivio per le scienze mediclie, iii, No. 21, p. 1; but see also 
Paul Mater, in Mitth. Zool. Stat. Neapel, xii, 2, 1896, p. 328, who has 
not been able to obtain Capparelli’s results). CHAPTER XVII. 
METHYLEN BLUE. 
286- Methylen blue is the chloride or the zinc chloride 
double salt of tetramethyltliionin, and is a “ basic ” colour in 
Ehrlich’s sense. It appears that some persons have con- 
founded it with methyl blue, which is either a basic colour 
derived from diphenylamine blue, or an acid colour obtained 
by the sulphonation of diphenylamine blue; neither of which 
colours has histologically any resemblance to methylen blue. 
Commercial methylen blue sometimes contains as an im- 
purity a small quantity of methylen red. This is not an 
undesirable factor; on the contrary, it sometimes affords 
differentiations of elements of tissues or of cells that cannot be 
produced by any other means. Samples containing methylen 
red are not so common now as formerly. The colour in this 
state is known as 'polychromatic methylen blue, and is em- 
ployed for staining certain cell-granules. It can be obtained 
from Grubber and Co. 
The colour to be employed for intra-vitam nerve staining 
should, on the contrary, be as pure as possible. Apathy has a 
note in Zeit. f. wiss. Mik., ix, 4, 1898, p. 466, to the effect 
that the best methylen blue for impregnation—in fact, the 
only one that will give exactly the results described by him 
(see § 292)—is that obtained from E. Merck, of Darmstadt, 
and quoted in his price list as “ medicinisches Methylenblau,” 
and described on the label as “ Anilin-blau, Methylen, chemisch 
rein und chlorzinkfrei.” It is therefore highly desirable, when 
ordering methylen blue, to specify for what purpose it is 
required. 
287. The Uses of Methylen Blue. —This colour seems to be 
in a fair way to become one of the most important reagents METHYLEN BLUE. 
203 
in the histological laboratory. Its importance as a stain for 
micro-organisms in tissues is well known to all pathologists. 
As a histological reagent it is used for sections of hardened 
central nervous tissue, in which it gives a specific stain of 
medullated nerves (post, Part II). It is a valuable specific 
reagent for plasma-cells (for which see also Part II). It 
possesses the property of washing out the stain of certain 
other anilins, with which it gives valuable double stains (next 
chapter). It stains a large number of tissues intra vitam, 
with little or no interference with their vital functions. And 
last, not least, it can be made to furnish stains of nerve-tissue, 
intercellular cement substances, lymph spaces, and the like, 
that are essentially identical with those furnished by a 
successful impregnation with gold or silver. The results are 
quite equal in most cases to those of gold or silver impregna- 
tion, and are obtained with far greater ease and certainty. I 
call especial attention to these processes, which seem to be 
effecting a revolution in histological technique. 
288. Staining in toto during Life.—Small and permeable aquatic 
organisms may be stained during life by adding to tbe water in which they 
are confined enough methylen blue to give a scarcely perceptible or at least 
very light tint to tbe water. If transparent organisms be taken, they may 
be examined alive without further manipulation at any desired moment. 
Microscopically examined, they will be found after a time to be partially 
stained—that is, it will be found that certain tissues have taken up the 
colour, others remaining colourless. If now you put back tbe animals into 
the dilute solution and wait, you will find on examination after a sufficient 
lapse of time that further groups of tissues have become stained. Thus it 
Avas found by Ehklich (Abh. k. Akacl. Wiss. Berlin, February 25, 1885), 
to Avhom the principle of the methods under consideration is due, that on 
injection of tbe colour into living animals axis-cylinders of sensory nerves 
stain, whilst motor nerves remain colourless. [Tbe motor nerves, however, 
will also stain, though later than the sensory nerves.] It might be supposed 
that by continuing the staining for a sufficient time, a point would be 
arrived at at which all the tissues Avould be found to be stained. This, 
however, is not the case. It is ahvays found that tbe stained tissues only 
keep the colour that they have taken up for a short time after they have 
attained the maximum degree of coloration of which they are susceptible ; 
as soon as that point is attained they begin to discharge the colour even 
more quickly than they took it up. And it is very often found that tbe 
elements which ha\-e stained first will have lost much or all of their colour 
by tbe time that those which stain later have attained their maximum colora- 
tion. It in a}' even happen, as I ha\’e observed, that the whole of tbe stainable 
tissues of an animal may run through the total gamut of coloration and 
decoloration until tbe animal has become as colourless as when first put into 204 
CHAPTER XV1I. 
the tinted, water, and that without any apparent change in its vital activities. 
The stain, therefore, that has thus been produced and lost is not a true 
histological stain, but a quasi-stain (see § 213). 
It follows that a total stain of all the tissues of an organism can hardly 
he obtained under these conditions, but that a Specific stain of one group or 
another of elements may be obtained in one of two ways. If the tissue to 
be studied be one that stains earlier than the others, it may he studied during 
life at the period at which it alone has attained the desired intensity of 
coloration, and the remaining tissues are not yet coloured at all, or not 
coloured enough to he an obstacle to observation. If it be one that stains 
later than the others, it may be studied during life at the period at which 
the earlier stained elements have already passed their point of maximum 
coloration and have become sufficiently decoloured not to be an obstacle to 
the observation of the later stained ones ; the latter being either at the point 
of maximum coloration or at a point of desired intensity either earlier or 
later than the maximum. Or the observer may fix the stain in either of 
these stages and preserve it for leisurely study by means of one of the 
processes given below under the heading “ Preservation of the Prepara- 
tions.” 
The proper strength of the very dilute solutions to be employed in the 
manner here considered must be made out by experiment for each object. I 
think the tint is practically a sufficient guide, but it may be stated that when 
in doubt a strength of 1 : 100,000 may be taken, and increased or diminished 
as occasion may seem to require. Zoja (Renclic. R. 1st. Lombardo, xxv, 
1892; Zeit.f. wiss. Mih., ix, 2, 1892, p. 208) finds that for Hydra the right 
strength is from 1 : 20,000 to 1 : 10,000. 
The stain is somewhat capricious. It is not possible to predict without 
trial which tissues will stain first in any organism. It is to be remarked 
that the stain penetrates very badly, which is perhaps the chief cause of its 
seeming capriciousness, and, I take it, the chief determining condition of 
the order in which tissues stain. Gland-cells generally stain early; then, 
in no definable order, other epithelium-cells, fat-cells, plasma-cells, “ Mast- 
xellen,” blood and lymph corpuscles, elastic fibres, smooth muscle, striated 
muscle. There are other elements that stain in the living state, but not 
when the staining is performed by simple immersion of intact animals in 
a dilute staining solution in the manner we are considering. Chief amongst 
these are nerve-fibres and ganglion-cells, which remain unstained in the 
intact organism,—most likely, so far as I can see, for the simple reason that 
the stain is not able to penetrate them. 
289. Staining1 Nervous Tissue during Life.—As was stated in 
the last section, it was made out by Ehrlich that by injecting 
a solution of methylen blue into the vessels or tissues of living 
animals there may be obtained a specific stain of axis- 
cylinders of sensory nerves. He held, and it has long been 
held by the majority of observers, that the stain so obtained 
is a product of a vital reaction of the tissues, and that it can- METHYLEN BLUE. 
205 
not be obtained with dead material. From the point of view 
maintained in § 213, the contrary would be the real state of 
the case. The stain is, of course, an iutra-vitam phenomenon 
in so far as it takes place during the life of the organism ; 
but I hold that the tissue itself does not take on the stain till 
it is dead or moribund. 
As said before, it was formerly held that the reaction 
could not be obtained with dead material. Dogiel, however 
{Arch. f. mih. Anat., xxxv, 1890, pp. 305 et seq.), found that 
muscle-nerves of limbs of the frog could be stained as much 
as from throe to eight days after the limbs had been re- 
moved from the animal. He concludes, indeed, that the 
reaction shows that the nerves were still living at that time. 
But it seems more natural to conclude with Apathy (Zeit.f. 
VM88. Mih., ix, 1, 1892, pp. 15 et seqq.) that nerve-tissue can 
be stained after life has ceased. 
Apathy has directly experimented on this point, and sums 
up the necessary conditions as follows :—The tissue need not 
be living, but must be fresh; nothing must have been ex- 
tracted from it chemically, and its natural state must not 
have been essentially changed by physical means. For 
example, the tissue must not have been treated with even 
dilute glycerin, nor with alcohol, though a treatment for a 
short time with physiological salt solution is not very hurtful; 
it must not have been coagulated by heat. 
Another common belief concerning the methylen-biue 
nerve reaction is that the presence of oxygen is necessary to 
the reaction. It is, therefore, the usual practice to dissect 
out the organ to be investigated after having exposed it to 
the action of metliylen blue by injection or immersion, and 
leave it for some time exposed to the air. Apathy has also 
investigated this point, and finds (loc. cit., p. 25) that the 
practice is in some cases correct, but the belief erroneous. 
His point of view is that the stain is a regressive one. It has 
been explained above that shortly after a tissue has attained 
the maximum degree of coloration of which it is susceptible 
it begins to give up its colour again to the surrounding liquid. 
The larger the volume of liquid with which the tissue is sur- 
rounded, the faster will this washing-out process go on; and 
in order that it may not go on with excessive rapidity, wash- 
ing out the stain from the nerve-fibres as well as from the 
O 206 
CHAPTKIt XVII. 
earlier stained elements (which alone it is desired to wash 
out, so ns to leave a differentiated specific stain of nervous 
elements), it is in many cases desirable to have the process 
go on in presence of as little liquid as possible. Another 
consideration that justifies the practice is that by exposure 
to air the preparations take up a trace of ammonia (derived 
from the ammonium carbonate of the air) ; and Apathy has 
experimentally established that this is an important factor 
in the sharpness of the stain. Oxygen has, according to 
him, nothing to do with it. 
290. Staining Nerve-tissue by Injection or Immersion.—The 
practice of the earlier workers at this subject was (following 
Ehrlich) to inject methylen blue into the vascular system or 
body-cavity of a living animal, wait a sufficient time for it to 
take effect on the organ to be stained, then remove the organ 
for further preparation and study. And there appears to 
have been a belief with some workers that it was an essential 
or at least a desirable condition to the production of the stain 
that it should have been brought about by injection of the 
colouring matter into the entire animal. It is now known 
that this is generally immaterial, and that the reaction can 
equally well be obtained by removing the organ and sub- 
jecting it to a bath of the colouring matter in the usual way. 
So that treatment with the colour by means of injection or 
by means of immersion in a solution may be taken to be, in 
general, a matter of convenience only. But it would also 
appear that in some cases the procedure by injection is pre- 
ferable, if not necessary. Burger (Mitth. Zool. Stat. Neapel, 
x, 1891, p. 206) found that for Nemertians it was necessary 
to inject the animals thoroughly, and leave them to dry, 
before the stain could be obtained. 
291. The Solutions employed.—The solutions used for in- 
jection are generally made in salt solution (physiological, or a 
little weaker); those used for staining by immersion are made 
either in gait solution or other “ indifferent ” liquid, or in pure 
water. Very various strengths of solution have been em- 
ployed. The earlier workers generally took concentrated 
solutions. Thus Arnstein (Anat. Anz., 1887, p. 125) injected 
1 c.c. of saturated solution into the vena cutanea magna of METHYLEN BLUE. 
207 
frogs, and removed tlie organ to be investigated after the 
lapse of an hour. Biedermann (Sitzb. d. k. Ah ad. Wiss. Wien, 
Math. Nat. CL, 1888, p. 8) injected 0‘5 to 1 c.c. of a nearly 
saturated solution in 06 per cent, salt solution into the thorax 
of crayfishes, and left the animals for fi*om two to four hours 
before killing them. Mayer (Zeit.f. wins. Mik., vi, 4, 1889, 
p. 428) took a strength of 1 :300 or 400 of 0’5 per cent, salt 
solution. This can be introduced into the system either by 
means of a syringe or other injecting apparatus, or by auto- 
injection through the heart. Even rabbits support this 
operation if artificial respiration be maintained. The solutions 
of Retzius are of the same strength. But the tendency of 
more recent practice is decidedly towards the employment of 
weaker solutions. Apathy (Zeit. f. miss. Mik., ix, 1, 1892, 
pp. 25, 26, et seq.) finds that it is not only superfluous, but 
positively disadvantageous, to take solutions stronger than 
1 : 1000. 
For staining by immersion similar solutions to those used 
for injecting may be employed, but they should, if anything, 
be still weaker. Dogiel (Arch. f. mik. Anat., xxxv, 1890, 
p. 305; Zeit.f. miss. Mik., vii, 4, 1891, p. 509) places objects 
in a few drops of aqueous or vitreous humour, to which are 
added two or three drops of a y1 to TU per cent, solution of 
methylen blue in physiological salt solution, and exposes 
them therein to the air. In thin pieces of tissue the stain 
begins to take effect in five or ten minutes, and attains its 
maximum in from fifteen to twenty minutes. For thicker 
specimens—retina, for instance—several hours may be neces- 
sary, the preparation being kept just moist by occasional 
treatment with a drop or two of indifferent liquid or methylen- 
blue solution, added by turns. The reaction is quickened by 
putting the preparations into a stove kept at 30° to 35° C. 
Rouget (Compt. Rend., 1893, p. 802) found it useful to modify 
the px-ocedure of Dogiel by employing a 00’5 per cent, solu- 
tion in 0'6 per cent, salt solution (for muscles of Batrachia). 
Allen (Quart. Journ. Mic. Sci:, 1894, pp. 461, 483) takes for 
embryos of the lobster a solution of 0T per cent, in salt 
solution, and dilutes it with 15 to 20 volumes of sea water. 
292. Apathy’s Methods.—As a good example of this kind 
of work, I subjoin a short account of the procedure recom- 208 
CHAPTER XVII. 
mended by Apathy (Zeit. f. wiss. Mik., ix, 1, 1892, p. 15) 
for Hirudinea. A portion of the ventral cord is exposed, 
and if it be considered desirable dissected out, but the sinus 
and pigmented connective tissue around it had better not be 
removed till the staining1 and fixation are completed. If, 
however, it be desired to stain as many ganglion-cells as 
possible, as well as fibres, the lateral nerves, as well as the 
connectives, should be cut through near a ganglion. The 
preparation is then treated with the stain. This is, for the 
demonstration chiefly of fibres in Hirudo and Pontobdella, 
either a 1 : 1000 solution in 0'5 to 075 per cent, salt solution, 
allowed to act for ten minutes; or a 1 : 10,000 solution allowed 
to act for an hour to an hour and a half; or a 1 : 100,000 
solution allowed to act for three hours (Lumbricus requires 
twice these times; Astacus and TJnio require three times; 
medullated nerves of Vertebrates four times). For the 
demonstration of ganglion-cells the stain is allowed to act 
three or four times as long (the connective and lateral nerves 
having been cut as directed above). 
The staining having been accomplished, the preparations 
from the 1 : 1000 solution are washed in salt solution for an 
hour; those from the 1 : 10,000 solution for a quarter of an 
hour; those from the 1 : 100,000 solution need not be washed 
at all. They are then treated with one of the ammoniacal 
fixing and differentiating liquids described below in the next 
section. This is done by pouring the liquid over them, and 
leaving them in it without moving them about in it for at least 
an hour, and by preference in the dark. The further treat- 
ment is as described in the next section. 
The object of the ammonia in these liquids is to differentiate 
the stain—to produce an artificial “secondary differentiation.” 
It acts by washing out the absorbed colour from certain 
elements, others resisting its action longer, much as HC1 
alcohol washes out a borax-cannine stain. In this case the 
elements that are washed out are the protoplasmic parts of 
nerve-fibres, and their “ interfibrillar ” and ‘‘perifibrillar” 
substance, the “ primitive fibrils ” still retaining the colour 
strongly. It is of theoretical interest to remark that accord- 
ing to Apathy the coloration thus obtained is a true stain of 
the “ primitive fibrils,” not an impregnation. The “ primitive 
fibrils ” are sharply stained of a violet-blue, showing no METHYLEN BLUE. 
209 
granular precipitate, and the “ interfibrillar” and “peri- 
fibrillar” substance, as well as nuclei, are either colourless or 
very lightly stained. The usual methods, on the other hand, 
give an “ inverse ” reaction, the “ primitive fibrils ” remain- 
ing colourless, whilst the interfibrillar substance and proto- 
plasm of the nerve-fibres are impregnated with a finely 
granular greenish-black or violet precipitate, and the nuclei 
are usually stained. 
293. Preservation of the Preparations.—There are consider- 
able difficulties in the way of obtaining permanent preparations 
of methylen blue stains, as the stain is so very unstable that, 
as above explained, it begins to discharge after a short time, 
even in the living and not yet totally impregnated tissue. 
The colour may, however, be fixed, and more or less permanent 
preparations be made by one or other of the following 
methods : 
Dogiel (Arch. f. mik. Anat., xxxiii, 4, 1889, pp. 440 et seq.), 
following Arnstein (Anat. Anzeig., 1887, p. 551), brings the 
preparations, in order to fix the colour, into saturated aqueous 
solution of picrate of ammonia, in which they are allowed to 
remain for half an hour or more, and are then removed, 
washed in fresh picrate of ammonia solution, and studied in 
dilute glycerin, or mounted permanently in glycerin saturated 
with picrate of ammonia. More recently (Zeit.f. wiss. Mik., 
viii, 1, 1891, p. 15) he has recommended an increased duration 
of the picrate of ammonia bath up to eighteen or twenty-four 
hours, and mounting, without washing out, in chemically pure 
glycerin, free from acid. There is a defect'in this process, 
namely, that picrate of ammonia has a very injurious action 
of a macerating nature on some tissues. This may, however, 
be avoided by adding to the fixing-bath 1 to 2 per cent, of a 
1 per cent, osmic acid solution. (If it be desired to harden 
the tissues so that sections may be cut, the proportion of 
osmium solution should be increased fourfold.) 
S. Mayer (Zeit.f. wiss. Mik., vi, 4, 1889, p. 422) preferred 
a mixture of equal parts of glycerin and saturated picrate of 
ammonia solution, which served to fix the colour and mount 
the preparations in. This was also in principle the method 
followed by Ketzius (Intern. Monatsschr. Anat. u. Pliys., 210 
CHAPTER XVII. 
Bd. vii, H. 8, 1890). Dogiel, after careful study, quite refuses 
to admit that this is in any way an improvement. 
Other workers have employed saturated solution of iodine 
in iodide of potassium (so Arnstein) or picro-carmine (so 
Feist, Arch. f. Anat. u. Entwickel., 1890, p. 116; cf. Ze.it. f. 
wiss. MiJc., vii, 2, 1890, p. 231), the latter having the advan- 
tage of preserving the true blue of the stain if it be not 
allowed to act too long, and the preparation be mounted in 
pure glycerin. 
Picric acid has been used by Lavdcwsky, but this too, after 
careful study, is rejected by Dogiel. 
Apathy (Zeit.fwiss. Mik., ix, 1, 1892, p. 30) has found, as 
stated above, that free ammonia is a capital factor in the 
differentiation of the stain. He brings preparations (after 
washing in salt solution if the staining have been performed 
with a strong methylen blue solution, or without washing if 
it have been done with a very dilute solution) either into a 
concentrated aqueous solution of picrate of ammonia free 
from picric acid, and containing five drops of concentrated 
ammonia for every 100 c.c. ; or, which is generally preferable, 
into a 1 to 2 per cent, freshly prepared solution of neutral 
carbonate of ammonia, saturated with picrate. They remain 
in either of these solutious, preferably in the dark, for at 
least an hour. They are then brought into a small quantity 
of saturated solution of picrate of ammonia in 50 per cent, 
glycerin, where they remain until thoroughly saturated. They 
are then removed into a saturated solution of the picrate in a 
mixture of 2 parts 50 per cent, glycerin, 1 part cold saturated 
sugar solution, and 1 part similarly prepared gum-arabic solu- 
tion. When thoroughly penetrated with this they are removed 
and mounted in the following gum-syrup medium (1. c., p. 37) : 
Picked gum-arabic . . .50 grins. 
Cane-sugar (not candied) . 50 ,, 
Distilled water . . . 50 ,, 
Dissolve ovei* a water-bath and add O05 grm. thymol. This 
mounting medium sets quickly and as hard as balsam, so that 
no cementing of the mounts is necessary. Farrant’s medium 
(with omission of the arsenious acid) will also do. In neither 
case should either ammonium picrate or methylen blue be 
added to the medium. 
Preparations preserved by these methods (I do not know METHYLEN BLUE. 
211 
whether it is the case with preparations preserved by Parker’s 
or Bethe’s method, next section) are extremely sensitive to the 
influence of light. Diffused daylight is less injurious to them 
than the light concentrated on them by the illuminating 
apparatus of the microscope during observation. Apathy finds 
that lamplight is particularly injurious, especially the intense 
lamplight used with high powers ; which he attributes partly 
to the yellow rays, partly to the heat. 
294. Methods for Sections.—None of the preceding1 methods 
can be said to be anything like perfectly satisfactory. They 
do not give preparations that will resist the operations neces- 
sary for imbedding in paraffin. The stain is generally not 
preserved in its true blue colour, but turns to a grey, varying 
in tone from reddish brown to bluish or greenish black. The 
preparations seldom keep even in that state for more than a 
very few months, and it is not satisfactoiy to be obliged to 
mount preparations only in aqueous media. A strong solution 
of platinum chloride is said to give a fixation that will resist 
the treatment necessary for imbedding either in celloidin or 
paraffin (see Feist, Arch. f. Anat. und Entw., 1890, p. 116; 
Zeit. f. iviss. Mik., vii, 2, 1-890, p. 281); but the precipitate 
it gives is a flocculent one, and the preparations are not very 
satisfactory. 
The method of Parker (Zool. Anzeig., No. 403, 1892, p. 375) 
is therefore a most welcome step forwards. The stain is fixed 
(1) (in the form of a finely grained purplish precipitate) by 
cold concentrated aqueous solution of corrosive sublimate. 
The preparations are dehydrated (2) in a solution composed 
of 1 grm. of sublimate and 5 c.c. of methylal (pure methylal 
washes out the stain to a certain extent). The methylal 
solution is now removed by means of a mixture (3) of two 
parts xylol, one part pure methylal, and one part of the 
dehydrating mixture (2). After a short time the preparations 
are placed in (4) a considerable quantity of xylol. Here they 
should remain till all the methylal is replaced by xylol and 
the corrosive sublimate completely washed out. This will 
take some four or five days, as sublimate is very little soluble 
in xylol. Mount in balsam, or if sections be desired, imbed 
in paraffin in the usual way. Sections should be fixed to the 
slide with Schallibaum’s collodion, and not with Mayer’s 212 
CHAPTER XVII. 
albumen, which discharges the colour. The preparations will 
keep for several weeks, but the finer details are likely to fade 
after a month. 
The times required for a ganglion of the ventral chain of a 
crayfish are for (1) ten minutes, (2) fifteen minutes, (3) ten 
minutes, (4) four or five days. 
Feist, in the paper quoted above, recommends Joliet’s 
gum-glycerin imbedding method; but in most cases the 
method of Parker will doubtless be preferable. 
The method of Bethe {Arch. f. mik. Anat., xliv, 1894, 
p. 585) is as follows :—A solution is made of— 
Molybdate of ammonium . . 1 grin. 
Distilled water .... 10 grins. 
Peroxide of hydrogen ... 1 grm. 
On adding the peroxide a yellow colour is produced. A 
drop of hydrochloric acid is added (white precipitate of 
molybdic acid which dissolves on agitation). After staining 
and rinsing in salt solution the preparations are put into the 
molybdic solution. The solution ought not to be more than 
eight days old, and it is well to use it cooled to zero. The 
preparations are left in it for two or three hours if they are 
small, or four or five hours if they are large (of a centi- 
metre in size). They are then washed for from half an hour 
to two hours in water, dehydrated in alcohol (which it is well 
to use cooled to zero), and cleared in clove oil or, better, in 
xylol. They may then be imbedded either in paraffin or 
celloidin in the usual way. 
This is for tissues of Vertebrates. For Invertebrates Bethe 
takes one grm. of molybdate, 10 c.c. of water, and 0’5 c.c. of 
peroxide. 
295. Methylen Blue Impregnation of Epithelia, Lymph-spaces, 
&c. (Dogiel, Arch. f. mik. Anat., xxxiii, 4, 1889, p. 440, et 
seq.).— Suitable pieces of tissue (thin membranes by pre- 
ference) are brought fresh into a 4 per cent, solution of 
methylen blue in physiological salt solution. After a few 
minutes therein they are brought into saturated solution of 
picrate of ammonia, soaked therein for half an hour or more, 
then washed in fresh picrate of ammonia solution, and exa- 
mined in dilute gtycerin. 
If it be wished only to demonstrate the outlines of endo- METHYLEN BLUE. 
213 
thelium cells, tlie bath in the stain should be a short one, not 
longer than ten minutes in general; whilst if it be desired to 
obtain an impregnation of ground-substance of tissue so as to 
have a negative image of juice-canals or other spaces, the 
staining should be prolonged to fifteen or thirty minutes, and 
it is advisable to remove the endothelial covering of the ob- 
jects operated on before putting them into the stain. 
If it be desired to preserve the preparations permanently, 
they had better be mounted in glycerin saturated with picrate 
of ammonia. (For an improvement in the method of preserva- 
tion given in a later paper see supra, § 293.) 
The effect is practically identical (except as regards the 
colour) with that of a negative impregnation with silver 
niti’ate. 
296. Mayer’s (S.) Impregnation Methods (Zeit. f. iviss. 
Mil:., vi, 4, 1889, p. 422).—Maker’s experiments were made 
contemporaneously with those of Dogiel, cover much the same 
ground, and give the same results. Mayer stained tissues for 
about ten minutes in a 1 : 300 or 400 solution of methylen 
blue in 05 per cent, salt solution, rinsed in salt solution, and 
put up in the glycerin-picrate of ammonia mixture given 
above, § 293. He finds it may be stated that by this method 
all the essential results of a nitrate of silver impregnation may 
he produced by means of methylen blue. The images are either 
positive or negative. In stratified epithelia and in endothelia 
(for instance, in the testicular canals of the rat, in the 
vascular system, in smooth muscle) cement-substance is 
stained. In the cornea, ground-substance is stained, giving a 
negative image of the corneal corpuscles ; and in like manner 
a negative image is sometimes obtained of the intra-sarco- 
lemmar nerve-endings in striated muscle of frogs and rats. 
But methylen blue sometimes, as does sometimes silver 
nitrate, gives positive images of the cornea. If the stain be 
brought about by injection of the colour into the vascular 
system, the positive impregnation is the more frequent; 
whilst if it have been brought about by the immersion of 
the cornea, a negative image is more frequently obtained. In 
medullated nerves, striking images of the cruciform figures in 
the constrictions of Ranvier are obtained, just as with silver 
nitrate. 214 
CHAPTER XVII. 
To sum up, almost any reaction that can be obtained with 
chloride of gold, or with nitrate of silver, can be obtained 
with methylen blue, and with much greater ease and 
certainty. 
297. Other Uses of Methylen Blue.—Methylen blue is also 
used, chiefly in conjunction with other colouring matters, for 
staining fixed and hardened tissues, especially preparations of 
central nervous system. These uses will be described in their 
proper places. CHAPTER XVIII. 
THE COAL-TAR PLASMA STAINS. 
298. As to Plasma Stains.—By a plasma stain is generally 
meant, rather vaguely, one that stains the extra-nuclear parts 
of cells and the formed material of tissues, or one of these. 
To be precise, the class ought to be subdivided, and we ought 
to speak of cytoplasm stains, granule stains, ground-substance 
stains, or the like. But the vague general sense of the 
term will be sufficient for the purposes of the present chapter. 
In the earlier editions of this work I fought against the 
needless employment of plasma stains. But it must now be 
admitted that with the increasing differentiation of the 
problems of histology the employment of plasma stains has 
become in very many cases a necessity. Histology no longer 
stops short at the study of the groupings of cells; it now in- 
quires into the inner differentiations of cells and of intercel- 
lular substances. 
Good plasma stains are therefore much wanted. Unfor- 
tunately such a thing can hardly be said to exist; for it is 
not enough to require of a plasma stain that it should stain 
extra-nuclear material. It is also desirable that it should 
do so in as selective a way as possible. Now all plasma 
stains are more or less diffuse stains. Many exhibit con- 
siderable selectivity, but it is by no means always easy to get 
them to display the particular selectivity that is desired. Those 
that do not display it are of little use, or of none at all. I have 
therefore suppressed a large number of formulae which appear 
to me to have little or no scientific value, and have not 
thought it worth while to cumber the text with references to 
the sources where they may be found. Most of them, if re- 
quired, will be found quoted in the previous editions. 216 
CHAPTER XVIII. 
299. Picric Acid.—I follow Flemming (Zeit. f. iviss. Mik., i, 
1884, p. 360) in pointing out that picric acid is the most gene- 
rally useful of all secondary stains. It gives useful plasma 
stains with most of the nuclear stains, and particularly with 
carmine and haematoxylin. It may be used with the most 
delicate of these stains, even the delicate coloration of alum- 
carmine being in no wise injured by it. The modus operandi is 
as simple as possible : it consists merely in adding picric acid 
to the alcohols employed for dehydrating the objects after 
staining with a nuclear stain. 
It should be borne in mind that picric acid has considerable 
power of washing out other anilin stains ; and that in com- 
bination with hydrochloric acid it very greatly enhances the 
power with which this acid washes out carmine stains. It 
should therefore not be added to the acidulated alcohol taken 
for differentiating borax-carmiue stains, or the like, but only 
to the neutral alcohol used afterwards. It does not otherwise 
affect any of the usual stains, and may be most highly recom- 
mended as a useful though frequently inelegant stain. It has 
the great quality, shared by very few plasma stains, that it 
can be used for staining entire objects. And as it is extremely 
penetrating, it is very much indicated for the preparation of 
such objects as small Arthropods or Nematodes, mounted 
whole. 
It can in some cases be employed by dissolving it in the 
solution of another dye (see Picro-carmine, Legal’s alum- 
carmine, § 228, &c.). 
300. Orange G.—This is the benzenazo-beta-naphthol- 
disulphonate of soda (to be obtained from Grubler and Co., and 
not to be confounded with about a dozen other colours that are 
on the market under the name of “ Orange/’ with or without a 
suffix). As indicated by its chemical description, this is an 
“ acid ” colour in Ehrlich’s sense; and it is also an acid colour 
in the usual sense, for its solution in water has an acid re- 
action. The solutions do not keep well, throwing down very 
quickly a pulverulent deposit. 
I think this is one of the most precise cytoplasm stains that 
I have met with. But it is not very useful, because the stain 
is a very pale one. But it may be found useful as a secondary 
stain to come after a haematein stain or other blue or red THE COAL-TAR PLASMA STAINS. 
217 
stain. I use a saturated solution in watei*, and allow' it to 
act for five or ten minutes (sections only). There is no pos- 
sibility of over-staining taking place, but some other anilin 
colours may be washed out by it. The chief use of this stain 
is in combination with other anilins, as in Flemming’s Orange 
Method, and the Ehrlich-Biondi-Heidenhain mixture, which 
see (§ 306). 
301. Flemming’s Orange Method (Arch.f. mile. Anat., xxxvii, 
1891, p. 249 ; ibid., p. 685; Zeit.f. iviss. Mile., viii, 2, 1891, p. 
223, and viii, 3, p. 343).—This is a substitution method. You 
stain (for as much as two or three days, or even weeks if 
you want a very powerful stain) in strong* alcoholic safranin 
solution diluted with anilin water (§ 278); rinse in distilled 
water; differentiate in absolute alcohol, containing at most 
0*1 per cent, of hydrochloric acid, until hardly any more 
colour comes away : stain for one to three hours in gentian 
(either a simple very strong aqueous solution, or, if you 
prefer, you may stain by the method of Gram, § 279) ; wash 
for a short time in distilled water; treat with concentrated, or 
at least fairly strong, aqueous solution of orange, which in 
virtue of its acid properties washes out most of the gentian. 
After at most a few minutes, whilst pale violet clouds are still 
being given off from the sections on agitation, bring them into 
absolute alcohol until hardly any more colour comes away, 
clear in clove or bergamot oil, and mount in damar or balsam 
before the last pale clouds of colour have ceased to come away 
(the orange should be the orange G, introduced into commerce 
by Meister, Lucius, and Briining, of Hochst, and also prepared 
by the Actiengesellschaft fur Anilinfabrication, Berlin ; it may 
be obtained from Grubler, § 216). 
This is not a triple stain in the sense of giving three 
different colours in the result; the orange does not act as a 
separate stain, but as an agent for the differentiation of the 
gentian stain. Chromatin and nuclei are purple-red; achro- 
matic spindle-fibrils grey to grey-brown, or, in very favorable 
cases, violet, and are very clearly brought out; attractive 
spheres, centrosomes, polar corpuscles, and “ Zwischenkorper ” 
reddish in light stains, brown-violet to black-brown in strong 
ones. 
N.B.—The description of the process given by Flemming as 218 
CHAPTER XVIir. 
above presumably applies to the case of loose sections. 
Working with sections fixed to the slide, I find that it does 
not apply quite exactly. An important difference is that I do 
not find that the orange washes out any of the gentian ; at 
all events, no perceptible clouds of colour come away. I have 
found it well to prolong the treatment with the orange to at 
least a quarter of an hour. The duration of the staining bath 
in the gentian, as given by Flemming, seems to me much too 
long for any objects; half an hour seems quite enough. 
I consider this a very fine stain when successful. The re- 
sults are as described by Flemming. But it is very capricious, 
one can never be certain of success. For this reason, and for 
others, I prefer for most objects the safranin and Kernschwarz 
stain described in the next chapter. 
302. Reinke’s Modification of Flemming’s Orange Method 
{Arch. f. mik. Anat., xliv, 2, 1894, p. 262).—Sections of 
material fixed in liquid of Hermann are put for twenty-four 
hours into a concentrated solution of potassium sulphite. 
They are washed with water and stained for an hour or two 
in safranin. They are then well washed with water, and 
stained for twenty-four hours in a “ neutral ” mixture of 
gentian and orange, prepared as follows. 
To a concentrated aqueous solution of gentian violet are 
added “ a few drops” of a like solution of orange G. The 
solution precipitates in part, owing to the formation of an im- 
perfectly soluble “neutral” colour; but becomes almost clear 
again if an excess of water be added. A drop of the mixture 
placed on blotting-paper should form a violet or brown spot 
with a narrow orange border. The solution is not to be 
filtered, but the sections are to be stained in it as it is (it is 
impossible to make out whether Reinke means the undiluted 
mixture, or the mixture made almost clear by addition of 
water). It is said that the “ neutral ” solution maybe pre- 
served for future use by adding to it one third of alcohol. 
After staining, you differentiate rapidly with alcohol, and 
clear with clove oil. 
I have tried this process, and obtained exactly the same 
final results as with Flemming’s process. I have not suffi- 
cient experience of it to be able to say whether it is any less 
capricious than Flemming’s process. THE COAL-TAR PLASMA STAINS. 
219 
303. Metanil Yellow (Metanilgelb). See Griesbach [Zeit.f. wiss. 
Mik., iv, 4, 1887, p. 448 ; see also Journ. Roy. Mic. Soc-, 1889, p. 464). It 
is said to have a certain affinity for various elements belonging to the group 
of the connective tissues. 
304. Sauregelb (Echtgelb), Tropaeolin O., Crocein, Gold Orange, 
(see Griesbach, Arch.f. mik. Anat., xxii, p. 132). 
305. Saurefuchsin (Acid Fuchsin, Fuchsin S, Acid Rubin, Rubin 
S, Saurerubin, Acid Magenta, Magenta S) —The denomination 
‘ Patentsaurerubin ” atti'ibuted to this colour by Kultschizky 
(Anat. Anz., viii, 1893, p. 357) is erroneous; the proper name 
of the colour used by Kultschizky is “ Rubin S, rein pat.,” see 
Ergebnisse cler Anatomie, iii, 1893, pp. 18, 19). The chemical 
description of this acid colour has been given (§ 268) : it is im- 
portant not to confound it with basic fuchsin, as seems to 
have been done by some writers. 
This is one of the best cytoplasm stains that I know of. I 
use a 0 5 per cent, solution in water, and allow it to act on 
sections for about five minutes. But it is a matter of chance 
whether one hits off exactly the desired effect in this way. 
The chief use of Saurefuchsin is as an ingredient in the 
Ehrlich-Biondi-Heidenhain stain, and Ehrlich’s Triacid, which 
see. It is also used as a specific stain for nerve tissue (see 
“Neurological Methods” in Part II). 
306. The Ehrlich-Biondi Mixture (or Ehrlich-Biondi-Hei- 
denhain Mixture) (Pfliiger’s Arch., xlii, 1888, p. 1; Zeit.f. 
wiss. Mih., v, 4, 1888, p. 520).—This well-known stain is 
somewhat troublesome to prepare. It may be obtained ready 
made from Grubler and Co. 
The receipt is as follows :—To 100 c.c. saturated aqueous 
solution of Orange add with continual agitation 20 c.c. satu- 
rated aqueous solution of Saurefuchsin (Acid Fuchsin) and 
50 c.c. of a like solution of Methyl Green. 
(According to Krause, 100 parts of water will dissolve about 20 of Saure- 
fuchsin [Rubin S.], 8 of Orange G, and 8 of Methyl Green.) The solutions 
must be absolutely saturated, which only happens after several days. 
Dilute the mixture with 60 to 100 volumes of water. The 
dilute solution ought to redden if acetic acid be added to it; 
and if a drop be placed on blotting-paper it should form a 220 
CHAPTER XV IIJ. 
spot bluish green in the centre, orange at the periphery. If 
the orange zone is surrounded by a broader red zone, the 
mixture contains too much fuchsin. 
According to M. Heidenhain’s instructions {“ Ueber Kern 
u. Protoplasma/’ in Festschr. Herrn. Geb. A. v. Kblliker 
gewidm., &c., 1892, p. 115; see Zeit. f. wiss. Mik., ix, 2, 1892, 
p. 202) the orange to be used should be “ Orange Gr; ” the 
Acid Fuchsin or Saurefuchsin should be “ Rubin S ” 
(“ Rubin ” is a synonym of Fuchsin); and the Methyl Green 
should be “ Methylgrun 00.And it is absolutely necessary 
that these ingredients be those prepared under those names 
by the Actienfabrik fitr Anilinfabrication in Berlin. 
The strong solutions directed to be taken readily precipi- 
tate on being mixed. To avoid this it is recommended by 
Squire (Methods and Formulse, &c., p. 37) to dilute them 
before mixing. 
Stain sections (N.B., sections only) for six to twenty-four 
hours. Dehydrate with alcohol, clear with xylol, and mount 
in xylol balsam. 
In the intention of the observers who have elaborated this 
stain, it is a progressive stain, and not a regressive one. It 
does not require any differentiation, and the sections should 
be got through the alcohol into xylol as quickly as possible 
in order to avoid any extraction of the colour. The great 
point of difficulty in working with this stain is to prevent the 
colour of the methyl green from coming out in the alcohol. 
If (Heidenhain, Arch. f. mik. Anat., xxxv, 1890, p. 173 ; 
Zeit. f. wiss. Mik., vii, 3, 1890, p. 357) the alcohol used for 
washing out be slightly alkaline, the stain of the fuchsin will 
become relatively pale in the result, and the stain of the 
methyl green relatively strong ; whilst a slightly acid reaction 
of the alcohol will produce the opposite effect. The energy 
of coloration of the fuchsin is often found to become weakened 
in kept solutions; it may be restored by adding very dilute 
acetic acid until a marked intensification of the red tint of the 
mixture is perceived. 
The precautions detailed above are not sufficient to ensure 
the correct working of the stain, particularly as regards the 
study of “ attraction-spheres ” and other achromatic elements. 
M. Heidenhain (UeberKern und Protoplasma,Engelmann, 1892, 
p. 116 ; see also Zeit. f. wiss. Mik., ix, 2, 1892, p. 202, a very 221 
THE COAL-TAR PLASMA STAINS. 
fall report) gives tlie following instructions :—Preparations 
made with the usual mixture are liable to fade; by acidifying 
the mixture a stronger and more sharply selective stain is 
obtained, which does not fade. But too much acid must not 
be added, as this would cause a staining of the interfilar 
substances. Heidenhain’s directions are as follows : 
Take some of the “commercial stock solution” (by which 
appears to be meant the undiluted mixture as given above), 
and dilute it with distilled water in the proportion of about 
6 parts of the mixture to 400 of water. Fill two beakers with 
distilled water, and add to each a few drops of the diluted 
solution, so as to bring them both up to the same tint. The 
coloured liquid thus obtained will show, at the same time, a 
red tint owing to the rubin, a yellow tone owing to the 
orange, and a greyish tone derived from the methyl green. 
Now add to one of the beakers, with continual agitation, drop 
by drop, a 1 : 500 solution of acetic acid until the colour of 
the liquid turns to a strong crimson, the former yellow tone 
disappearing, and the grey of the methyl greeu becoming les& 
marked. The two beakers serve as standards for the degree 
of acidity that should be given to the staining bath. The 
dilute solution that you made by diluting the original stock 
solution * is now acidified with the dilute acetic acid, added 
drop by drop with continual agitation, and from time to time 
a few drops are taken from it and added to a beaker of 
distilled water until there is obtained the crimson tint of the 
first test-beaker. Addition of acid should then cease, and the 
staining bath is ready. If the preparations should not turn 
out quite satisfactory a small further quantity of acid may be 
added. 
Before staining, sections should be treated for a couple 
of hours with OT per cent, acetic acid, then for ten to 
fifteen minutes with officinal tincture of iodine, and be rinsed 
with alcohol before bringing into the stain, in which they 
should l’emain for twelve to eighteen hours. The treatment 
with acid is necessary in order to ensure having the sec- 
tions acid on mounting in balsam. The primary object of the 
iodine is to remove any sublimate from the preparations 
(Heidenhain’s descriptions refer to sublimate objects, but 
Ehrlich-Biondi mixture will work with chrom-osmium ob- 
* “ Das aus der Starnmlosung durch Yei'diinnung gewonnene Quantum.” 222 
CHAPTER XVIIT. 
jects) ; but it also enhances the power of staining of the 
chromatin with methyl gTeen, and produces a more selective 
staining of protoplasmic elements. 
Nor will these precautions always suffice to ensure correct 
results. In Arch. f. mih. Anat., xliii, 3, 1894, p. 430, M. 
Heidenhain adds that it is so highly important to attain and 
maintain exactly the right degree of acidity, that further 
precautions are necessary. The solution must not be filtered, 
for filtration may render it less acid. If it has been kept for 
some time a little more acid must be added; for it will have 
dissolved traces of glass, which is an alkaline body. Heiden- 
hain therefore recommends that it be preserved in rubber 
bottles. 
The following instructions for acidifying the Ehrlich - 
Biondi mixture, also due to M. Heidenhain, are from a 
paper of Warburg’s (quoted from Zeit. f. wiss. Mih., xi, 3, 
1894, p. 383). To 2 c.c. of the Biondi mixture (1 : 30) (by 
this is presumably meant the original mixture as given above, 
but diluted with only 30 volumes of water instead of 60) add 
40 c.c. of distilled water, 3 c.c. of 05 per cent, solution of 
Saurefuchsin, and 0'2 c.c. of one-fifth per cent, solution of 
acetic acid (or, according to Grouven, op. cit., xii, 3, 1896, 
p. 379, four drops). [Grouven here speaks of the mixture as 
<e Triacid” (see next section): there is a deplorable confusion 
in the nomenclature of these stains.] 
The characters of the stain when successful are much as 
follows :—The nuclear staining is very sharp and good, the 
chromatic elements being coloui*ed of a somewhat slaty blue. 
Cytoplasm is of a more or less violet or more or less orange 
red, and caryoplasm is of the same colour as the cytoplasm, 
but of a lighter tone. Cell membranes, nuclear membranes, 
achromatic fibrillar structures, the Nebenkern when present— 
in a word, all the denser protoplasmic structures, are stained 
of the same colour as the hyaloplasm, but darker. After a 
very careful study of this admired stain, I find I cannot 
recommend it for any but the most special objects. It must 
in the first place, I think, be acknowledged that a reagent 
that can neither be prepared nor preserved without the 
minute precautions detailed above, is something much too 
great and good for human nature’s daily food. The stain is 
a very fine one when successful, but the most minute pre- THE COAL-TAR PLASMA STAINS. 
223 
cautions will not ensure its being successful. It is very 
capi’icious, it seldom gives the same result twice running. 
The coi-rect result should be a precise chromatin stain, com- 
bined with a precise stain of the plastin element of cytoplasm 
by the Saurefuchsin. Now the least defect or excess of 
acidity causes the plasma stain of the Saurefuchsin to be- 
come a diffuse one, instead of being sharply limited to the 
plastin element. And the methyl green, being very little 
resistent to alcohol, goes out of the chromatin with such 
rapidity during the dehydration, that there is always danger 
of the chromatin stain being lost altogether. For this reason 
the stain will only work with very thin sections : to be quite 
sure of good results, the sections should be of not more than 
3 fi in thickness, and if they are over five the desired results 
are almost hopeless. The preparations keep very badly; the 
majority of mine at any rate have become spoilt sooner or 
later, sometimes after only a few days. I admit that the 
method has its raison d’etre for the very special objects for 
which it was imagined,—for the researches on cell-granula- 
tions for which Ehrlich employed the three colours, or for 
the researches on the plastin reticulum of cytoplasm, for 
which Martin Heidenhain employed the mixture; and for 
similar objects. But to recommend it and to use it, as has been 
done by many workers, as a general stain for sections, applic- 
able to ordinary work,is nothingbut mischievous exaggeration. 
Far from having the qualities that should be possessed by a 
normal section-stain, the Ehrlich-Biondi mixture is highly 
unfitted for ordinary work. 
307. Ehrlich’s Triacid Mixture.—According to a custom 
which, I believe, originated with Ehrlich himself, and which 
would, perhaps, be better honoured in the breach than the 
observance, the name of Triacid (“ Triacidlosung ”) has been 
given to a mixture of the same three dyes as in the Ehrlich- 
Biondi mixture, but in such proportions that the “ acid ” 
colours therein have a larger share assigned to them. The 
denomination is improper, for the mixture contains only 
two “ acid ” colours, methyl green being a strongly “ basic ” 
colour. The following is a recent formula of Ehrlich, com- 
municated to Reinbach (quoted from Zeit.f. iviss. Mile., xi, 3, 
1894, p. 259). 224 
CHAPTER XVJII. 
Orange G, sol. sat. aq. . . . 120 
Saurefuchsin „ . . .80 
Methyl green „ . . .100 
Distilled water .... 300 
Absolute alcohol . . . .180 
Glycerin . . . . .50 
Care must be taken that the solutions be absolutely satu- 
rated before mixing. The mixture must never be shaken, 
the quantity necessary for use at any time must be carefully 
taken off from the top of the stock by means of a pipette. 
With these precautions the mixture will keep for years. 
I have not made up this mixture, but have examined a tri- 
acid solution procured from Grubler and Co. Its qualities 
and defects appear to be very much those of the Ehrlich- 
Biondi mixture. It appears to be a more powerful, but perhaps 
less delicate stain, and the methyl green appears to have more 
resistance to alcohol, so that it may perhaps give better 
results for ordinary work. I have obtained some powerful 
stains of spindles with it. 
As noted in the last section, the modified form of the 
Ehrlich-Biondi mixture there given has been spoken of as 
“ Triacid. 
308. Rawitz’ Adjective Plasma Stains.—It has been discovered by 
Rawitz that by means of appropriate mordants certain basic anilins, which 
by the usual methods of regressive staining are pure chromatin stains, may 
be made to afford a pure plasma stain—one not affecting chromatin at all. 
He has succeeded with safranin, fuchsin, methyl violet, gentian violet, and 
emerald green. The process is as follows :—Sections of material fixed in liquid 
of Flemming or other chromic mixture (I find that liquid of Hermann will 
also give the reaction) are put for twenty-four hours into a 20 per cent, 
solution of tannin in water (this solution must he made in the cold). They 
are then washed and put for two or three hours into a solution of tartar 
emetic of from 1 per cent, to 2'5 per cent, strength, kept at a temperature of 
37° C., or for twenty-four hours into the same solution at the normal tempe- 
rature. They are then carefully washed, and are stained for twenty-four 
hours in one of the above-named dyes, and either differentiated in the usual 
way with alcohol, or by treating them for from two to twenty-four hours 
with a 2'5 per cent solution of tannin ; they are then cleared, and mounted 
in the usual way. 
Successful preparations show an “ inversion ” of the stain ; the nuclear 
chromatin remains perfectly colourless, while cytoplasm and intercellular 
substances are stained. According to Rawitz, spindles are well stained, and 
“ centrosomes ” and “ attraction spheres ” are stained with a sharpness that 
he has not been able to attain to by any other method. THE COAL-TAR PLASMA STAINS. 
225 
The instructions given by Bawitz (Sitzb. Ges. naturf. Freunde, Berlin, 
1894, p. 174; Zeit. f. wiss. Mik., xi, 4, 1895, p. 503 ; and his Leit- 
faden f. hist. Untersuchungen, Jena, 1895, p. 76) are not sufficient to 
ensure success. Bawitz at the time of publishing these instructions had only 
tided the process on the testis of Salamandra. I find that the times neces- 
sary vary very much with different objects, and that the results also vary 
very greatly with different objects. I have only succeeded in obtaining a 
pure “ inversion ” by much abridging the baths, especially the staining bath. 
And I have only succeeded in obtaining a pure “ inversion ” of the stain 
with testicular material. As to the value of the process, which Bawitz 
commends warmly, I am inclined to regard it as a technical curiosity. I have 
found it to give, in some instances, an extraordinarily intense stain of polar 
coi'puscles, and think it may possibly be of some use in this respect with 
testicular material. With sections of tissues in general I do not think it 
will give tolerable results at all; for it stains intercellular substances with 
such intensity that it is frequently difficult to distinguish anything at all in 
the sections. It also seems to me that the tissues suffer under the treatment 
to a remarkable degree. It is one of the dirtiest stains I have ever seen; 
every slightest speck of dirt that may be present in the preparations is 
singled out by the dye, and coloured with remarkable intensity. Many of 
my preparations have not given the “ inversion,” but show the chromatin 
stained in the usual way, and a plasma stain besides. 
Theoretically this curious “ inversion ” of the stain is very interesting. 
I can at present suggest no further explanation of it than that put forth in 
§ 214. 
309. Congo Red (Congoroth) see Griesbach, in Zeit.f. wiss. Mih., iii, 
3, 1886, p. 379).—Also an “ acid” colour, in the sense in which that appel- 
lative is given to Saurefuchsin, &c. The aqueous solution, however, has a 
neutral or alkaline reaction. It becomes blue in presence of the least trace 
of free acid (hence Congo is a valuable reagent for demonstrating the presence 
of free acid in tissues; see the papers quoted, 1. c.). A stain much of the 
same nature as Saurefuchsin, and like it seems to be at present chiefly useful 
in staining axis-cylinders. See the chapter on Nerve-Tissue, in Part II. 
It may also be used for staining some objects during life. See ante, § 213. 
I have tried it as a general plasma stain, and cannot recommend it, as the 
stain does not keep. Note that this colour is not to be confounded with 
other Congos, as Congo yellow, or Brilliant Congo. 
310. Benzopurpurin.—According to Griesbach (1. c.), another “acid” 
colour very similar in its results to Congo red. It has been warmly com- 
mended as a plasma stain, to follow a hsematein stain or the like. I am 
bound to say that after some trouble I have been unable to obtain any results 
whatever with it. See, however, Zschokke (ibid., v, 4, 1888, p. 466), who 
recommends Benzopurpurin B, and says that weak aqueous solutions 
should be used for staining, which is elfected in a few minutes, and alcohol 
for washing out. Deltapurpurin, a more purple red, has, it is said, 
similar properties, and may be used in the same way. The solution (aqueous) 
should be moderately concentrated, and allowed to act for a minute or twc. 226 
CHAPTER XVIII. 
311. Neutral Red (Neutralroth) (Ehrlich, Allg. vied. Zeit., 1894, 
pp. 2, 20; Zeit.f. wiss. Mik., xi, 2, 1894, p. 250; Galeotti, ibid., p. 193). 
A recently discovered colour, which may be pi’ocured from Griibler and Co. 
Up to the present it has only been employed for intra-vitavi staining. Tad- 
poles kept for a day or two in a solution of 1: 10,000 or 100,000 absorb so 
considerable a quantity of the colour that all their tissues appear of a dark 
red. The stain is limited to cytoplasmic granules (Ehrlich), and to the 
contents of mucus-cells (Galeotti). 
312. Biebrieher Scharlach (Biebrich Scarlet), a diffuse bright red 
stain, mav be useful as a contrast stain. See Griesbach, Arcli. f. mik. Anat., 
xxii, p. 132). 
313. The Eosins, found in commerce under the names of 
Eosin, Saffrosin, Primerose Soluble, Phloxin, Bengal Rose, Eryth- 
rosin, Pyrosin B, Rose B a l’Eau, etc., are all plithalein colours. 
The preparations indicated by these names are not quite 
identical in their properties, but vary according to the different 
modes of manufacture. Most of them are soluble both in 
alcohol and in water, but some only in alcohol (“Primerose a 
VAlcool ”). 
They are all diffuse stains, formerly much used as contrast 
stains, less so now. 
chief use is in combinations or mixtures, to be given 
further on. For Bengal Rose see Griesbach, Zool. Anz., 
No. 135, 1883, p. 172. 
Eosin is a specific stain for red blood-corpuscles, and hence 
is much used in the study of blood, for which see in Part II. 
It is also specific for certain granules of leucocytes, for 
which also see Part II. 
The yolk of some ova takes the stain strongly, so that it is 
useful in some embryological researches. 
I at one time used eosin as a contrast stain to gentian 
violet, but have given up that combination, as I now get better 
results in other ways. 
314. Methyl Green and Eosin (Calberla, Morpli. Jahrb., iii, 1877, 
Heft 3, p. 625).—Mix 1 part of eosin with 60 parts of methyl green, and 
dissolve the mixture in warm 30 per cent, alcohol. 
Sections stain in this solution in five or ten minutes; they should be 
quickly washed in successive alcohols, and mounted in balsam or glycerin. 
Methyl Green and Eosin (List, Zeit.f. wiss. Mile., ii, 1885, p. 147).— 
Stain for a few minutes in a mixture of three parts of absolute alcohol with 
one part of aqueous solution of eosin (0'5 per cent.), wash, and stain for five 
minutes in 0‘5 per cent, aqueous solution of methyl green. Wash, de- THE COAL-TAR PLASMA STAINS. 
227 
hydrate, clear, and mount in balsam. The preparations do not keep well in 
glycerin. 
The method may he varied (1. c., p. 150) by diluting the methyl green 
solution with fifty volumes of water, and staining for twenty-four hours. 
It may also be varied by diluting the original methyl green solution with 
three volumes of absolute alcohol. 
The preparations should not be left in the alcohol used for dehydrating 
after the colour of the eosin has begun to reappear. 
315. Methylen Blue and Eosin (Chenzinsky, quoted from Zeit. f. 
wiss. Mile., xi, 2, 1894, p. 260). 
Methylen blue, sol. sat. in water . . . .40 
Eosin, 0"5 per cent, in 70 per cent, alcohol . . 20 
Distilled water, or glycerin . . . . .40 
This solution will only keep for about eight days. 
It has been recommended by good workers as a specific stain for blood. 
I find it will work with sections, but do not recommend it for that purpose. 
The eosin is diffuse in the cytoplasm. 
The mixture of Pianese (Zeit.f'. wiss. Mile., xi, 3, 1894, p. 345) contains 
the same ingredients in the same proportions, with the addition of a con- 
siderable proportion of carbonate of lithia. 
See also the mixture of Bremer, Arch.f. mile. Anat., xlv, 1895, p. 433 ; 
or Zeit.f. wiss. Mile., xii, 3, 1896, p. 380. 
316. Light Green (Lichtgriin S. F.). An acid colour, soluble 
in water or alcohol, and a valuable plasma stain. 
Benda {Verh. physiol. Ges. zu Berlin, Dec. 18th, 1891, Nos. 
4u. 5; see also Zeit.f. wiss. Mik., viii, 4, 1892, p. 516) pro- 
ceeds as follows (testes of Mammalia) :—Sections stained for 
twenty-four hours in anilin-water safranin solution, then for 
about half a minute in a solution of 0‘5 grm. “ Lichtgriin 
F. S.” or Saureviolett (Grriibler) in 200 c.c. of alcohol, 
dehydrated, and mounted in balsam. Chromatin red, archi- 
plasina bright green (or violet), centrosomata and “ Spitzen- 
knopf ” (of spermatozoa) sometimes green, sometimes red. 
The cliromatoid “ Nebenkorper ” of Hermann stains red. 
I have carried out this process both with the Lichtgriin 
and the Saureviolett. It gives one of the most beautiful stains 
known to me. The Saureviolett gives perhaps the more bril- 
liant preparations and seems to be rather easier to carry out, 
as it may be allowed to act rather longer than the Lichtgriin. 
The process in either form is a rather delicate one to carry 
out, and requires very thin sections. The Lichtgriin stain 
unfortunately does not keep very well; I find my preparations 
much faded after two years, but they will keep sufficiently for 
many months at all events. 228 
CHAPTER XVIII. 
317. Malachite Green (syn. Solid Green, Victoria Green, New 
Green, Benzoyl Green, Fast Green). A basic colour, which has been 
used as a plasma stain for the ova of Ascaris by van Beneden and Neyt 
(see in Part II, “ Cytological Methods ”) These authors used it for glycerin 
preparations. I have tried it for balsam sections, and find that it cannot be 
used, as the stain is not sufficiently resistent to alcohol. 
318. Iodine Green (Hoffmann’s Griin”), see Griesbach, Zool. Anz., 
No. 117, vol. v, 1882, p. 406.—The high praise accorded to this stain by 
Griesbach has not been justified by the experience of other workers. The 
colour is now no longer manufactured for industrial purposes, but may be 
obtained of excellent quality from C. A. F. Kuhlbaum’s Chemische Fabrik, 
Berlin, S.O. {Zool. Anz., No. 130, 1883, p. 56). 
319. Thiophen Green (Thiopbengrtin), see Krause, Intern. Monats- 
sclir.f. Anat., &c., iv, 1887, Heft 2. 
320. Anilin Green is said to have a special affinity for mucous gland 
cells, and other qualities; but I have not been able to identify the colour 
mentioned by authors. Recent chemical treatises make no mention of any 
dye bearing this name, which is, therefore, presumably an erroneous attri- 
bution. 
321. Quinolein Blue (Cyanin, Chinolinblau; v. Ranvier, Traite, 
p. 102).—Quinole’in is said to have the property of staining fatty matters 
an intense blue. 
It is useful for staining Infusoria, which in dilute solution it stains 
during life. On this point see the methods of Certes (post, Part II). 
322. Indulin and JNTigrosin.—The Indulins are a group of dyes 
related to the base violanilin. They may be either “ basic ” colours, or 
“ acid ” colours, according as they have or have not by supbouation been 
converted into suphonic acids. The soluble indulins of commerce are 
generally mono- and disulphonic acids (Benedikt and Knecht, Chemistry 
of Coal-tar Colours, p. 187). They occur under the brands (not strictly 
synonyms) of Indulin, Nigrosin, Indigen, Coupier’s Blue, Fast 
Blue It, Fast Blue B, Blackley Blue, Guernsey Blue, Indigo sub- 
stitute. According to Behrens the name Indulin is generally given to a 
bluish brand, and that of Nigrosin to a blacker one. 
Nigrosin, used with sublimate material, I find stains both nuclei and 
cytoplasm, the chromatin strongly (see § 281). I do not consider it a very 
good plasma stain. It will not give the stain at all with chromo-osmium 
material. 
According to Calberla (Morph. Jahrh., iii, 1877, p. 627), Indulin dis- 
solves into a dark blue solution in warm water or in dilute alcohol. For 
staining, the concentrated aqueous solution should be diluted with six 
volumes of water. Sections will stain in the dilute solution in five to 
twenty minutes; they may be washed in water or in alcohol, and examined 
either in glycerin or oil of cloves. THE COAL-TAE PLASMA STAINS. 
229 
The peculiarity of this stain is said by Calberla to be that it never stains 
nuclei; the remaining cell-contents and intercellular substance are stained 
blue. In its general effects it resembles quinolein blue, and is exactly the 
opposite of methyl green. The stroma of tendinous tissue, for instance, 
stains of a fine blue, the connective tissue that surrounds the bundle hardly 
at all, and the tendon-corpuscles of Ranvier remaining perfectly colourless, 
stand out as white stellate figures on a blue ground. As far as I have been 
able to control Calberla’s assertions, they appear to me correct. Used on 
Flemming material, Indulin seems to me a pure but weak plasma stain, 
leaving nuclei entirely unaffected, and possessing therefore opposite qualities 
to those of Nigrosin (see § 281). For Flemming material it will evidently 
be well to employ as strong a solution as possible. I see no special good 
in it. 
323. Ehrlich’s Indulin-Aurantia-Eosin, or Acidophilous 
Mixture, or Mixture C, or Mixture for Eosinophilous Cells (from 
the formula kindly sent me by Dr. Griibler).—Indulin, aurantia, 
and eosin, of each two parts; glycerin, thirty parts. This gives 
a very thick, syrupy solution. To use it, cover-glass prepara- 
tions may be floated on to it; or sections on slides may have a 
few drops poured on to them, the slide being laid flat till the 
stain has taken effect (twenty-four hours for Flemming 
material). This mixture was imagined for the purpose of 
obtaining a specific stain of certain granules of leucocytes. 
It has been pointed out by jSTikiforow [Zeit. f. iviss. Mik., viii, 
2,1891, p. 189 ; and xi, 2, 1894, p. 246) that it is also available 
for staining sections. I find this is the case. With Flemming 
material it gives a powerful and good stain, which is much 
more resistent to alcohol than that of the Ehrlich-Biondi 
mixture, and is therefore much more adapted to ordinary 
work. The stain with my objects is a blue one, in general 
effect like a Inematein stain, the eosin and aurantia only 
coming into play with certain elements. Chromatin in my pre- 
parations is of a very dark blue, cytoplasm being of a lighter 
blue (except where it has taken the stain of the aurantia or 
eosin). It will thus be seen that the Indulin in this com- 
bination behaves in a manner quite opposed to its behaviour 
when used alone (see last section). The stain is said to keep 
well. 
324. Safranin and Indigo-Carmine (Kossinski, Zeit. f. wiss. Mik., 
vi, 1, 1880, p. 61).—Stain sections ten to twenty minutes in saturated 
aqueous solution of indigo-carmine, wash with water and with alcohol, and 
stain with safranin (05 per cent, in dilute alcohol), dehydrate and mount. 230 
CHAPTER XVIII. 
Safranin and Nigrosin is a combination also recommended by the same 
author. Stain for three to five minutes in 0*1 per cent, aqueous solution of 
nigrosin, and proceed as before. 
N.B.—These times are stated for sublimate or alcohol material. I find 
they do not apply at all to chrom-osmium material. With this neither nigrosin 
nor indigo-carmine will give a stain. 
325. Anilin Blue.—Under this title are comprised various 
derivatives of the base rosanilin. They occur under the 
names Spirit Soluble Blue (Bleu Alcool), Gentian Blue 6 B, Spirit- 
Blue 0., Opal Blue, Bleu de Unit, Bleu Lumiere, Parma Blue, 
Bleu de Lyon. Receipts of the older authors for staining with 
“Anilin blue” should I think, be disregarded, as it would 
probably now be impossible to obtain the colours used by 
them or even to ascertain what colour was meant by them. 
The only one of the above-mentioned colours of which I 
have any personal knowledge, or that appears really valuable, 
is Bleu de Lyon. (Some authors give the names Bleu de Nuit 
and Griinstichblau as synonyms of Bleu de Lyon.) I find 
that though there is nothing very specific in its action, it is 
a very good plasma stain. It is a fairly true plasma stain, 
for though in a strong stain it will stain chromatin, 
yet in a light stain it will stain cytoplasm first, and thus 
works very well with carmine or safrauin as a nuclear stain, 
leaving the chromatin of a perfectly- pure red. I find it 
gives very good differentiations of nerve tissue, and of car- 
tilage (as has already been pointed out by Baumgarten and by 
Jacoby). The older mode of using it (Maurice and Schulgin) 
was to stain in bulk with it after borax-carmine, using a very 
dilute alcoholic solution. Baumgarten and Jacoby stain 
sections in a 0'2 per cent, alcoholic solution. I have ex- 
perimented with safranin as the chromatin stain, and obtained 
some fair results. But I do not think the combination can 
be recommended. For if you stain first with the blue, the 
safranin will extract it if allowed to act for more than a short 
time. And if you stain first with the safranin, the blue will 
extract it very quickly. The stain is a somewhat opaque one, 
and the results when obtained appear to me to be not com- 
parable for delicacy with those given by Wasserblau (next §). 
326. Methyl Blue .—Under this title are comprised some 
other derivatives of the base rosanilin. They are acid THE COAL-TAR PLASMA STAINS. 
231 
colours, being mostly salts of triphenylrosanilin- and triphenyl- 
pararosanilin-trisulplionic acid. Here belong the dyes sold 
as Methyl Blue, Cotton Blue, Water Blue (Wasserblau), Methyl 
Water-Blue, China Blue (Chinablau), Soluble Blue. 
Amongst these Water Blue (Wasserblau), appears to possess 
some useful properties. According to Mitrophanow (quoted 
from Zeit.f. wiss. Mik. v, 4, 1888, p. 513), used in concen- 
trated aqueous solution it gives a very good double stain 
with safranin. It is very resistent to alcohol. Using- the 
Wasserblau first, and then the safranin, I have had some in- 
teresting results, and as the process is easy to carry out I 
think it may be recommended. The Wasserblau must be 
used first, as if used after the safranin it will destroy the 
stain in a short time. With chrom-osmium material, twelve 
to twenty-four hours in the blue, and four or five in the 
safranin, may not be too much. 
Mann (op. cit., xi, 4, 1894, p. 490) has used it in conjunc- 
tion with eosin for staining ganglion cells. For the somewhat 
complicated details of the process, see the place quoted. 
327. Anilin Blue-black.—A preparation cited under this name has been 
recommended by Be van Lewis and others for nervous tissue. Unfortunately 
these authors have not given the chemical description of the colour used by 
them, so that it is not possible to ascertain whether they mean the Blue- 
black B of the oxyazo series, or the Anilin black of Lightfoot, also 
known under the names of Nigranilin and Noir Colin. 
328. Carmine Blue (Bleu Carmin Aqueux, prepared by Meister, 
Lucius and Brunig, at Hochst-a-M.). Jansens (La Cellule, is, 1, 1893, 
p. 9) has shown that this colour possesses a special affinity for the parts of 
cytoplasm that are undergoing cuticular differentiation. He uses it in 
alcoholic solution acidified. The stain will hear mounting in balsam. 
329. Violet B (S. Mayek, Sitzb. d. 1c. Tc. Ahad. d. wiss. Wien, iii Abtli., 
February, 1882).—This colour is a methyl violet prepared by Bindschedler 
and Busch of Bale, and by the Aktienfabvik fur Anilinfarben at Berlin. 
Used in solutions of 1 grm. of the colour to 300 grms. of 0'5 per cent, salt 
solution, and with fresh tissues that have not been treated with any reagent 
whatever, this colour gives a stain so selective of the elements of the 
vascular system that favorable objects, such as serous membranes, appear as 
if injected. The preparations do not keep well; acetate of potash is the 
least unsatisfactory medium for mounting them in. 
330. Saureviolett, see § 316. 232 
CHAPTER, XV1I1. 
331. Benzoazurin may be made to give either a diffuse or a nuclear 
stain, according to Martin (see Journ. Roy. Mic. Soc., 1890, p. 114). 
332. Baumgarten’s Fuchsin and Methylen Blue (Zeit.fwiss. Mih., 
i, 1884, p. 415).—Stain sections (of chromic objects) for twenty-four hours 
in a stain made by adding 8 to 10 drops of concentrated alcoholic solution 
of fuchsin to a watch-glassful of water. Rinse with alcohol, and stain for 
four or five minutes in concentrated aqueous solution of methylen blue, wash 
out with alcohol for five to ten minutes, and clear with clove oil. Nuclei 
red, tissues blue, the fuchsin having been driven out of the tissues by the 
methylen blue, a result which is not attained by washing with alcohol alone, 
either pure or acidified. 
333. Artificial Alizarin (Rawitz, Anat. Anz., xi, 10, 1895, p. 294). 
Rawitz has worked out a process of obtaining a double stain (chromatin and 
cytoplasm being stained of different colours) by means of artificial Alizarin, 
or Alizarin-cyanin. The process is an adjective one, requiring the use of 
special mordants only to be obtained from the colour manufacturers, and is 
extremely complicated and very lengthy, taking at least several days. As 
the results, to judge from the description of Rawitz, do not seem to possess 
a single point of superiority over those that may be obtained in half an hour 
by use of two or more colours in succession, or in combination according to 
known methods, the process would seem to be superfluous. CHAPTER XIX. 
OTHER ORGANIC STAINS AND COMBINATIONS. 
334. Kernschwarz (Platner, Zeit.f. tviss. Mik., iv, 3, 1887, 
p. 350; Journ. Roy. Mic. Soc., 1888, p. 675).—Kernschwarz 
is a black liquid of unknown composition, prepared in Russia. 
It maj be obtained from Griibler (address § 216). Platner’s 
method of using it was as follows : — Sections (sections only) 
may be stained in a tolerably strong dilution of the concen- 
trated liquid, and washed out (it may be for some hours) in 
an alkaline aqueous liquid. Dilute ammonia will do, but it is 
better to take a not quite saturated solution of carbonate of 
lithium (you may take a saturated solution, and dilute it with 
three, four, or more volumes of water). The result is a nuclear 
stain in the cytological sense; nuclear figures of division are 
stained deeply, resting chromatin less deeply or not at all, 
cytoplasm unstained or faintly grey. A peculiaiuty of this 
stain, on which much stress was laid by Platner in his first 
announcement of the colour, is that it stains also the Nebenkern 
(which safranin and the other basic anil ins do not do when 
employed in the usual way). 
Platner, on the whole, recommended this colour as a 
chromatin stain. As stated in former editions, I do not find 
that it possesses any very brilliant qualities as a chromatin 
stain. But I consider that employed as a plasma stain it has 
very important qualities. It is one of the most useful plasma 
stains that I am acquainted with. I do not say one of the 
most beautiful, for the sober neutral-tint stain it gives is not 
aesthetically seductive. I use it as follows : 
Sections (of chrom-osmium material—sublimate material 
does not give good results) are fixed on slides and treated 
with Kernschwarz until they become of a somewhat yellowish 
grey. If the material is fresh, the required depth of stain 
may be obtained in a few minutes, and in that case it may be 
well to first dilute the Kernschwarz with about ten volumes 234 
CHAPTER XIX. 
of water, so as not to run the risk of overstaining. If the 
material is not fresh, that is if it has been kept for some 
months, it will be necessary to stain for twenty-four hours in 
the undiluted liquid. The differentiation in an alkaline fluid 
prescribed by Plainer is omitted. After staining, I rinse in 
water, and stain for twenty-four hours or more in a chromatin 
stain, safraniu by preference, but gentian violet, Victoria blue, 
or a hsematein stain will do. I extract the safranin with 
alcohol, acidulated or neutral according to ch*cumstances, 
followed by clove oil, and mount in the usual way. This 
process gives a nearly pure double stain. In nuclei the 
chromatin and nucleoli are of a dark red (which appears rather 
brownish because the Kernschwai’z has stained the plastin 
sheath of these elements), whilst the plastin element of cyto- 
plasm is of a rich purple grey. 
The enchylema or hyaloplasm of nuclei and cytoplasm is 
colourless, or only lightly stained; spindles are faintly 
stained ; spindle-relics, both equatorial and polar (“ Neben- 
kerne,” or “ Spheres” of some authors) are deeply and accu- 
rately stained. As I have explained elsewhere (La Cellule, 
xi, 1, 1895, p. 31, and xi, 2, 1896, p. 257) this method is the 
best I have been able to discover for the study of these 
elements. But I have also found it to be excellent for the 
study of very various tissues, and as it is perfectly safe and 
very easy to execute, I think it may be recommended as a 
method of very general applicability. 
The stain of Kernschwarz is stated to be a good one for 
preparations that it is desired to photograph. 
335. Orchella (Orseille) (Wedl, Arch. f. path. Anat., lxxiv, p. 143 ; 
Journ. Boy. Mic. Soc., ii, 1879) : for an account of this substance vide 
Cooley’s Cyclopaedia, sub voce “ Archil; ” and see Fol, Lelirb., p. 192, 
and former editions of this work. 
336. Orcein (Israel, Virchow’s Archiv, cv, 1886, p. 169 ; Journ. Boy. 
Mic. Soc., 1887, p. 514, and Israel, Pralcticum der path. Hist., 2 Aufl., 
Berlin, 1893, p. 72).—Orcein (not “ orcin ”) is a vegetable dye obtained 
from the tinctorial lichen, Lecanora parella, and is not to be confused with 
orcin, another derivative of the same lichen. It is said to unite in itself 
the staining properties of the basic and acid stains, and also the combination 
of two contrast colours. Israel stains sections in a solution containing 
2 grms. of orcein, 2 grms. of glacial acetic acid, and 100 c.c. of distilled 
water, washes in distilled water, and passes rapidly through absolute alcohol OTHER ORGANIC STAINS AND COMBINATIONS. 
235 
to thick cedar oil, in which the preparations remain definitively mounted. 
Nuclei blue, protoplasm red. 
For the specific staining of elastic and connective tissue by means of this 
reagent, a subject which has been carefully worked out by Tanzek and 
Unna, see the paragraphs on the Connective Tissues in Part II. 
As far as I can see, this dye is not likely to be of any use 
in ordinary work. I have not obtained a good solution in the 
acetic acid. Using-a 1 percent, solution in absolute alcohol, 
I have obtained a faint plasma stain, the most conspicuously 
stained element of the cells being the Nebenkern, which is 
well brought out. The colour may perhaps be useful for the 
study of this element. 
337. Purpurin, see Ranviek’s Traite technique, p. 280; Duval’s- 
Precis de Technique histologique, p. 221; and Grekacher’s formula in 
Arch.f. mile. Anat., xvi, 1879, p. 470. 
338. Indigo.—Indigo is employed in histology in the form of solutions 
of so-called indigo-carmine, or sulpindigotate of soda or potash. The 
simple aqueous solution gives a diffuse stain, and is therefore not capable of 
being usefully employed alone. It is, however, of use when employed to 
bring about a double stain in conjunction with carmine. Though it has 
no selective preference for nuclei or protoplasm, it possesses to a high 
degree the property of imparting different hues and intensity of stain to 
different tissues ; and the nuclei being brought out by carmine, prepara- 
tions are obtained of a diagrammatic clearness that is not afforded by 
carmine alone. Some processes for double-staining with carmine are 
mentioned below. I have tried to use indigo-carmine in combination with 
safranin, and have not succeeded, but see § 324. 
Indigo-carmine is found in commerce. The reader who may desire to 
prepare it himself will find the necessary directions in Arch. f. mile. Anat., 
x, 1874, p. 32, and in Journ. Boy. Micr. Soc., ii, 1879, p. 614. 
Thiersch’s Oxalic Acid Indigo-carmine (see Arch.f. mile. Anat., i, 
1865, p. 150). 
Carmine Combinations. 
339. Seiler’s Carmine followed by Indigo-Carmine (Am. Quart. 
Mic. Journ., i, 1879, p. 220; Journ. Boy. Mic. Soc., ii, 1879, p. 613).— 
Stain in borax-carmine, wash out with HC1 alcohol, wash out the acid, and 
after-stain in an extremely dilute alcoholic solution of indigo-carmine (two 
drops of saturated aqueous solution added to an ounce of alcohol and 
filtered). 
I find this method sjives good results when applied to sections, but very- 
bad results if it be attempted to stain in the mass with the indigo. The 
indigo over-stains the superficial layers before it has penetrated to the 236 
CHAPTER XIX. 
deeper layers. The instructions given refer to sublimate material or the 
like; I find chrom-osmium material will not take the stain at all. 
340. Merkel’s Carmine and Indigo-Carmine in One Stain (Mer- 
kel, Unters. a. cl. cmat. Anst. Rostock, 1874; Month. Mic. Journ., 1877, 
pp. 242 and 317). 
Authors (Merkel, loc. cit.; Norris and Shakespeare, Amer. Journ. 
Med. Sc., January, 1877 ; Merkel, Mon. Mic. Journ., 1877, p. 242; Marsh, 
Section Cutting, p. 85; Bayerl, Arch. f. mik. Anat., xxiii, 1885, pp. 36, 
37; Macallum, Trans. Canad. Instit., ii, 1892, p. 222 ; Journ. Roy. Mic. 
Soc., 5, 1892, p. 698) are unanimous in stating that successful preparations 
show a most richly differentiated and yet very precise colouring. According 
to Bayerl, the stain is quite specifically elective for red blood-corpuscles, 
which are stained of an apple-green. The ground substance of cartilage and 
hone stains blue, their cells red. 
But the results are very uncertain. Merkel’s formula, as 
has been pointed out by Paul Mayee (Mitth. Zool. 8tat. 
Neapel, xii, 2, 1896, p. 320) is not only highly irrational, and 
inconvenient to employ, but gives an alkaline fluid that may 
be injurious to tissues. I agree with him that it should be 
suppressed. 
341. Mayer’s Carmine (or Haematein) and Indigo-Carmine in 
one stain.—In the place quoted in the last section, Mayer 
states that he obtains very good results by taking a solution 
of OT gramme of indigo-carmine in 50 e.c. of distilled water, 
or 5 per cent, alum solution, and combining it with from four 
to twenty volumes of carmalum or lisemalum. 
342. Carmine and Anilin Blue (or Bleu Lumiere, or Bleu de 
Lyon).—Duval (Precis de technique microscopique, 1878, p. 
225) proceeds as follows :—Stain with carmine “ in the or- 
dinary way; ” dehydrate, and stain for a few minutes (ten 
minutes for a section of nerve-centres) in an alcoholic solu- 
tion of anilin blue (ten drops of saturated solution of anilin 
blue soluble in alcohol to ten grammes of absolute alcohol, for 
sections of nerve-centres). Clear with turpentine, without 
further treatment with alcohol, and mount in balsam. 
The sections should appear of a fine dark violet when taken 
from the anilin; they are extremely transparent under the 
microscope—nerve-cells and axis-cylinders reddish violet, 
blood-vessels bluish violet, and so sharply marked out that 
the preparations have the aspect of injections. The con- OTHER ORGANIC STAINS AND COMBINATIONS. 
237 
nective elements are stained of a nearly pure blue, so that it 
is easy to distinguish them from the nervous elements. 
Applicable to all kinds of tissues, but especially to sections 
of nerve-centres. 
Recent authors recommend, instead of anilin blue, bleu de 
Lyon, dissolved in 70 per cent, alcohol acidulated with acetic 
acid (Maurice and Schulgin), or bleu lumiere; which has 
hardly any effect on nuclei. 
The solutions of both these colours should be extremely 
dilute for sublimate material, but strong for chrom-osmium 
material. It is possible to use them for staining in bulk. 
Baumgarten (Arch.f. mik. Anat., xl, 1892, p. 512) stains 
sections (of material previously stained in borax-carmine) for 
twelve hours in a0'2 per cent, solution of bleu de Lyon in 
absolute alcohol, and washes out for about half that time 
before mounting in balsam. He recommends the process for 
cartilage and nerve-centres. 
343. Carmine and Malachite Green.—Maas (Zeit.f. wiss. Zool., x, 
4, 1890, p. 527 ; Zeit. f. wiss. Mik., viii, 2, 1891, p. 205) recommends borax- 
carmine followed by weak alcoholic solution of malachite green, with a final 
washing out with stronger alcohol, see also § 317. 
344. Carmine and Picro-nigrosin (Pianese) (see Journ. Roy. Mic„ 
Soc., 1892, p. 292). 
345. Carmine and Picric Acid. See § 299. 
Hsematein or Hsemcitoxylin Combinations. 
346. Hsematoxylin and Picric Acid.—This excellent combina- 
tion has been treated of above (§ 299). 
347. Hsematoxylin and Eosin.—This is a well-known and 
popular combination, though some workers prefer the com- 
bination of hasmatoxylin with Benzopurpnrin (§ 310). Objects 
may be stained with haematoxylin (either in the mass or as 
sections) and the sections stained for a few minutes in eosin. 
I think it is better to take the eosin weak, though it has 
been recommended (Stohr, see Zeit. f. wiss. Mik., i, 1884, 
p. 583) to take it saturated. Either aqueous or alcoholic 
solutions of eosin may be used. 
Hickson (Quart. Journ. Mic. Sci., 1893, p. 129) gives the 
following instructions for staining sections on the slide :— 238 
CHARTER XIX. 
One hour in a strong solution of eosin in 90 per cent, alcohol, 
wash with alcohol, and stain for twenty minutes in a weak 
solution of haematoxylin. 
This method is most particularly recommendable for 
•embryological sections, as vitellus takes the eosin stain ener- 
getically, and so stands out boldly from the other germinal 
layers in which the blue of the haematoxylin dominates. 
List (Zeit.f. iviss. Milt., ii, 1885, p. 148) stains for twenty- 
four hours in a solution of three or four drops of Renaut’s 
ha3matoxylic glycerin (§ 259) in 250 c.c. of water, and then 
for a few minutes in a mixture of one part of 0-5 per cent, 
aqueous solution of eosin with three parts of absolute alcohol. 
See also Busch (Verli. Berl. Phys. Ges., 1887) ; Gierke (Zeit. 
f. wiss. Mik., i, 1884, p. 505). 
It should be noted that sections should be very well washed 
before being passed from eosin into haematoxylin or the 
reverse, as eosin very easily precipitates haematoxylin. 
348. Renaut ’ s Haematoxylic Eosin (Foe’s Lehrbuch, p. 196). 
—Renaut lias given from time to time several formulas for 
this stain. This one, communicated to Fol by Renaut, is the 
latest, and I suppress the others. 
Take— 
Concentrated aqueous solution of potassic eosin 
(eosine a la potasse) . . . . . 30 c.c. 
Saturated solution of hasmatoxylin in alcohol 
(ought to have been kept some time and to 
have precipitated). . . . . .40 c.c. 
Saturated solution of potash alum in glycerin 
(of a density of about 1'26) . . . 130 c.c. 
Mix, and let the mixture stand five or six weeks in a vessel 
•covered with a sheet of paper pierced with holes until the 
alcohol is evaporated, then filter. 
For staining, the solution may be used as it is or diluted. 
Staining goes on very slowly, and at first the colour is not 
held by the tissues, but disappears on washing. After some 
days or weeks, however, it becomes localised and fixed in the 
tissues. You may then mount in balsam, taking care to 
employ alcohol charged with a sufficient quantity of eosin. 
But it is frequently preferable to proceed by mounting the 
objects in the staining fluid diluted with one to tw'o volumes OTHER ORGANIC STAINS AND COMBINATIONS. 
239 
of glycerin. After a few weeks this mounting medium will 
have become perfectly colourless through the absorption of 
the colour by the tissues. 
The stain has a specific action on the cells of salivary and 
gastric glands. Mucus-cells become pale blue; salivary 
ferment-cells (crescent-cells of Gianuzzi) intense rose. 
See also Comptes Rendus, 1879, p. 1039 Ire ser., and Arch, 
de Physiol., 1881, p. 640. 
Everard, Demoor and Massart (Ann. Inst. Pasteur, vii, 
1893, p. 166), prepare a similar mixture as follows :—A 
solution is made with 1 grm. of eosin, 25 grm. of alcohol, 75 
of water, and 50 of glycerin. Then 20 grm. of alum are 
dissolved by the aid of heat in 200 grm. of water, the solu- 
tion is filtered, and after twenty-four hours there is added to 
it 1 grm. of hsematoxylin dissolved in 10 grm. of alcohol. 
This solution is allowed to stand for eight days, then filtered 
again, and combined with an equal volume of the eosin 
solution. 
349. Hsematoxylin and Benzopurpurin (Zschokke).—For 
this and the combination with Delta Purpurin, see ante, 
§310. 
350. Hsematein and Saurefuchsin.—Get a sharp chromatin 
stain with haemalum, then stain for five or ten minutes in 
0'5 per cent, aqueous solution of Saurefuchsin, dehydrate and 
mount. I would only advise this for sublimate material, not 
for chrom-osmium material. 
351. Hsematoxylin and Saurefuchsin and Orange.—Proceed 
as above, using for the second stain the following mixture : 
Saurefuchsin, 1 grin.; orange G, 6 grm.; rectified spirit, 
60 c.c.; water, 240 c.c. (from Squire’s Methods and Formulas, 
p. 42). I consider this a very good combination. Ten minutes is 
rather too much for sublimate sections, and not enough for 
chrom-osmium sections. 
The process described by Cavazzani (Riforma Med., 
Napoli, 1893, p. 604, Zeit. f. iviss. Mik., xi, 3, 1894, p. 344) 
is far too complicated to be recommendable. 
352. Hsematoxylin and Safranin.—In this combination, which was 
used to such good effect by Kabl in his classical researches on nuclei {Morph. 240 
CHAPTER XIX. 
Jahrb., x, 1884, p. 215), the safranin is the chromatin stain, and the hsema- 
toxylin the plasma stain. You stain very lightly with hsematoxylin, so 
lightly that the stain would not be of any use by itself (Itabl used very 
dilute Delafield’s solution, for twenty-four hours); wash out first with water, 
and then with alcohol acidulated with HC1, then stain for some hours in 
(Pfitzner’s) safranin, and wash out with pure alcohol. Rabl certainly was 
not far wrong when he wrote, “ This method is unequalled by any other.” 
It was certainly one of the best known at that time ; but better plasma 
stains are now known, and for most purposes the method may be considered 
to be out of date. 
Fol (Festschr. II. Virchow gewidmet, &c., i, 1891, p. 481 ; Zeit. f. wiss. 
Mik., ix, 2, 1892, p. 227) prefers using the hsematoxylin and safranin 
combined in one solution. You take— 
Aq. dest ca. 100 
Bohruer’s hsematoxylin 25 
Safranin, usual 1 per cent, water and alcohol 
solution .... .... 20 
Stain sections in a few drops of the mixture for from one to three 
minutes, wash in water, dehydrate, and mount; or before dehydrating treat 
with a weak alcoholic solution of picric acid, or, for some cases, of orange. 
This mixture was specially used for staining marrow in an investigation 
into the development of blood. CHAPTER XX. 
METALLIC STAINS (IMPREGNATION METHODS). 
353. The Characters of Impregnation Stains.—By impregna- 
tion is understood a mode of coloration in which a colouring 
matter is deposited in certain elements of tissues in the form 
of a more or less finely granular or sometimes even flocculent 
precipitate—the impregnated elements becoming in conse- 
quence opaque. By staining, on the other hand, is understood 
a mode of coloration in which the colouring matter is retained 
by the tissues in a state of solution, showing no visible solid 
particles under the microscope, the stained elements remain- 
ing in consequence transparent. But it should be understood 
that it is not correct to draw a hard and fast line between 
the two kinds of coloration. Some of the metallic salts 
treated of in this chapter give, besides an impregnation, in 
some cases a true stain. And some of the dyes that have 
been treated of in preceding chapters give, besides a stain, 
a true impregnation. Methylen blue, for instance, will give 
in one and the same preparation an impregnation and a stain; 
and critical examination of most fairly successful gold chlo- 
ride preparations will show that the coloration is in places of 
the nature of a finely divided solid deposit, in others a per- 
fectly transparent stain. 
Thus is justified the alternative title that this chapter has 
always borne—Metallic Stains, or Impregnation Methods. 
354. Negative and Positive Impregnations.—Impregnations 
are distinguished as negative and positive. In a negative 
impregnation intercellular substances alone are coloured of a 
deep black or brown or violet, according to the method 
employed, the cells themselves remaining colourless or very 
lightly tinted. In a positive impregnation the cells are 
Stained and the intercellular spaces are unstained. (This 242 
CHAPTER XX. 
explanation is the more needful as a directly contrary state- 
ment is made in a recent Lehrbuch.) 
Negative impregnation is 'primary because it is brought 
about by the direct reduction of a metal in the intercellular 
spaces. Positive impregnation is secondary (in the case of 
silver nitrate) because it is brought about by the solution in 
the liquids of the tissues of the metallic deposit formed by a 
primary or negative impregnation, and the consequent stain- 
ing of the cells by the new solution of metallic salt thus 
formed. These secondary impregnations take place when the 
reduction of the metal in the primary impregnation is not 
sufficiently energetic (see on these points His, Schweizer Zeit. 
f. Heilk., ii, Heft 1, p. 1 ; Gierke, Zeit. f. wiss. Mik., i, p. 393; 
Ranvier, Traite, p. 107). 
355. Nature of the Metallic Deposit.—There still exists consider- 
able obscurity as to the nature of the black or brown deposit formed in the 
intercellular spaces in cases of primary impregnation with a silver salt; 
v. Recklinghausen held that the silver salt combined with a hypothetical 
intercellular cement-substance (Kittsubstanz), forming a compound that 
blackens under the influence of light. Other authors refuse to believe in 
the intercellular cement, and hold either that the coloured lines represent 
stained cell-membranes, or that the metallic salt combines with the albu- 
minous and saline liquids that surround the cells, and is precipitated in 
simple intercellular spaces. Schwalbe {Arcli. f. mile. Anat., vi, 1870, p. 5), 
thinks that two cases should be distinguished ; the blacle lines that are 
obtained by the action of very weak solutions for a very short time being due 
to a true precipitate formed by a reduction of metal in the intercellular 
liquids, the brown lines that are obtained by exposing tissues for a longer 
period to the action of more concentrated solutions being due to the forma- 
tion of a compound of metal and cement-substance that becomes brown on 
exposure to light. (For the history of these questions see Gierke’s 
Fdrberei zu mileroskopisclien Zwecken.) Joseph (Sitzb. d. k.preuss.Akad. 
d. Wiss. Berlin, 1888; Zeit. f. wiss. Mile., xi, 1, 1894, p. 42, et seq.) has 
pointed out that the deposit can in no case consist of metallic silver, for it 
is dissolved by sodium hyposulphite, but it may be an albumino-nitrate or 
an oxide of silver. 
356. Action of Light on Solutions of Metallic Salts.—Stock 
solutions of metallic salts are generally kept in the dark, or 
at least in coloured bottles, under the belief that exposure 
to light spoils them by precipitating the metal in a state 
of reduction. It has been pointed out in § 33 that in the 
case of osmic acid not light, but dust is the reducing agent; METALLIC STAINS (IMPREGNATION METHODS). 
243 
and that solutions may be exposed to light with impunity if 
dust be absolutely denied access to them. I have now good 
evidence to the effect that the same is the case with other 
metallic solutions; and the interesting point is raised whether 
such solutions are not positively improved for impregnation 
purposes by exposure to light ! My able friend Dr. Lindsay 
Johnson has been examining this question both from a histo- 
logical and from a photographic point of view, and writes me 
as follows: 
“ One may (I find by experiment) state as a rule without 
exception that all the solutions of the chlorides and nitrates 
of the metals will keep indefinitely in clean white stoppered 
bottles in the sunlight; and as far as osmium, uranium, gold 
and silver, and platinum are concerned, actually improve or 
ripen by a good sunning. All photographers tell me their 
papers salt more evenly by old well-sunned silver nitrate than 
by a fresh solution kept in the dark; and I go so far as to 
say that this is one of the reasons why gold stains ai’e so un- 
satisfactory.” I have not been able myself to perform any 
experiments for the purpose of confirming the hypothesis of 
ripening by sunning, but I must say that it appears to me 
very plausible and deserving of further inquiry. 
357. State of the Tissues to he impregnated.—It has been 
pointed out in earlier chapters that the majority of histo- 
logical stains are not obtained with fresh tissues, but with 
tissues that have been more or less, charged with metallic 
salts, or otherwise changed in their composition by the action 
of fixing and preservative reagents. The contrary is the case 
with metallic impregnations: perfectly fresh tissues—that is, 
such as are either living or at all events have not been treated 
by any reagent whatever—are those in general that impregnate 
with the greatest ease and precision. Most impregnations 
will not succeed at all with tissues that are not fresh in the 
sense above explained. 
Silver. 
358. Silver Nitrate : Generalities.—This is the most com- 
monly used salt of silver. The general principles of its 
employment are so well stated by Ranvier (Traite, p. 105) 
that I cannot do better than abstract his account. 244 
CHAPTER XX. 
Silver nitrate may be employed either in solution or in the 
solid state. The latter method is the less frequently em- 
ployed, but is easy and gives good results. It is useful for 
the study of the cornea and of fibrous tissue, but is not suit- 
able for epithelia. For the cornea, for instance, proceed as 
follows :—The eye having been removed, a piece of silver 
nitrate is quickly rubbed over the anterior surface of the 
cornea, which is then detached and placed in distilled water; 
it is then brushed with a camel’s-hair brush in order to remove 
the epithelium. The cornea is then exposed to the action of 
light. On subsequent examination it will be found that the 
silver nitrate, which was dissolved by the liquid that bathes 
the surface of the cornea, has traversed the epithelium and 
soaked into the fibrous tissue, on the surface of which it is 
reduced by the action of light. The cells of the tissues will 
be found unstained. 
Silver nitrate is generally employed in solution in the fol- 
lowing manner :—A 1 per cent, solution is taken, to which 
two, three, or four volumes of water are added according to 
circumstances. The mode of employment varies in its details 
according to circumstances, a point which is very important 
to observe. In the case of a membrane such as the epiploon,, 
the membrane must be stretched like a drum-head over a 
porcelain dish,* and washed with distilled water, in order to 
remove the albuminates and white blood-corpuscles that are 
found on its surface; it is then washed with the solution of 
silver nitrate. In order to obtain a powerful stain it is neces- 
sary that this part of the operation be performed in direct 
sunlight, or at least in a very bi’illiant light. As soon as the 
tissue has become white, and has begun to turn of a blackish 
grey, the membrane is removed, washed in distilled water, 
* The Hoggans’ Histological Rings will be found much more con- 
venient, They are vulcanite rings made in pairs, in which one ring just 
fits into the other, so as to clip and stretch pieces of membrane between them. 
They will be found described and figured in Journ. Roy. Mic. Soc., ii, 1879, 
p. 357, and in Robin’s Journ. de VAnat., 1879, p. 54. They may be 
obtained, in sets of various sizes (that of seven eighths of an inch being the 
most convenient for 3x1 slides), of Burge and Warren, 42, Kirby Street, 
Hatton Garden, London, E.C., price ten shillings the dozen pairs. 
This useful little apparatus has lately been re-invented by Eternod (Zeit. 
f. wiss. Mile., iv, 1, 1887, p. 39), and is made according to his designs by 
Demaurex, Bandagiste, Fusterie, Geneva (Switzerland). METALLIC STAINS (IMPREGNATION METHODS). 
245 
and mounted on a slide in some suitable examination 
medium. 
If the membrane were left in the water the cells would 
become detached, and would not be found in the finished 
preparation. 
If the membrane had not been stretched as directed the 
silver would be precipitated not only in the intercellular 
spaces, but in all the small folds of the surface, and the forms 
of the cells would be disguised. 
If the membrane had not been washed with distilled water 
before impregnation there would have been formed a deposit 
of silver on every spot on which a portion of an albuminate 
was present, and these deposits might easily be mistaken for 
a normal structure of the tissue. It is thus that very often 
impurities in the specimen have been described as stomata of 
the tissue. 
If the solution be taken too weak—for instance, 1 : 500 or 
1 :1000, or if the light be not brilliant—a general instead of 
an interstitial stain will result; nuclei will be most stained, 
then protoplasm, and the intercellular substance will contain 
but very little silver. 
In general, in a good “ impregnation ” the contents of 
cells, and especially nuclei, are quite invisible. 
Ranvier notes that when tissues are to be impregnated by 
immersion they should be constantly agitated in the silver- 
bath in order to avoid the formation on their surfaces of de- 
posits of chlorides and albuminates of silver, which would 
give rise to deceptive appearances. 
Impregnation with silver may be followed by treatment 
with picro-carmine (or other carmine stain), which will bring 
out the nuclei, provided the impregnation has not been 
overdone. 
It should be noticed that impi’egnations only succeed with 
fresh tissues, and cannot be made to succeed with tissues 
preserved in any way. 
359. Silver Nitrate: the Solutions to be employed (Ranvier). 
—The solutions generally employed by Ranvier vary in 
strength from 1 : 300 to 1 : 500. Thus 1 : 300 is used for the 
epiploon, pulmonary endothelium, cartilage, tendon; whilst 
a strength of 1 :500 is employed for the study of the phrenic 246 
CHAPTER XX. 
centre, and for that of the epithelium of the intestine. For 
the impregnation of the endothelium of blood-vessels (by in- 
jection) solutions of 1 : 500 to 1 : 800 are taken. 
M. Duval (Precis, p. 229) recommends solutions of 1, 2, or 
at most 3 per cent. 
Y. Recklinghausen used, for the cornea, a strength of from 
1 : 400 to 1 : 500 {Die Lymphgefdsse, &c., Berlin, 1862, p. 5). 
Robinski {Arch, de Physiol., 1869, p. 451) used solutions 
varying between 0'1 and 0'2 per cent., which he allowed to 
act for thirty seconds. 
Reich {Sitzb. d. wien. Akad., 1873, Abth. 3, April; Zeit.f. 
wiss. Mik., i, p. 397) takes solutions of from 1 :600 to 1 :400 
for the study of the endothelium of vessels by injection. 
Rouget {Arch, de Physiol., 1873, p. 603) employed solutions 
as weak as 1 : 750, or even 1 : 1000, exposing the tissues to 
their action several times over, and washing them with water 
after each bath. 
The Hertwigs take, for marine animals, a 1 per cent, solu- 
tion {Jen. Zeit.f. Naturk., xvi, pp. 313 and 324). 
The Hoggans {Journ. of Anat. and Physiol., xv, 1881, 
p. 477) take, for lymphatics, a 1 per cent, solution. 
Tourneux and Hermann (Robin’s Journal de l’Anat., 1876, 
p. 200), in their fine studies on the epithelia of Invertebrates, 
employed a solution of 3 :1000 strength, and in some cases 
weaker solutions. The tissues were allowed to remain in the 
silver-bath for one hour, and were washed out with alcohol 
of 36° strength. 
Hoyer {Arch. f. mik. Anat., 1876, p. 649) takes a solution 
of nitrate of silver of known strength, and adds ammonia to 
it until the precipitate that is formed just redissolves, then 
dilutes the solution until it contains from 0-75 to 050 per 
cent, of the salt. 
This ammonio-nitrate solution is intended principally for 
the impregnation of the endothelium of vessels by injection, 
but can also be used for the impregnation of membranes by 
pouring on. It has the advantage of impregnating absolutely 
nothing but endothelium or epithelium; connective tissue is 
not affected by it. It is also said to give a sharper locali- 
sation of the stain than the ordinary solutions. 
Dekhuysen {Anat. Am., iv, 1889, No. 25, p. 789; Zeit.f. 
wiss. Mik., vii, 3, 1890, p. 351) has applied to tissues of METALLIC STAINS (IMPREGNATION METHODS). 
247 
terrestrial animals the method of Harmer for marine animals 
(see below, § 363). He washes a portion of mesentery of a 
frog in a 1*3 per cent, solution of nitrate of potash, and 
brings it for from three to six minutes into a 025 per cent, 
solution of silver nitrate containing 3 per cent, of nitric acid. 
After that time it is brought into pure 3 per cent, nitric acid, 
thence after a few minutes into 96 per cent, alcohol, and then 
into clove oil, in which it is reduced in diffused light in a few 
minutes. The method is stated to have the advantage of 
giving an excellent fixation of tissues, and of allowing a good 
nuclear after-stain with haematoxylin, safranin, or methyl 
green. 
Regaud (Journ. Anat. et Pliys., xxx, 1894, p. 719; Zeit.f. 
wiss. Mik., xii, 1, 1895, p. 74) recommends for the study of 
lymphatics a process devised by Renaut. One per cent, so- 
lution of nitrate of silver is mixed either with three or four 
parts of a mixture of eighty parts of saturated solution of 
picric acid and twenty of 1 per cent, osmic acid solution, or 
with four parts of a mixture of forty parts of picric acid 
solution and twenty of osmic acid solution, with or without 
the addition of 1 per cent, of acetic acid to the mixture. 
360. Other Salts of Silver.—Alferow {Arcli. de Physiol., 1874; 
Laboratoire d’histologie du College de France, 1874, p. 258; Duval, 
Precis, p. 230) recommends the soluble silver salts of organic acids, viz. the 
picrate, lactate, acetate, and citrate, as giving better results than the nitrate. 
He employs them in solutions of 1: 800, and adds to the solution employed 
for staining a small quantity of the acid of the salt taken (10 to 15 drops 
of a concentrated solution of the acid to 800 c.c. of the solution of the salt). 
The object of the free acid is to decompose the precipitates formed by the 
action of the silver salt on the chloi’ides, carbonates, and other substances 
existing in the tissues, leaving only the albuminate, which is a more insis- 
tent compound. . ; 
361. Silver Nitrate : Reduction.—Reduction maybe effected 
in other media than distilled water. 
Y. Recklinghausen washed his preparations in salt solution 
before exposing them to the light in distilled water (Arch. /. 
path. Anat., xix, p. 451). Physiological salt solution (O'75 
per cent.) is commonly used for these washings. 
Muller (Arch./. path. Anat., xxxi, p. 110), after impregna- 
tion by immersion for two or three minutes in a 1 per cent, 
solution of nitrate of silver in the dark, adds to the solution 248 
CHAPTEB XX. 
a small quantity of 1 per cent, solution of iodide of silver 
(dissolved by the aid of a little iodide of potassium). After 
being agitated in this mixture the preparations are washed 
with distilled water, and exposed to the light for two days in 
a 1 per cent, solution of nitrate of silver (see also Gierke, in 
Zeit. f. wiss. Mik., i, 1884, p. 396). 
Rouget (Arch. de Physiol., 1873, p. 603) reduces in glyce- 
rin. 
Sattler {Arch. f. mik. Anat., xxi, p. 672) exposes to the 
light for a few minutes in water acidulated with acetic or 
formic acid. Thanhoffer {Das Mikroskop, 1880) recom- 
mends this method. He employs a 2 per cent, solution of 
acetic acid. 
Krause brings his preparations, after washings, into a light 
red solution of permanganate of potash. Reduction takes 
place very quickly, even in the dark. The method does not 
always succeed (see Gierke, in Zeit. f. wiss. Mik., i, 1884, 
p. 400). 
Oppitz brings his preparations for two or three minutes 
into a 0‘25 or 0'50 per cent, solution of chloride of tin. 
Reduction takes place very rapidly (Gierke, 1. c.). 
Jakimovitch {Journ. de VAnat., xxiii, 1888, p. 142 ; Journ. 
Boy. Mic. Soc., 1889, p. 297) brings nerve preparations, as 
soon as they have become of a dark brown colour, into a 
mixture of formic acid 1 part, amyl alcohol 1 part, and water 
100 parts. The objects exposed to the light in this mixture 
for two or three days at first become brighter, a part of the 
reduced silver being dissolved ; hence the mixture must be 
renewed from time to time. When all the silver has dissolved, 
a darker colour is permanently assumed. The nerve-cells 
are left in this mixture for five to seven days. 
362. After-blackening.—Legros (Journ. de VAnat., 1868, 
p. 275; washes his preparations after reduction in hyposulphite 
of soda, which prevents after-blackening. According to 
Duval {Precis, p. 230) they should be washed for a few 
seconds only in 2 per cent, solution, and then in distilled 
water. 
363. Silver Impregnation of Marine Animals.—On account of 
the considerable quantity of chlorides that bathe the tissues METALLIC STAINS (IMPREGNATION METHODS). 
249 
of marine animals, these cannot be treated directly with ni- 
trate of silver. 
Hertwig {Jen. Zeit., xiv, 1880, p. 324) recommends fixing 
them with a weak solution of osmic acid, then washing with 
distilled water until the wash-water gives no more than an in- 
significant precipitate with silver nitrate, and then treating 
for six minutes with 1 per cent, solution of silver nitrate. 
Harmer (Mitth. Zool. Stat. Neapel, v, 1884, pp. 44 to 56) 
has discovered that many marine animals will live for some 
time (half an hour) in a 5 per cent, solution of nitrate of 
potash in distilled water. By washing them in this way they 
may be freed from the greater part of their chlorides, and 
may then be treated with silver nitrate in the usual way. This 
method gave good results with Loxosoma and Pedicellina, with 
Medusa, Hydroids, Sagitta and Appendicularia. 
Voshaer has been able by this means to demonstrate the 
epithelium of Chondrosia and Tlienea, which Sollas was unable 
to see; and Meyer has obtained good results with Annelids 
and ova of Teleostea. Few animals resist the action of nitrate 
of potash so well as Loxosoma and Pedicellina, but die in the 
solution in a few minutes. Their tissues, however, suffer but 
little change, and give good impregnations. Harmer thinks 
that for these animals other solutions having the same density 
as sea water might be substituted for the nitrate of potash, 
and recommends a 4’5 per cent, solution of sulphate of soda. 
See also Dekhuysen’s method, ante, § 359. 
364. Impregnation of Nerve Tissue.—For this subject, which 
includes the important bichromate-and-silver method of Golgi, 
see Part II. 
365. Double-staining Silver-stained Tissues.—The nuclei of 
tissues impregnated with silver may be stained with the usual 
reagents, provided that solutions containing free ammonia be 
avoided, as this would dissolve out the silver. These stains 
will only succeed, however, with successful negative impreg- 
nations, as nuclei that have been impregnated will not take 
the second stain. 
Impregnation with silver may be followed by impregnation 
with gold. In this case the gold generally substitutes itself 
for the silver in the tissues, and though the results are sharp 
and precise, the effect of a double stain is not produced. 250 
CHAPTER XX. 
Gold. 
366. The Characters of Gold Impregnations.—Gold chloride 
differs from nitrate of silver in that it generally gives 'positive 
(§ 354) impregnations only. It only gives negative images, 
so far as I know, when caused to act on tissues that have first 
received a negative impregnation with silver, the gold sub- 
stituting itself for the silver. In order to obtain these images 
you first impregnate very lightly with silver; reduce; treat 
for a few minutes with a 05 per cent, solution of gold chloride, 
and reduce in acidulated distilled water. 
This process, however, is in but little use, and except for 
the staining of cytoplasm for cytological researches and for 
certain special studies on the cornea, and on connective tissue, 
the almost exclusive function of gold chloride is the impreg- 
nation of nervous tissue. For this tissue, gold chloride 
exhibits a remarkable selectivity, in virtue of which it justly 
ranks as a most valuable reagent for the study of nerve end- 
organs and the distribution of nerves. 
For all the objects above named gold chloride is capable of 
furnishing preparations that for beauty and clearness cannot 
be surpassed, if even they can be equalled by any other means. 
A successful gold preparation shows at a glance, with dia- 
grammatic clearness, a wealth of minute detail which perhaps 
can only be painfully glimpsed by other means. But not 
every gold preparation is successful. I think there is no use 
in blinking the fact that very few are successful (one of the 
most experienced authorities in the matter told me lately that, 
as to nerve end-orgaus at all events, one preparation in ten 
thousand is successful). I took up in the first edition of this 
work the doubtless unpopular position that “ with all possible 
precautions gold chloride is uncertain in its action, and that 
the results obtained by means of it need to be controlled by 
the employment of other methods/’ and illustrated that posi- 
tion at considerable length. 
That this position was the correct one is now generally 
admitted. It is acknowledged to be abundantly evident that 
the very best gold preparations give images that are only 
worthy of credence as to what they show, and furnish abso- 
lutely no evidence whatever as to the non-existence of any- METALLIC STAINS (IMPREGNATION METHODS). 
251 
thing that they do not show; for you can never be sure that 
the imbibition of the salt has not capriciously failed, or its 
reduction capriciously stopped at any point. That the images 
frequently do stop capriciously short in the representation of 
reality there is abundant evidence. One such case has been 
treated by me ex professo in Recueil Zool. Suisse, i, 1884, 
p. 685 (Les organes chordotonaux des Dipteres, et, la methods du 
chlorure d’or). 
The authors of some of the methods about to be described 
claim for them that they give premanent preparations. I 
warn the reader against indulging in the hope that, with all 
possible precautions, his preparations will retain all their 
beauty for more than a few weeks. A successful gold pre- 
paration is certainly a thing of beauty, but it is, unfortunately, 
by no means a joy for ever. The able histologist whose ex- 
perience I have taken the liberty of quoting above tells me 
that “as to permanence, they are— 
‘ Like the snowfall on the river.’ ” 
Still, the greater the care taken in preparation, and 
particularly the greater the care taken to ensure thorough 
reduction of the gold, the longer will be the life of the pre- 
parations. 
Careful attention to the devices to this end detailed in 
the following paragraphs will do much ; and possibly Lindsay 
Johnson’s suggestion (supra, § 356) of the utility of “sun- 
ning” the solutions before use may prove an unexpected help. 
367. As to the Commercial Salts of Gold.—It is necessary to 
remind .the histologist that “ all is not gold that glitters.” 
Many things are not what they seem, and gold chloride is 
one of them, as will appear from the following quotation from 
Squiee’s Methods and Formulae, &c. (p. 43), an excellent 
authority on the chemistry of histological reagents : 
“ Commercial chloride of gold is not the pure chloride, 
AuC13, but the crystallised double chloride of gold and 
sodium, containing 50 per cent, of metallic gold. 
“ Commercial chloride of gold and sodium is the above 
crystallised double chloride mixed with an equal weight of 
chloride of sodium, and contains 25 per cent, of metallic 
gold.” 252 
CHAPTER XX. 
368. The State of the Tissues to be impregnated.—Gold 
methods may be divided into two groups : the one, chiefly 
concerned with the study of peripheral nerves or nerve end- 
organs, is characterised by employing either 'perfectly fresh 
tissues or tissues that have been subjected to a special treat- 
ment by organic acids; the other, concerned with the study 
of nerve-centres, is characterised by the employment of tissues 
hardened in the usual way. This latter group naturally falls 
to be considered in Part II. 
The hitherto classical rule, that for researches on nerve- 
endings the tissues should be taken perfectly fresh, seems 
not to be valid for all cases. For Drasch (Sitzb. h. h. ATcad. 
Wiss. Wien, 1881, p. 171, and 1884, p. 516; and Abhand. 
math.-phys. Cl. d. K. Sach. Ges. d. IFfss., xiv, No. 5, 1887; 
Zeit. /. iviss. Mih., iv, 4, 1887, p. 492) finds that better results 
are obtained with tissues that have been allowed to lie after 
death for twelve, twenty-four, or even forty-eight hours in a 
cool place. He even suspects that the function of the organic 
acids in the methods inspired by Lowit’s method is to bring 
the tissues into somewhat the state in which they are naturally 
found at a certain moment of post-mortem process—a state, 
namely, in which the nerves have a special susceptibility for 
impregnation with gold. 
369. Cohnheim’s Method (Virchow’s Arch., Bd. xxxviii, 
pp. 346—349 ; Strieker’sHandb.,p. 1100).—This, the archetype 
of the gold methods, was as follows :—Fresh pieces of cornea 
(or other tissue to be operated upon) are put into solution of 
chloride of gold of 0'5 per cent, strength until they are 
thoroughly yellow, and then exposed to the light in "water 
acidulated with acetic acid until the gold is thoroughly re- 
duced, which happens in the course of a few days at latest. 
They are then mounted in acidulated glycerin. 
The method in this, its primitive form, often gave splendid 
results, but was very uncertain, giving sometimes a nuclear 
or protoplasmic stain, sometimes an extra-cellular impregna- 
tion similar to that of nitrate of silver. And the preparations 
thus obtained are anything but permanent. 
370. Lowit’s Method.—The principle of this process is that, 
in order to facilitate the penetration of the gold and its sub- METALLIC STAINS (IMPREGNATION METHODS). 
253 
sequent reduction in the tissues, the tissues are made to swell 
up by treatment with formic acid before being brought into 
the gold-bath, and formic acid is employed to assist the re- 
duction after impregnation. 
The following directions as to this method, which may 
serve as a type of the modern methods of research on nerve- 
endings, are taken from Fischer’s paper on the corpuscles of 
Meissner {Arch. f. mih. Anat., xii, 1875, p. 366). 
Lowit’s method was first published by him in the Wien. 
Sitzgsber., Bd. lxxi, Abth. 3, 1875, p. 1. 
Small pieces of fresh skin are put into dilute formic acid, 
(one volume of water to one of the acid of 1*12 sp. gr.), and 
remain there until the epidermis peels off. They then are 
put for fifteen minutes into gold chloride solution (li per 
cent, to 1 per cent.), then for twenty-four hours into dilute 
formic acid (1 part of the acid to 1—3 of water), and then for 
twenty-four hours into undiluted formic acid. (Both of these 
stages are gone through in the dark.) Thin sections are then 
made and mounted in dammar or glycerin. Successful pre- 
parations show the nerves alone stained, but it is not possible 
always to control the results. 
371. Ranvier’s Formic Acid Method {Quart. Journ. Mic. Sci. 
[N.S.], lxxx, 1880, p. 456).—The method of Lowit has been 
modified by many workers by omitting the final treatment 
with undiluted formic acid, and also in some other details. 
Ranvier proceeds as follows :—Reflecting that the action of 
the one third formic acid in which Lowit placed his tissues 
must be hurtful to the final ramifications of the nerves, he 
combines the formic acid with a fixing agent designed to 
antagonise its altering action, and takes for this purpose the 
chloride of gold itself. The tissues are placed in a mixture 
of chloride of gold and formic acid (4 parts of 1 per cent, gold 
chloride to 1 part of formic acid) which has been boiled and 
allowed to cool (Ranvier’s Traite, p. 826). They remain in 
this until thoroughly impregnated (muscle twenty minutes, 
epidermis two to four hours); the reduction of the gold is 
effected either by the action of daylight in acidulated water, 
or in the dark in dilute formic acid (1 part of the acid to 
4 parts of water). 
The object of boiling the mixture of gold chloride and 254 
CHAPTER XX. 
formic acid is this, that “ by boiling in the presence of the 
acid the gold acquires a great tendency to reduction, and 
for this reason its selective action on nervous tissues is 
enhanced.” 
372. Ranvier’s Lemon-juice Method (Traite,p. 813).—Instead 
of combining the formic acid with gold chloride in order to 
mitigate its action, recourse may be had to a less injurious 
acid than formic acid. Ranvier finds that of all acids lemon- 
juice is the least hurtful to nerve-endings. He therefore soaks 
pieces of tissue in fresh lemon-juice, filtered through flannel, 
until they become transparent (five or ten minutes in the case 
of muscle). They are then rapidly washed in water, brought 
for about twenty minutes into 1 per cent, gold chloride solu- 
tion, washed again in water, and brought into a bottle con- 
taining 50 c.c. of distilled water and two drops of acetic acid. 
They are exposed to the light, and the reduction is complete 
in twenty-four to forty-eight hours. The preparations thus 
obtained are good for immediate study, but are not permanent 
on account of their over-blackening with time, the reduction 
of the gold being incomplete. In order to obtain perfectly 
reduced, and therefore permanent, preparations, the reduction 
should be done in the dark in a few cubic centimetres of dilute 
formic acid (1 part acid to 4 of water). The reduction is 
complete in twenty-four hours. 
373. Viallanes’ Osmic Acid Method (Hist, et dev. des Insectes, 
1883, p. 42).—The tissues are treated with osmic acid (1 per 
cent, solution) until they begin to turn brown, then with one 
fourth formic acid for ten minutes; they are then put.into 
solution of chloride of gold of 1 : 5000 (or even much weaker) 
for twenty-four hours in the dark, then reduced in the light 
in one fourth formic acid. According to my expei’ience this 
is a very excellent method, both the fixation by osmic acid 
and the great dilution of the gold solution being features 
likely to be of advantage in many cases. 
374. Other Methods.—The numerous other methods that 
have been proposed differ from the foregoing partly in 
respect of the solutions used for impregnation, but chiefly METALLIC STAINS (IMPREGNATION METHODS). 
255 
in respect of details imagined for the purpose of facili- 
tating the reduction of the gold, and rendering it as complete 
as possible. 
Thus Bastian modified Cohnheim’s original method by- 
employing a solution of gold chloride of a strength of 1 to 
2000, acidulated with HC1 (1 drop to 75 c.c.), and perform- 
ing the reduction in a mixture of equal parts of fonnic 
acid and water kept warm, heat being an agent that 
furthers reduction. 
Henocque (Arch, de VAnat. et de la Physiol., 1870, p. Ill) 
impregnates in a 0'5 per cent, solution of gold chloride, 
washes in water for twelve to twenty-four hours, and re- 
duces, with the aid of heat, in a nearly saturated solution 
of tartaric acid. The tartaric acid solution must be con- 
tained in a well-stoppered bottle. The best temperature 
for reduction is 40° to 50° 0. Reduction is effected very 
rapidly, sometimes in a quarter of an hour. 
This process has been described as the method of Chrscht- 
schonowic (Arch.f. mik. Anat., vii, 1872, p. 383). 
Hoyer (Arch.f. mik. Anat., ix, 1873, p. 222) proceeds as 
follows :—(For corneal nerves.) The double chloride of gold 
and potassium has the following advantages over the simple 
gold chloride. It is more easy to be obtained of unvarying 
composition, it is more perfectly neutral, and its solutions are 
more perfectly stable. It is used in solutions of the same 
strength as chloride of gold, viz. 0*5 per cent. Corneae 
must be very thoroughly imbibed with the solution. Small 
corneae (rabbit, guinea-pig) require half to one hour, human 
corneae two to five hours (in an acidulated solution). It is 
better to err on the side of too prolonged immersion rather 
than the contrary. In order to demonstrate the intra-epithe- 
lial ramifications of nerves, the gold is partially reduced by 
exposure for sixteen to twenty-four hours in (one or two 
ounces of) distilled water, and there is added to the water one 
or two drops of a pyrogallic acid developing solution, such 
as is used in photography (vide Gerlach, Die Photographic 
als Hiilfsmittel der mikroskopischen Forschung, Leipzig, 1863). 
Or instead of treating them with the developing solution, the 
corneae may be removed to a warm concentrated solution of 
tartaric acid, and remain there at the temperature of an in- 
cubating stove until the gold is fully reduced. 256 
CHAPTER XX. 
I have myself used the double chloride of gold and sodium 
with good results. 
Ciaccio (Journ. de Microgr., vii, 1883, p. 38; Journ. Roy. 
Mic. Soc. [N.S.], iii, 1883, p. 290) prefers the double chloride 
of gold and cadmium. 
For Gerlach’s method for hardened nerve-centres see 
Strieker’s Handb., 1872, p. 678. It may be considered to be 
now superseded by Golgi’s bichromate of silver process. 
Flechsig (Die Leitungsbahnen im Gehirn, 1876; Arch. f. 
Anat. u. Phys., 1884, p. 453) reduces in a 10 percent, solution 
of caustic soda. 
Nesteroffsky treats impregnated preparations with a drop 
of sulphydrate of ammonium, and finishes the reduction in 
glycerin (quoted from Gierke’s Fdrberei z. mik. Zwecken). 
Bohm reduces in Pritchard’s solution. 
Pritchard’s solution consists of amyl alcohol, 1 per cent.; 
formic acid, 1 per cent.; and water, 98 per cent. 
Manfredi treats fresh tissues as follows (Arch, per le Sci. 
med., y, No. 15) :—Gold chloride, 1 per cent., half an hour; 
oxalic acid, 0-5 per cent.; they are then warmed in a water- 
bath to 36°, allowed to cool, and examined. Mount in gly- 
cerin. Sunny weather is necessary. 
He treats tissues previously hardened in 2 per cent, solu- 
tion of bichromate of potash, as follows [ibid.). They are put 
for half an hour into solution of arsenic acid, or into 1 per 
cent, acetic acid. They are then put into 1 per cent, gold 
chloride for half an hour, washed in water, and reduced in 
sunlight in 1 per cent, arsenic acid solution, which is changed 
for fresh as fast as it becomes brown. Mount in glycerin. 
Sunny weather is necessary. 
Boccardi (Lavori Instit. Fisiol. Napoli, 1886, i, p. 27; Journ. 
Roy. Mic. Soc., 1888, p. 155) recommends oxalic acid of 01 
per cent, or of 025 to 03 per cent., or a mixture of 5 c.c. pure 
formic acid, 1 c.c. of 1 per cent, oxalic acid, and 25 c.c. of 
water. Objects should remain in this fluid in the dark not 
longer than two to four hours. 
Kolossow (Zeit. f. wiss. Mik., v, 1, 1888, p. 52) impregnates 
for two or three hours in a 1 per cent, solution of gold chloride 
acidulated with 1 per cent, of HC1, and reduces for two or 
three days in the dark in a 0‘01 per cent, to 0'02 per cent, 
solution of chromic acid. METALLIC STAINS (IMPREGNATION METHODS). 
257 
Underwood (Journ. Brit. Dental Ass., xi, 1890, p. 696; 
Journ. Boy. Mic. Soc., 1890, p. 815) gives the following :— 
Wash sections in bicarbonate of soda (strength not given) ; 
treat them for half an hour to an hour with 1 per cent, solu- 
tion of chloride of gold, neutralised if acid by bicarbonate of 
soda; wash in water; reduce until the sections turn crimson 
(about an hour) in 1 per cent, formic acid kept “faii’ly hot ” 
on a water-bath, in the dark; wash for half an hour in cold 
water; dry the sections (sic) and mount them in glycerin 
jelly (“specimens mounted in balsam always go wrong”). 
This is for sections of decalcified teeth. 
Geberg (Intern. Monatsschr., x, 1893, p. 205) states that' 
previous treatment of tissues for twenty-four hours with lime- 
water (Arnstein’s method) greatly helps the reduction. 
Bernheim (Arch. f. Anat. u. Phys., Phys. Abth., 1892, 
Supp., p. 29; Zeit. f. wiss. Mik., x, 4, 1893, p. 484) adds to 
Lowit’s dilute formic acid a piece of sulphite of sodium 
(must be fresh and smell strongly of sulphurous acid). 
Dr. Lindsay Johnson writes to me that besides the “ sun- 
ning ” of the impregnating solution recommended above 
(§ 356), the following precautions should be taken :—“ The 
tissue must be well washed in distilled water, and the gold 
carefully acidulated with a neutral acetate or formiate, or 
acetic or formic acid, at least twenty-four hours before using; 
and then afterwards the tissue must be washed until no re- 
action occurs to test-paper.” 
For the details of the application of the methods of which 
the principles have been set forth above, and for those of the 
important processes of impregnation of central nerve organs, 
the reader is referred to those chapters of Part II which, 
treat of nerve-tissues and organs. 
375. Ulterior Treatment of Impregnated Preparations.—Pre- 
parations may be mounted either in balsam or in acidulated 
glycerin (1 per cent, formic acid). 
Theoretically they ought to be permanent if the reduction 
of the metal has been completely effected. 
In practice, all are doomed to destruction in course of time 
by after-blackening, and few will be found to survive more 
than a few months. Kanvier states that this can be avoided 
by putting the preparations for a few days into alcohol, which 258 
CHAPTER XX. 
possesses tbe property of stopping the reduction of the gold. 
But this must be taken to mean that by this device the period 
of usefulness of the preparations may be prolonged for some 
time, not indefinitely. 
Blackened preparations may be bleached with cyanide or 
ferrocyanide of potassium. Bedding employs a weak solution 
of ferrocyanide ; Cybulsky a 0'5 per cent, solution of cyanide. 
But the results are far from being perfectly satisfactory. 
Preparations may be double-stained with the usual stains 
(safranin and methyl green being very much to be recom- 
mended), but nuclei will only take the second stain in the 
case of negative impregnation. 
376. Impregnation of Marine Animals.—For some reason that 
I am unable to explain, the tissues of marine animals do not 
readily impregnate with gold in the fresh state. It is said by 
Fol that impregnation succeeds better with spirit specimens. 
Other Metallic Stains. 
377. Osmic Acid and Pyrogallol.—Everybody knows that osmic 
acid stains tissues. Most people, I should think, would be heartily glad if 
it did not. Meanwhile, to make the best of this willy-nilly stain, you may 
sometimes find it useful to treat the tissues with weak pyrogallic acid, 
which will very quickly turn them of a line greenish black, sometimes 
giving useful differentiations. 
This method was first published by me in 1887 {La Cellule, t. iv, fasc. 1, 
p. 110), and I have since republished it twice, with qualified recommenda- 
tion. I have found it occasionally very useful, but have hesitated to recom- 
mend it for general work because I have usually found the stain too 
energetic and too little discriminating. It frequently happens that every- 
thing in a preparation comes out blacker than ink. 
At the time of the publications mentioned above I had only- 
tried the reaction with tissues impregnated with 'pure osmic 
acid. Since then Hermann (Arch. f, mik. Anat., xxxvii, 4, 
1891, p. 570) tried it with tissues fixed in his platino-aceto- 
osmic mixture (§ 65). With this modification of the process 
incomparably better results are obtained, as I can testify. 
Hermann’s procedure is as follows :—The tissues are put for 
one or two days into the platino-aceto-osmic mixture, washed 
thoroughly in water, and hardened in successive alcohols ; 
after which, to obtain the black reaction, they are put for METALLIC STAINS (IMPREGNATION METHODS). 
259 
twelve to eighteen hours into raw pyroligneous acid. This 
acid ought (Ergebnisse der Anat., ii, 1893, p. 28) to be as raw 
as possible, and to be of a dark brown colour and evil-smelling. 
According to my experience, the particular procedure of 
Hermann is not that which gives the best results. I now pro- 
ceed as follows : 
Either the mixture of Hermann or the mixture of Flemming 
may be used for fixing. You may leave the tissues therein 
for twelve or twenty-four hours if you think that that is 
desirable in the interest of a complete fixation; but in the 
interest of the stain alone, half an hour is enough and is pre- 
ferable. It is not only useless but hurtful to put the prepara- 
tions into alcohol after fixation, for it is desirable that the 
tissue should be in as fresh a state as possible on coming into 
the pyrogallol. In consequence it is not possible to obtain 
the best results by treating paraffin sections. The tissues are 
therefore brought in bulk, directly after fixing, into pyrogallol. 
You may use Hermann’s pyroligneous acid if you like, but I 
consider that a weak solution of pyrogallol is preferable. In 
pyrogallol it seems to me that a finer differentiation is obtained, 
and that the preservation of the tissues is sensibly better. 
The tissues may remain in either of these liquids for twenty- 
four hours, but for small objects an hour or less is sufficient. 
An alcoholic solution of pyrogallol may be taken if desired, 
and this may be indicated in certain cases. I have not 
obtained the reaction with tannin used on chrom-osmium 
material. 
The method as thus modified is important. There is obtained 
a black stain, which is at the same time a plasma stain and 
a nuclear stain, chromatin being so far stained that it is not 
necessary to have recourse afterwards to a special chromatin 
stain. This is one of the best methods that I know of for the 
study of Nebenkerne. With Invertebrates it sometimes gives 
very elegant differentiations of nervous tissue. It is a very 
easy method, and if pyrogallol be used a very safe one (with 
pyroligneous acid not so safe). But it is not quite a first-class 
method, for the plasma stain obtained is not quite of the 
highest order, the cytoplasmic hyaloplasm or enchylema being 
too frequently stained at the same time as the reticulum. 
Although, as said, this method enables one to dispense with 
a special second chromatin stain, yet it is frequently veryadvan- 260 
CHAPTER XX. 
tageous to use one. I greatly recommend safranin (stain 
very strongly, twenty-four hours at least, and start the extrac- 
tion with acid alcohol). 
This method has been attributed to von Maehrenthal. A modification 
of this method is said by Azoulay to give a specific stain of the medullary 
sheath of nerves, see his process under “ Neurological Methods ” in Part II. 
See also a similar process for medullated nerve by Heller and Gumpertz, 
quoted Zeit. f. wiss. Mile., xii, 3, 1896, p. 385. 
The communications of Kolossow (Zeit. f. wiss. Mile, ix, 1, 1892, p. 38, 
and ix, 3, 1893, p. 316) do not appear to me to constitute a useful contribu- 
tion to the subject. 
378. The method of Brosicke (Centralb. f. d. med. TFiss., 1879, p. 873 ; 
Zeit.f. wiss. Mile., i, 1884, p. 409) consists in treating osmium objects for 
twenty-four hours with a solution of one part of oxalic acid in fifteen parts 
of water. This gives a Burgundy-red stain. It is necessary that the 
objects be washed and brought into the oxalic acid as soon as possible after 
the treatment with osmium, as if the osmium has once begun to blacken 
them, the oxalic acid is powerless afterwards to redden them. I consider 
this method to be superseded by the pyrogallol process. 
379. Perchloride of Iron.—This reagent, introduced by Polaillon 
{Journ. de VAnat., iii, 1866, p. 43), sometimes gives most useful results, 
especially in the study of peripheral nerve-ganglia, in which it stains the 
nervous tissue alone, the connective tissue remaining colourless. The 
method consists in impregnating in perchloride of iron, and reducing in 
tannic, gallic, or pyrogallic acid. 
The Hogg an s, who have done very good work with this reagent, proceed 
as follows (Journ. Queleett Club, 1876; Journ. Boy. Mic. Soc., ii, 1879, 
p. 358) :—The tissue (having been first fixed with silver nitrate, which is 
somewhat reduced by a short exposure to diffused light) is dehydrated in 
alcohol, and treated for a few minutes with 2 per cent, solution of per- 
chloride of iron in spirit. It is then treated with a 2 per cent, solution of 
pyrogallic acid in spirit, and in a few minutes more, according to the depth 
of tint required, may be washed in water and mounted in glycerin. 
Fol (see ante, § 67) fixes in perchloride solution, and treats the prepara- 
tions for twenty-four hours with alcohol containing a trace of gallic acid. 
Polaillon (l. c.) reduces in tannic acid. 
This method is not applicable to chromic objects. 
I should add that in my own experience I have found it very useful in 
certain special cases. 
380. Pyrogallate of Iron (Roosevelt, Med. Bee., ii, 1817, p. 84; 
Journ. Boy. Mic. Soc., 1888, p. 157).—A stain compound of 20 drops of 
saturated solution of iron sulphate, 30 grms. water, and 15 to 20 drops pyro- 
gallic acid. METALLIC STAINS (IMPREGNATION METHODS). 
261 
381. Palladium Chloride (F. Schultze, see ante, §§66 and 102). 
Prussian Blue (see Leber, Arch.f. Ophthalm., xiv, p. 300; Rattier, 
Traite, p. 108). Cupric Sulphate (see Leber, ibid.). Lead Chromate 
(see Leber, ibid.). Sulphides (see Landois, Centralb. f. d. med. PFiss., 
1885, No. 55; and Gierke, in Zeit. f. iviss. Mile., i, 1884, p. 497). 
Molybdate of Ammonium (Merkel ; Krause) (see Gierke, Zeit. f. 
iviss. Mile., i, 1884, p. 96). Oxychloride of Ruthenium (Nicolle and 
Cantacuzene) (see Ann. Inst. Pasteur, vii, 1893, p. 331). Impregna- 
tion with Pats, Altmann’s Method (see post, “ Corrosion”). CHAPTER XXL 
EXAMINATION AND PEE SERRATION MEDIA. 
382. Introductory.—I comprehend under this heading all 
the media in which an object may be examined. The old 
distinction of “ indifferent ” liquids, and those which have 
some action on tissues, appears to be misleading more than 
helpful; inasmuch as it is now well understood that no 
medium is without action on tissues except the plasma with 
which they are surrounded during the life of the organism ; 
and this plasma itself is only “ indifferent ” whilst all is in 
situ ; as soon as a portion of tissue is dissected out and trans- 
ferred to a slide in a portion of plasma the conditions become 
evidently artificial.. 
It does not appear necessary to create a separate group for 
mounting media, as all preservative media may be used for 
mounting. 
For directions as to making permanent mounts in fluid 
media see the early sections of Chap. XXII. 
383. Water.—To preserve it from mould, a lump of thymol or camphor 
should be kept in the supply. Water may be employed without inconveni- 
ence, and sometimes (on account of its low index of refraction) with great 
advantage for the examination of all structures that have been fixed with 
osmic or chromic acid, or some salt of the heavy metals; but it is by no 
means applicable to the examination of fresh tissues,—that is, tissues that 
have not been so fixed. It is important that the beginner should bear in mind 
that water is very far from being an “ indifferent ” liquid; many tissue-ele- 
ments are greatly changed by it (nerve-end structures, for instance), and some 
are totally destroyed by its action if prolonged (for instance, red blood-cor- 
puscles). 
384. Theory of Indifferent Liquids.—In order to render 
water inoffensive to such tissues as these it must, firstly, have 
dissolved in it some substance that will give it a density 
equal to that of the liquids of the tissue, so as to prevent the 
occurrence of osmosis, to which process the destructive action 
of pure water is mainly due. Salt solution is a medium sug- 
gested by this necessity. But salt solution by no means EXAMINATION AND PRESERVATION MEDIA. 
263 
fulfils all the conditions implied in the notion of an “ indif- 
ferent ” liquid. In so far as it possesses a density approach- 
ing to that of the liquids of the tissues, one cause of osmosis 
is eliminated; but there remains another, due to the dif- 
ference of composition of the liquids within the tissues and 
that without. Cell contents are a mixture of colloids and 
crystalloids; salt solution contains only a crystalloid, whose 
high diffusibility causes it to diffuse over into the colloids of 
the tissues. In order to reduce the consequent osmotic pro- 
cesses to a minimum, it is necessary that the examination 
medium contain, in addition to a due proportion of salt or 
other crystalloid, also a due proportion of colloids. By 
adding, for instance, white of egg to salt solution this end 
may be attained; and, as a matter of fact, the liquids re- 
commended as indifferent are found invariably to contain 
both crystalloid's and colloids. Thus (as stated by Frey) 
vitreous humour contains 987 parts of water to about 4’6 of 
colloid matters and 7#8 of crystalloids (common salt). In 
1000 parts of liquor amnii are contained about 3‘8 parts of 
colloid matter (albumen), 5‘8 of salt, and 3’4 of urea. In 
blood-serum, 8’5 of colloids and 1 of crystalloid substance 
are found. 
385. Salt Solution (“ normal salt solution,” “ physiological 
salt solution ”).—O’75 per cent, sodium chloride in water. 
Carnoy recommends the addition of a trace of osmicacid. 
386. Pictet’s Liquid (Mitth. Zool. Stat. Neajiel, x, 1, 1891, 
p. 89).—Five to ten per cent, solution of chloride of man- 
ganese. According to my experience, this solution is ex- 
cellent, and very often advantageously takes the place of 
Cfnormal salt solution.” The proportions given are for 
marine animals, and for terrestrial animals will generally be 
found much too high. For these from 1 per cent, to 3 per 
cent, will be nearer the mark. 
387. Iodised Serum.—Iodised serum was first recommended 
by Max Schultze (Virchow's Archiv, xxx, 1864, p. 263). I 
take the following- instructions concerning it from Ranvier 
(Traite, p. 76). 
The only serum that gives really good results is the amniotic 264 
CHAPTER XXI. 
liquid of mammals. A gravid uterus of a sheep or cow 
having been obtained (in large slaughterhouses such can be 
obtained without difficulty), an incision is made through the 
wall of the uterus and the foetal membranes. A jet of serum 
issues from the incision, and is caught in a flask prepared for 
the purpose. Flakes of iodine are then added, and the flask 
is frequently agitated for some days. Two points should be 
noted. A perfectly fresh amnios must be taken, for the 
merest incipience of putrefaction will spoil the preparation. 
The flask should have a wide bottom, so that the serum may 
form only a shallow layer in it; otherwise the upper layers 
will not be sufficiently exposed to the action of the iodine. 
Another method is as follows :—Serum is mixed with a 
large proportion of tincture of iodine; the precipitate of 
iodine that forms is removed by filtration, and there remains 
a strong solution of iodine in serum. This should be kept in 
stock, and a little of it added every two or three days to the 
serum that is intended for use. 
Ranvier explains that at the outset serum dissolves very 
little iodine ; but if an excess of iodine be kept constantly 
present in the solution, it will be found that after two or 
three weeks iodides are formed, and allow fresh quantities of 
iodine to dissolve; so that after one or two months a very 
strongly iodised serum is obtained. It should be dark brown. 
Such a solution is the most fitting for the purpose of iodising 
fresh serum in the manner directed above, and for making 
the different strengths of iodised serum that are required for 
different purposes. In general, for maceration purposes, a 
serum of a pale brown colour should be employed. 
388. Aqueous Humour, Simple White of Egg.—Require no 
preparation beyond filtering. They may be iodised if 
desired. 
389. Artificial Iodised Serum (.Frey, Le Microscope, p. 131). 
Distilled water . . . . .135 grms. 
White of egg . . . . . 15 „ 
Sodium chloride . . . . . 0120 grm. 
Mix, filter, and add— 
Tincture of iodine .... 3 grms. EXAMINATION AND PRESERVATION MEDIA. 
265 
There is formed a precipitate, which is removed by filtering 
through flannel; and a little iodine is added to the filtrate. 
390. Kronecker’s Artificial Serum (from Vogt et Yung, 
Traite d’Anat. comp, prat., p. 473 : I have been unable to 
discover the original source). 
Common salt ...... 6 grms. 
Caustic soda ...... 0’06 grm. 
Distilled water .... 1000 grms. 
391. Migula’s Glycerized Blood-serum (see the paper in Zeit. 
f. wiss. Mik., vii, 2, 1890, p. 172; also Journ. Roy. Mic. Soc., 
1890, p. 804). 
392. Syrup .—An excellent medium for examining many 
structures in the fresh state. To preserve it from mould, 
chloral hydrate may conveniently be dissolved in it (1 to 5 
per cent.). I have used as much as 7 per cent., and found no 
disadvantage. 
Carbolised Syrup.—Carbolic acid may be employed instead 
of chloral; 1 per cent, is sufficient. 
Either of these syrups maybe used as a mounting medium, 
but they are not to be recommended for that purpose, as there 
is always risk of the sugar crystallising but. 
A good strength for syrup is equal parts of loaf sugar and 
water. Dissolve by boiling. 
393. Acetate of Alumina (Gannal’s Solution, Beale, How to WorTc, 
&c., p. 58). 
Acetate of alumina ..... 1 part. 
Water ........ 10 parts. 
394. Acetate of Potash (Max Schultze, Arch. mik. Anat., vii, 1872, 
p. 180).—A nearly saturated solution in water. It is used by letting a drop 
run in under the cover-glass to the object, which is in water. After twenty- 
four hours the mount may be closed. The index of refraction is lower than 
that of glycerin. 
This medium has been frequently recommended as having the property 
of preventing the blackening of objects that have been treated with osmium ; 
hut it seems extremely doubtful whether this is really the case. 
395. Chloral Hydrate.—5 per cent, in water (Ladowsky, 
Arch. f. mile. Anat., 1876, p. 359). 266 
CHAPTER XXI. 
Or, 2‘5 per cent, in water (Brady, British Gopepods). 
Or, 1 per cent, in water (Munson, Journ. Boy. Mic. Soc.. 
1881, p. 847). 
396. Alcohol.—Not very recommendable for mounting, as if taken weak 
it is not a very efficient preservative, and if taken strong it attacks the 
cement of mounts. 
Carpenter (The Microscope) recommends a strength of 1 part to 5 of 
water. 
The chief use of alcohol for preservation purposes is of 
course for preserving specimens in till wanted for further 
preparation and study. See, on this point, the remarks in 
Chap. I, § 3. 
397. Formaldehyde. See §§ 86 and 109. 
Mercurial Liquids. > 
398. Gilson’s Fluid (Caenoy’s Biologie cellulaire, p. 94). 
Alcohol of 60 per cent. . . .60 c.c. 
W ater . . . . . 30 „ 
Glycerin . . . . . 30 ,, 
Acetic acid (15 parts of the glacial 
to 85 of water) . . . . 2 ,, 
Bichloride . . . . .0*15 grm. 
A really excellent medium for the study of fine cellular 
detail with well-fixed objects. 
399. Gage’s Albumen Fluid (Zeit. f. wiss. Mik., 1886, p. 223). 
White of egg . . . .15 c.c. 
Water . .• . • . . . 200 ,, 
Corrosive sublimate . . . 0-5 grm. 
Salt ...... 4 grms. 
Mix, agitate, filter, and preserve in a cool place. Recom- 
mended for the study of red blood-corpuscles and ciliated cells. 
400. Pacini’s Fluids (Journ. de Mic., iv, 1880; Journ. Boy. Mic. Soc. 
[N.S.], ii, 1882, p. 702, and previous editions of this work).—These anti- 
quated formulae are quite superfluous for the study of fixed tissues. They 
consist essentially of corrosive sublimate of from one half to one third per 
cent, strength, with the addition of a little salt or acetic acid. 
. .. ' ' ■ ' ' ' . •• ' 
401. Harting’s Fluid.—See Micro. Diet., art. “ Preservation,” p. 640. EXAMINATION AND PRESERVATION MEDIA. 
267 
402. Goadby’s Fluids (Micro. Diet., art. “ Preservation,” or pre- 
vious editions of this work).—They are quite unsuited for histological pur- 
poses. 
403. Owen’s Fluid (see Vogt et Yung, Traite d’Anat. comp.pratique, 
p. 19, or previous editions of this work).—It is quite superfluous for tissues 
that have been duly fixed. 
Other Fluids. 
404. Chloride and Acetate of Copper (Kipart et Petit’s fluid, 
Brebissonia, 1880, p. 92; Carnot’s Biol, cell., p. 95). 
Camphor water (not saturated) . 75 grms. 
Distilled water . . . .75 ,, 
Crystallised acetic acid ... 1 grm. 
Acetate of copper . . . . 0*30 ,, 
Chloride of copper .... 0'30 „ 
This is certainly a most valuable medium for work with 
delicate fresh tissues. It may be used in combination with 
methyl green, which it does not precipitate. The most delicate 
elements are perfectly preserved in it; the addition of a drop 
of osmic acid or corrosive sublimate does not cause the least 
turbidity, and enhances its fixing action. 
405. Tannin (Caenoy, l. c.). 
Water ..... 100 grms. 
Powdered tannin . . . 0'50 grm. 
406. Piero-carmine.—Picro-carmine has been recommended by Ranvier 
as a medium for teasing fresh tissues in, in the belief that it possesses suffi- 
cient fixing action to preserve the form of cells. Carnoy finds that cells live 
in it for a considerable time, and become gorged with water and deteriorated 
to a considerable degree. Unfortunately, too, picro-carmine cannot be com- 
bined with a good fixing agent, as it is precipitated by alcohol and by acids, 
and especially by osmic acid. 
407. Methyl Green.—See under Staining Agents. The 
aqueous solution is very useful as an examination medium for 
fresh tissues. It should be taken fairly concentrated, in 
which state it has sufficient fixing power, which is enhanced 
by the addition of a trace of osmic acid. 
408. Wickersheimer’s Fluid (Zool. Anz., 1879, p. 670; cf. Journ. 
Roy. Mie. Soc., 1882, p. 427; id., 1880, p. 355; and Entomol. Nachr. 
1880, p. 129).—This once famous fluid appears to be quite unsuccessful for 
histological purposes. 
409. Meyer’s Salicylic Vinegar Preservative Solutions (Arch, 
mik. Anat., xiii, 1876, p. 868).—“Salicylic vinegar” is a 2 68 
CHAPTER XX[. 
solution of 1 part of salicylic acid in 100 parts of pyroligneous 
acid. The pyroligneous acid should be of 1‘04 specific gravity, 
and should be of a pale yellow colour. This product is found 
in commerce, and may be obtained from Herrn J. M. Andreae, 
Droguerie-Handlung, Frankfurt-a.-M. 
1st Fluid : 
One vol. salicylic vinegar to 10 vols. of the following dilute 
glycerin, viz. glycerin 1 vol., water 2 vols. 
For various larvae, Hydrae, Nematodes, &c. 
2nd Fluid : 
One vol. salicylic vinegar to 10 vols. of the following dilute 
glycerin, viz. glycerin 1 vol., water 4 vols. 
For Infusoria. 
410. Noll’s Salicylic Vinegar and Gum Medium (Zool. Anz., 
1883, p. 472).—A mixture of equal vols. of Meyer’s second 
fluid (ante, last formula) and Farrant’s medium (post, § 413.) 
This mixture never becomes turbid, and does not dry up. 
The covers may be luted with asphalt or any other cement. 
The fluid answers admirably for delicate Crustacea and their 
larvae; the preparations do not shrink, and are not too much 
cleared. It also answers well for hardened and stained pre- 
parations of Hydroids, small Medusae, and other Ccelen- 
terates. 
411. Deane’s Medium (see Micro. Diet., art. “ Preservation ”).—Appears 
to be now superfluous. 
412. Hoyer’s Gum with Chloral Hydrate or Acetate of Potash 
(Biol. Centralb., ii, 1882, pp. 23-4; Journ. Roy. Mic. Soc. 
[N.S.], iii, 1883, pp. 144-5).—A high 60 c.c. glass with a 
wide neck is filled two thirds full with gum arabic (in pieces), 
and then either a solution of chloral (of several per cent.) 
containing 5—10 per cent, of glycerin is added, or acetate of 
potash or ammonia. The gum with frequent shaking dissolves 
in a few days, and forms a syrupy fluid, which is slowly 
filtered for twenty-four hours. The clear filtered fluid will 
keep a long time, but if spores of fungi begin to develop a 
little chloral can be added and the fluid refiltered. The 
solution with chloral is for carmine or hsematoxylin objects— 
that with acetate for anilin objects. 
413, Farrant’s Medium (Beale, How to Work, &c., p. 58). EXAMINATION AND PRESERVATION MEDIA. 
269 
Picked gum arabic . . . .4 ounces. 
Water . . . . . . 4 ,, 
Glycerin . . . . . . 2 „ 
To be kept in a stoppered bottle with a lump of camphor. 
This medium is quoted by Frey as consisting of equal parts 
of gum, glycerin, and saturated aqueous solution of arsenious 
acid. 
The Micrographic Dictionary gives the following directions: 
—Gum arabic 1 ounce, glycerin 1 ounce, water 1 ounce, 
arsenious acid If grains ; dissolve the arsenious acid in the 
water, then the gum (without heat), add the glycerin, and 
incorporate with great care to avoid forming bubbles. 
Another method for making this medium is given by A. F. 
Stanley Kent in Journ. Boy. Mic. Soc., 1890, p. 820. 
414. Gum and Glycerin Medium (Langerhans’ formula, a 
modification of Farrant’s medium, Zool. Anzeig., ii, 1879, 
p. 575). 
Gummi arab. ...... 5‘0 
Aquae ....... 5‘0 
To which after twelve hours are added— 
Glycerini ....... 5-0 
Sol. aquosa acid, carbol. (5'100) . . 10'0 
Marine animals may be preserved in this by simply run- 
ning in a drop under the cover, and next day or later adding 
what is necessary to make up for evaporation, and closing 
the mount. Shrinkage is very slight, and most colours keep 
well. 
415. Faris’s Glycero-gum (The Microscope, x, 1890, p. 59 ; 
Journ. Roy. Mic. Soc., 1890, p. 414).—Gum arabic 2 ounces, 
glycerin 1*5 ounces, water 1*5 ounces, thymol 1 grm. Mix, 
dissolve with heat, and filter. 
416. Gum and Glycerin Jelly (Shimer, The Microscope, ix, 
1889, p. 138; Journ. Boy. Mic. Soc., 1890, p. 411).—Equal 
parts of glycerin jelly (Fol’s second formula, post, § 432), 
Farrant’s solution, and glycerin. 
417. Cole’s Gum and Syrup Medium. See § 179. 
418. Apathy’s Gum and Syrup Medium (see Chap. IX, § 
293).—This medium is recommended by Apathy in a general 270 
CHAPTER XXI. 
way, and not merely for the special purpose for which it is 
quoted in § 293. It sets very havd, and, combined with a 
paper cell (see § 453), may be used for ringing glycerin 
mounts. 
419. Fabre-Domergue’s Glucose Medium [La Nature, No. 823, 
9, Mars, 1889, supp.). 
Glucose syrup diluted to twenty- 
five degrees of the areometer 
(sp. gr. 1-1968) . . . 1000 parts. 
Methyl alcohol .... 200 ,, 
Glycerin ..... 100 ,, 
Camphor, to saturation. 
The glucose is to be dissolved in warm water, and the other 
ingredients added. The mixture, which is always acid, must 
be neutralised by the addition of a little potash or soda. 
This medium is said to preserve without change almost all 
animal pigments. 
420. Brun’s Glucose Medium (from Fabre-Domergue’s Pre- 
miers Principes du Microscope et de la Technique microscopique, 
Paris, 1889, p. 123). 
Distilled water .... 140 parts. 
Camphorated spirit . . . 10 ,, 
Glucose . . . . . 40 ,, 
Glycerin . . . . 10 „ 
Mix the water, glucose, and glycerin, then add the spirit, 
and filter to remove the excess of camphor which is pre- 
cipitated on mixing. I am indebted to Dr. HENNEGuyfor 
calling my attention to this liquid, which is an important one. 
It is preferable to glycerin because it preserves the colour of 
preparations stained with anilin dyes, methyl green included. 
421. Levulose as a Mounting Medium.—Levulose is recommended 
as a mounting medium by Behrens, Kossel, u. Schiefferdecker (Das 
Mikroslcop u. d. Metli. d. mik. TJnters., Braunschweig, 1889). It is un- 
crystallisable, and preserves well carmine and coal-tar stains (hgematoxylin 
stains fade somewhat in it). The index of refraction is somewhat higher 
than that of glycerin. Objects may be brought into it out of water. 
Glycerin Media. 
422. Glycerin.—Glycerin diluted with, water is frequently 
employed as an examination and mounting medium. Dilution EXAMINATION AND PRESERVATION MEDIA. 
271 
with water is sometimes advisable from an optical point of 
view, on account of the increased visibility that it gives to 
many structures by lowering the index of refraction of the 
glycerin. But from the point of view of efficacious preserva- 
tion it is always advisable to use undiluted glycerin, the 
strongest that can be procured. 
Long soaking of tissues in glycerin of gradually increased 
strength is a necessary preliminary to mounting in all cases 
in which it is desired to obtain the best possible preparations, 
and to ensure that they shall keep well. If this soaking is 
done on the slide (the cover being removed and the object 
treated with fresh glycerin every one or two days), it is well 
to take the precaution recommended by Beale, of luting the 
edges of the cover so as to make the preparation air-tight, as 
glycerin is so highly hygroscopic that a drop of it exposed to 
the air rapidly diminishes in strength to a very considerable 
degree. In order to facilitate the removal of the cover in 
this process, the slide may be gently warmed by passing* it 
two or three times through the flame of a spirit lamp. No 
preparation can be considered to be made secundum artem 
until every part of the object has been thoroughly impregnated 
with strong pure glycerin. 
The shrinking that frequently occurs when delicate struc- 
tures are brought into glycerin may generally be cured by 
this treatment; cells which first appear hopelessly collapsed 
gradually swell out to their normal forms and dimensions. 
For closing glycerin mounts, the edges of the cover should 
first (after having been cleansed as far as possible from 
superfluous glycerin) be painted with a layer of glycerin jelly; 
as soon as this is set a coat of any of the usual cements may 
be applied. This has of course -been for the last twenty years 
one of the commonplaces of histological technic, but that 
has not prevented somebody from recently describing the 
process at great length as new. 
Glycerin dissolves carbonate of lime, and is therefore to be 
rejected in the preparation of calcareous structures that it is 
wished to preserve. 
423. Extra-refractive Glycerin.—The already high index of refraction 
of glycerin (Price’s glycerin, n = 1'46) may be raised to about that of 
crown glass by dissolving suitable substances in the glycerin. Thus the 272 
CHAPTER XXI. 
refractive index of a solution of chloride of cadmium (CdCl2)* in glycerin 
may be T504; that of a saturated solution of sulphocarbolate of zinc in 
glycerin may be T501; that of a saturated solution of Schering’s f chloral 
hydrate (in crusts) in glycerin is T510; that of iodate of zinc in glycerin 
may be brought up to T56.£ The clearing action of glyeerin may thus be 
greatly increased, and the full aperture of homogeneous objectives brought 
to bear on objects mounted in one of the above-named solutions. 
The sulphocarbolate of zinc solution§ may be prepared by taking equal 
parts by weight of Price’s glycerin and sulphocarbolate of zinc crystals, 
mingling the two, and applying sufficient heat to boil the glycerin. The 
solution can be made in about an hour, but no fear need be had about 
boiling too long, as the longer this is done the less liability will there be 
for the solution to deposit crystals on the bottom of the bottle when cooled, 
which it will do if the temperature is only kept up long enough to dissolve 
the crystals. Filter while hot. The index may be brought up to T525 if 
desired by evaporating the solution somewhat, or by adding more carbolate. 
424. Barff’s Boroglyceri.de (see Journ. Roy. Mic. Soc., 1882, p. 124). 
—This preparation may be obtained (price Is. per bottle) from The Kreo- 
chyle Company, Viaduct House, Farringdon Street, E.C., or all whole- 
sale chemists. 
425. Glycerin and Alcohol Mixtures.—These most useful 
fluids afford one of the best means of bringing delicate objects 
gradually from weak into strong glycerin. The object is 
mounted in a drop of the liquid, and left for a few hours or 
days, the mount not being closed. By the evaporation of the 
alcohol the liquid gradually increases in density, and after 
some time the mount may be closed, or the object brought 
into pure glycerin or glycerin jelly. 
1. Calberla’s Liquid : 
Glycerin . . . . . .1 part. 
Alcohol . . . . . . 1 „ 
Water . . . . . . 1 ,, 
A most valuable examination fluid. As already pointed out 
(p. 5), this liquid is in many cases to be preferred to alcohol 
for keeping fixed objects in until required for dissection or 
other further preparation. 
2. I strongly recommend the following for very delicate 
objects : 
* Journ. Roy. Mic. Soc., ii, 1879, p. 346. 
t Ibid. (N.S.), i, 1881, p. 943. 
J Ibid., p. 366. 
§ Ibid., iii, 1880, p. 1051. EXAMINATION AND PEESERVATION MEDIA. 
273 
Glycerin 1 part. 
Alcohol . . . . . . 1 „ 
Water . . . . .2 parts. 
3. ILentsch’s Liquid : 
Glycerin 1 part. 
Alcohol . . . . . .3 parts. 
Water . . . . . . 2 ,, 
4. -Jager’s Liquid (quoted from Vogt and Yung’s fraite 
d’Anat. comp, prat., p. 16): 
Glycerin 1 part. 
Alcohol . . . . . . 1 „ 
Sea water . . . . . .10 parts. 
426. Deane’s Glycerin Jelly (from Frey’s Le Microscope, p. 
231).—120 grammes glycerin, 60 grammes water, 30 grammes 
gelatin. Dissolve the gelatin in the water, and add the gly- 
cerin. This, and the following glycerin jellies, must of course 
be used warm. 
427. Lawrence’s Glycerin Jelly (Davies, Preparation and 
Mounting of Microscopic Objects, p. 84).—“ He takes a quan- 
tity of Nelson’s gelatin, soaks it for two or three hours in cold 
water, pours off the superfluous water, and heats the soaked 
gelatin until melted. To each fluid ounce of the gelatin, 
whilst it is fluid but cool, he adds a fluid drachm of the white 
of an egg. He then boils this until the albumen coagulates 
and the gelatin is quite clear, when it is to be filtered through 
fine flannel, and to each ounce of the clarified solution add 6 
drachms of a mixture composed of 1 part of glycerin to 2 
parts of camphor water.” 
428. Beale’s Glycerin Jelly (How to Work, &c., p. 57).— 
Gelatin or isinglass, soaked, melted, and clarified if desired, 
as in the last formula. To the clear solution add an equal 
bulk of strong glycerin. 
429. Brandt’s Glycerin Jelly (Zeit.f . iviss. Mik., ii, 1880, p. 
69; Journ. Roy. Mic. Soc., iii, 1880, p. 502).—Melted gelatin 
1 part, glycerin 11 parts. 
The gelatin to be soaked in water and melted in the usual 
way. After incorporating the glycerin, the mixture is to be 274 
CHAPTER XXI. 
filtered. This is a point of vital importance, as the gelatin of 
commerce is always mixed with particles of dust and minute 
threads. Swedish does not allow the fluid to 
pass through sufficiently, and flannel produces more threads 
than before. The following simple apparatus is found effec- 
tive. A wide-necked bottle is broken in two, and the upper 
part taken. The neck is stopped with a cork having two holes 
bored in it. In the first hole a glass tube, about 20 cm. long, 
is inserted so as to project a little into the inside of the bottle, 
and on the outside it is bent sharply to one side and drawn 
out into a point of about to 2 mm. diameter. In the 
second hole a funnel-shaped filter is inserted so that the coni- 
cal part is inside the bottle and the tube projects a few centi- 
metres beyond the cork and the neck of the bottle. The 
appai'atus is then placed so that the wide opening of the 
bottle and of the funnel is uppermost, and some spun glass 
is pressed into the lower conical part of the filter. In using 
the apparatus the funnel is filled with glycerin gelatin, and 
the bottle with hot water, which runs off slowly through the 
tube in the first hole and is constantly replenished. 
Some drops of carbolic acid should be added to the fluid 
product of the filtering. For mounting, use warm by melt- 
ing a small portion on the slide, the object having been pre- 
viously soaked for some time in a small bottle of the medium 
warmed with a suitable apparatus. 
430. Kaiser’s Glycerin Jelly (Bot. Cent., i, 1880, p. 25; 
Journ. Boy. Mic. Soc., iii, 1880, p. 504).—One part by weight 
finest French gelatin is left for two hours in 6 parts by weight 
distilled water, 7 parts of glycerin are added, and for every 
100 grammes of the mixture 1 gramme of concentrated car- 
bolic acid. Warm for ten to fifteen minutes, stirring all the 
while, until the whole of the flakes produced by the carbolic 
acid have disappeared. Filter whilst warm through the finest 
spun glass laid wet in the filter. Use for mounting as above. 
I prepared some of this jelly many years ago, and find it is 
still perfectly clear. 
431. Seaman’s Glycerin Jelly (Amer. Mon. Mic. Journ., ii, 
1881, p. 45; Journ. Roy. Mic. Soc. [N.S.], i, 1881, p. 534).— 
Dissolve isinglass in water, so that it makes a stiff jelly when 
at the ordinary temperature of the room, add one tenth as EXAMINATION AND PRESERVATION MEDIA. 
275 
much glycerin, and a little solution of borax, carbolic acid, 
or camphor water. Filter whilst warm through muslin, and 
add a little alcohol. 
432. Fol’s Glycerin Jellies (Lelirb., p. 138). 
1. Melt together one volume of Beale’s jelly (§428) and 
one half to one volume of water, and add 2 to 5 per cent, of 
salicylic acid solution, or carbolic acid or camphor. 
2. Gelatin . . . . . .30 parts. 
Water . . . . . 70 ,, 
Glycerin . . . . . 100 ,, 
Alcoholic solution of camphor . 5 ,, 
Prepare as before, adding the camphor last. 
3. Gelatin ...... 20 parts. 
Water .... . . 150 ,, 
Glycerin ..... 100 ,, 
Alcoholic solution of camphor . .15 ,, 
433. Squire’s Glycerin Jelly (Squire’s Methods and 
Formulse, &c., p. 84).—Soak 100 grms. of French gelatin in 
chloroform water, drain when soft, and dissolve with heat in 
750 grms. of glycerin. Add 400 grms. of chloroform water 
with which has been incorporated about 50 grms. of fresh 
egg-albumen; mix thoroughly, and heat to boiling-point for 
about five minutes. Make up the total weight to 1550 grms. 
with chloroform water. Filter in a warm chamber. 
434. Gilson’s Chloral Hydrate Jelly (kindly communicated 
by Prof. Gilson).—1 vol. of gelatin, melted secundum artem, 
and 1 vol. of Price’s glycerin. Mix, and add 1 vol. of chloral 
hydrate (i. e. add crystals of chloral until the volume of the 
mixture has increased by one half); warm till dissolved. 
This gives a very highly refractive aqueous mounting medium, 
which is found useful for opaque tissues that it is desired not 
to dehydrate. 
A similar medium is published by Geoffroy, Journ. de 
Botan., 1893, p. 55 (see Zeit. f. wiss.Mik., ix, 4, 1893, p. 476). 
He dissolves, by the aid of as little heat as possible, 3 to 4 
grms. of gelatine in 100 c.c. of 10 per cent, aqueous solution 
of chloral hydrate. 
435. Stephenson’s Biniodide of Mercury and Iodide of Potas- 276 
CHAPTER XXI. 
sium (Journ. Roy.Mic. Soc. N.S., ii, 1882, p. 167).—A solution 
of the two salts in water. “ This is very easily prepared by 
adding- the two salts to the water until each shall be in excess; 
when this point of saturation has been reached the liquid will 
be found to have a refractive index of 1*68, by far the highest 
aqueous solution known to me. Its advantages from an 
optical point of view are considerable, and it may be used of 
any strength; commencing with pure water, with a refractive 
index of 1*33, we can go on progressively to 1-465, which 
represents glycerin, still on to 1'54 (Canada balsam), again 
onwards to 1'624, which represents bisulphide df carbon, to 
1-658, which represents the monobromide of naphthalin, to 
1-662, the equivalent of a solution of sulphur in bisulphide of 
carbon, until, undiluted, it finally reaches its own maximum 
of l-680; thus we have the representatives of all these media 
and an infinite number of others in this one fluid.” 
This fluid is very dense, its specific gravity being 3*02. It 
is highly antiseptic. 
“Its refractive index being T68, the visibility of diatoms, 
when mounted in it, is represented by the number 25 as com- 
pared with 11 in Canada balsam, in other words, the image 
is nearly two and a half times as strong 
For muscular fibre, on the other hand, a strong solution is 
not suitable, since the high refractive power of the object ap- 
proaches that of the medium, but as every other medium, of 
a lower index than 1’68 can, by dilution, be represented by 
it, any degree of visibility down to that of water can be 
obtained. 
“ For marine animals a weak solution is probably well 
adapted, as about a 1 per cent, solution (5 minims to the 
ounce) will give the specific gravity of sea water, with no 
appreciable difference in the refractive index.” 
Covers should be sealed with white wax, and the mounts 
finished with two or three coatings of gold-size and one of 
shellac. 
I have experimented both with strong and with weak solutions. They 
are not adapted, I find, for the purposes of a permanent mounting medium. 
Tissues are well preserved, hut the preparations are ruined by a precipitate 
which forms in the fluid. But as a temporary examination medium I have 
occasionally found this solution valuable. Its optical properties are wonder- 
ful ; it allows of the examination of watery tissues, without any dehydra- EXAMINATION AND PRESERVATION MEDIA. 
277 
tion, in a medium of refractive index surpassing that of any known resinous 
medium. 
436. Monobromide of Naphthalin. — See Journ. Roy. Mic. Soc., 
1880, p. 1043 (Abbe and van Heueck), and Zool. Anz., 1882, p. 555 (Max 
Flesch). 
437. Thompson’s High Refractive Medium.—See Journ. Roy. 
Mic. Soc., 1892, p. 902. 
Resinous Media. 
438. Resins and Balsams.—Resins and balsams consist of a 
vitreous or amorphous substance held in solution by an essen- 
tial oil. By distillation or drying in the air they lose the 
essential oil and pass into the solid state. It is these solidi- 
fied resins that should, in my opinion (and that, I believe, of 
the best microscopists), be employed for microscopical pur- 
poses ; for the raw resins always contain a certain proportion 
of water, which makes it difficult to obtain a clear solution 
with the usual menstrua, is injurious to the optical properties of 
the medium and to its preservative qualities, and, further, 
especially hurtful to the preservation of stains. I therefore 
recommend that all solutions be made by heating gently the 
balsam or resin in a stove until it becomes brittle when cold, 
and then dissolving in an appropriate menstruum. Solid 
resins are now easily found in commerce. 
Fol (Lehrb., pp. 138-9) is of a different opinion. 
Solutions made with volatile menstrua, such as xylol and 
chloroform, set rapidly, but become rapidly brittle. Solutions 
made with non-volatile media, such as turpentine, set much 
less rapidly, and pass much less rapidly into the brittle state. 
The former should, therefore, be employed whenever it is 
desired to have a mount that sets hard rapidly; but the 
latter should be employed whenever it is above all desired to 
have a mount that will prove as durable as possible. 
According to my experience, there is no such thing as a 
faultless resinous mounting medium for histological purposes. 
Solutions of gum Damar in xylol are very beautiful from the 
physical point of view, and frequently afford a better defini- 
tion of delicate detail than Canada balsam does. But I am 
convinced that no Damar solution is perfectly stable. A review 278 
CHAPTER XXI. 
of some old Damar mounts has shown that the majority of 
them have developed granules that have deteriorated the 
preparations to a greater or less extent. (These granules are 
in the worst cases large enough to at once attract attention 
even with low powers; at other times they are so small that 
they can only be seen with the highest powers, and in this 
case may be mistaken for normal elements of cells.) Xylol 
balsam and benzol balsam mounts are in the same case, but 
to a less degree. Chloroform balsam keeps much better, so 
far as granules are concerned. But it becomes very brown 
with age, and has the defect that it is injurious to stains made 
with coal-tar colours. Seiler’s alcohol-balsam keeps remai-k- 
ably well, but it also will not preserve the coal-tar stains or 
hasmatein stains. I use it for carmine stains. For coal-tar 
stains I now generally use turpentine colophonium. It gives 
very good definition of delicate detail, and keeps perfectly. 
(Dr. Paul Mayer, however, writes me that turpentine solu- 
tions are not at all good for hasmatein stains). Tui'pentine 
colophonium has a rather low index of refraction for objects 
that require much clearing. For these I very frequently use 
oil of cedar wood in preference to any resinous medium. It 
gives perfect definition of all elements of the right index of 
visibility, and it keeps perfectly. With time it thickens 
sufficiently to hold the cover in place ; or, if desired, prepara- 
tions may be luted with Bell’s cement. After using an oil 
immersion objective on a fresh mount, it is always easy to 
change the cover by floating it up with a drop of the oil 
placed at the side. 
Another reason for preferring turpentine-colophonium 
where possible is that it does not shrink in drying nearly so 
much as the media made with volatile solvents. Workers 
who use benzol balsam, for instance, generally mount sections 
with strips of paper interposed between the slide and the 
cover, in order to prevent the sections from being crushed 
by the cover as it is drawn down in the process of drying. 
With turpentine media this is not necessary. 
Still another motive is that turpentine media preserve the 
index of visibility of the preparations much longer than do 
media made with volatile menstrua. Preparations made with 
these last become so transparent in course of time that much 
fine detail is often lost. (Such mounts may, however, be EXAMINATION AND PRESERVATION MEDIA. 
279 
revivified without removing the cover by putting them for a 
day or two into a tube of benzol; the benzol penetrates the 
balsam, and brings it down to a lower refractive index.) 
The visibility of minute structures is proportional bo the 
difference between the refractive indices of the object and of the 
medium in which it is mounted. The majority of the elements 
of soft tissues are of an index of refraction somewhat superior 
to that of Canada balsam. It follows that by lowering the 
index of the balsam, increased visibility is obtained, and the 
desideratum in any case is to find a medium just low enough 
to give good visibility, and yet not so low as to seriously cut 
down the N.A. of the objectives employed. 
439. Choice of a Mounting Medium.—For the foregoing 
reasons I recommend turpentine-colophonium for general 
work; whilst for cases in which a more highly refractive 
medium is desired, I would recommend oil of cedar for coal- 
tar stains, and Seiler’s alcohol-balsam for carmine stains. 
For haematein stains perhaps xylol-balsam, though those of 
my haematein preparations of which I have full notes have 
kept very well in either cedar oil or turpentine-colophonium. 
Xylol-balsam is certainly a very fine medium. I have merely 
wished to point out that it is not perfectly safe on the score 
of the possible formation of granules. (P. Mayer, in litt., 
is of the same opinion.) 
440. Canada Balsam.—Prepare with the solid balsam as above 
described, § 438. The usual menstrua are xylol, benzol, 
chloroform, and turpentine. Dissolve the solid balsam in one 
of these to the required consistence. The turpentine solution 
is to be preferred only in cases where it is desired to have a 
medium that sets very slowly, or in view of the better pre- 
servation of certain stains. (The objection to turpentine as a 
solvent is that it does not always give a homogeneous solution 
with Canada balsam as it does with colophonium.) For most 
other purposes the xylol solution is the best. If time be an 
object, a benzol solution should be preferred, as it sets much 
quicker than the xylol solution. 
Heys states that if the chloroform solution be poured into 
long, thin, half-ounce phials, corked up, and set aside for at 
least a month, the medium will be clearer and set much 280 
CHAPTER XXI. 
quicker than if the balsam is mixed with the chloroform at 
the time it is required for use {Trans. Mic. Soc., Jan., 1865, 
p. 19 ; Beale, p. 51). 
Sahli (Zeit. f. wiss. Mik., 1885, p. 5) recommends oil of 
cedar as a menstruum. 
Martinotti {Zeit. f. wiss. Mik., iv, 2, 1887, p. 159) says 
that he has obtained some beautiful solutions with oil of 
spike {“ essence d’aspic rectifiee,” of Duroriez, Paris). Un- 
fortunately, he says, this medium will not preserve safranin 
stains. 
441. Seiler’s Alcohol Balsam (Proc. Amer. Soc. Mic., 1881, 
pp. 60-2 ; Journ. Boy. Mic. Soc. [N.S.],ii, 1882, pp. 126-7).— 
“ Take a clear sample of Canada balsam and evaporate it in 
a water- or sand-bath to dryness; i. e. until it becomes brittle 
and resinous when cold. Dissolve this while warm in warm 
absolute alcohol and filter through absorbent cotton.” 
The advantage of this medium is stated to be that objects 
may be mounted in it direct from absolute alcohol, without 
previous treatment with an essential oil or other clearing 
agent; Seiler considers that by this means “ shrivelling is 
avoided, as well as the solution of fat in the cells.” 
The process of mounting direct from alcohol is not very 
easy to carry out, and I cannot recommend it for general 
work. But used in the ordinary way, after clearing by an 
essence, only xylol or the like, Seiler’s solution is for some 
purposes admirable. 
As stated above, I find that it is one of the most stable solu- 
tions known to me. (My stock, made up fifteen years ago, is 
still perfectly limpid, and has not sensibly darkened in colour.) 
It works pleasantly enough (if care be taken not to breathe 
on it during the process of mounting, as this may easily cause 
cloudiness). The definition is very fine, and the preservation 
of the preparations almost invariably perfect; my oldest pre- 
parations only show a few granules of little importance. Of 
course it has serious limitations. It cannot be used with the 
soluble coal-tar colours, and I find that it does not always 
preserve heematein stains, so that its chief employment is for 
carmine stains. 
442. Damar (Gum Damar, or Dammar, or d’Ammar). — The EXAMINATION AND PRESERVATION MEDIA. 
281 
menstrua are the same as for balsam, and the solution should 
be prepared in the same way. The most beautiful of all 
these mounting media is the solution of damar in xylol. Heat 
is not necessary to make the solution. 
Minute directions (which I think unnecessary) for preparing 
a working solution are given by Martinotti in Zeit. f. wiss. 
Mih., iv, 2, 1887, p. 156, and in Malpighia, ii, 1888, p. 270; 
cf. also Journ. Roy. Mic. Soc., 1889, p. 163. 
Flemming, Ppitzner, and a writer signing C. J. M., all 
employ a mixture of benzol and turpentine (see Arch. mih. 
Anat., xix, 1881, p. 322 ; Sci. Gossip, 1882, p. 257 ; Journ. 
Roy. Mic. Soc. [N.S.], iii, 1883, p. 145; Morphol. Jahrb., vi, 
1880, p. 469; Journ. Roy. Mic. Soc. [N.S.], ii, 1882, p. 583). 
Max Flesch notes hereon (Zool. Jahresher.fi. 1880, p. 51) 
that at Wurzburg the ordinary dammar varnish of artists is 
employed. 
-James {Engl. Aleck., 1887, p. 184; Journ. Roy. Alic. Soc., 
1887, p. 1061) also gives some, I think, superfluous formulas 
for damar solutions; and still another new method is given, 
op. cit., 1890, p. 680. 
A formula for a damar and mastic medium is given by 
Squire in his Alethods and Formulse, &c., p. 84. 
See further details concerning these solutions in former 
editions- 
I quite acknowledge the special beauty of definition ob- 
tained by means of damar solutions; but I am convinced that 
not one of these solutions can he depended on for really per- 
manent preservations. Sooner or later, sometimes after a few 
weeks or days, or it may be only after months or years, the 
granules mentioned in § 438 will make their appearance. 
443. Colophonium.—A solution of colophonium in turpen- 
tine was first recommended by Kleinenberg. I find it to be 
most highly recommendahle. 
This medium sets very slowly, so that ample time is afforded 
for arranging objects in it. Kleinenberg warns against the 
employment of absolute alcohol as a solvent; the prepara- 
tions are beautiful at first, but soon become spoiled by the 
precipitation of crystals or of an amorphous substance. 
The turpentine solution keeps perfectly limpid, gives very 
good definition, and is altogether so excellent a medium that 282 
CHAPTER XXI. 
I am surprised that it is not more used. It should be recom- 
mended to beginners. And, as stated in § 438, I consider 
that for many purposes it is perhaps the best and most reliable 
medium known. To make the solution, I add small lumps of 
colophonium to a quantity of rectified oil of turpentine kept 
in a stove, and when a sufficiently thick solution has been 
obtained, filter twice, the filtering being done in the stove. 
About a fortnight is required for the whole process. The 
solution should not be too thick, as it thickens somewhat with 
age. The palest sorts of colophonium should of course be 
selected. 
444. Venice Turpentine for Mounting (Vosseler, Zeit. f.wiss. 
Mik., vi, 3, 1889, p. 292, et seq.).—Vosseler strongly recom- 
mends this medium as having considerable advantages over 
Canada balsam or damar. Commercial Venice turpentine is 
mixed in a tall cylinder glass with an equal volume of 96 per 
cent, alcohol, allowed to stand in a warm place for three or 
four weeks, and decanted. It is stated that preparations 
may be mounted in this medium without previous clearing 
with essential oils or the like. The index of refraction being 
lower than that of the above-named balsams, delicate details 
are more distinctly brought out. Stains keep well in the 
medium, and Vosseler states that he possesses preparations 
made fifteen years ago that are perfectly well preserved. 
This medium is also recommended by Suchannek {ibid., vii, 
4, 1891, p. 463). He advises that it be prepared with equal 
parts of Venice turpentine and neutral absolute alcohol (ob- 
tained by treating commercial absolute alcohol with calcined 
cupric sulphate and quicklime). The mixture should be 
agitated frequently and kept in a tile stove for a day or two 
until clear and sufficiently inspissated. 
445. Copal Varnish.—I have seen tissues very instructively mounted 
in this medium. I have tried it myself, and failed. “ Berry’s Hard 
Finish,” which is an easily obtainable copal varnish, has been highly 
praised for mounting purposes (see Journ. Roy. Mic. Soc., 1887, p. 1064). 
446. Cedar Oil.—I most highly recommend this oil, both as 
a temporary examination medium, and as a mounting medium. 
See § 438. 
447. Castor Oil.—This was recommended as a mounting medium for EXAMINATION AND PRESERVATION MEDIA. 
283 
certain delicate tissues (sections of eyes of Cephalopods) by Grenacher 
(Abhandl. naturf. Ges. Halle-a.-S., Bd. xvi; Zeit. f. wiss. Mik., 1885, 
p.244). This was with the idea that its low refractive index w=T49, 
whilst Canada balsam n = T54) would give a useful augmentation of visi- 
bility for the more refractive elements of the tissues. 
With the objects with which I have experimented I have not found this 
to be the case. 
448. Photographic Negative Varnish (for mounting large sections 
without cover-glasses).—See Weigebt, Zeit. f. wiss. Mik., iv, 2, 1887, 
p. 209. 
449. Styrax and Liquidambar.—See Journ. Roy. Mic. Soc., 1883, 
p. 741 ; ib., 1884, pp. 318, 475, 655, and 827 ; and the places there quoted. 
Also Bull. Soc. Beige de Mic., 1884, p. 178; and Fol, Lehrb., p. 141. 
These are very highly refractive media, which is just what is not wanted in 
general in histology. 
450. Sandarac (Lavdowsky, from Ref. Handbook Med. Sci., Supp. p. 
438).—Gum sandarac 30 grs., absolute alcohol 50 c.c. This may, if desired, 
be diluted with an equal volume of absolute alcohol, and used for clearing 
sections. CHAPTER XXII. 
CEMENTS AND VARNISHES. 
451. Introduction.—Thanks to the efforts of the dilettanti 
to outshine one another with neatly gaudy “rings/’ micro- 
scopical literature contains a goodly show of receipts for 
cements and varnishes. I have collected such as appear 
likely to be useful, rejecting all that relates merely to 
ornament. 
Two, or at most three, of the media given below will 
certainly be found sufficient for all useful purposes. For 
many years I have used only one cement (Bell’s). I recom- 
mend this as a cement and varnish; gold size may be found 
useful for turning cells ; and Miller’s caoutchouc cement may 
be kept for occasions on which the utmost solidity is required. 
Marine glue is necessary for making glass cells. 
Carpenter lays great stress on the principle that the cements 
or varnishes used for fluid mounts should always be such as 
contain no mixture of solid 'particles ; he has always found 
that those that do, although they might stand well for a few 
weeks or months, yet always become porous after a greater 
lapse of time, allowing the evaporation of the liquid and the 
admission of air. All fluid mounts should be ringed with 
glycerin jelly before applying a cement; by this means all 
danger of running in is done away with. See § 453 and 454. 
The above passage stands as it stood, italicised as here, in the first and 
second editions. It was translated and amplified, in a special paragraph, in 
the Traite des Meth. techniques. I may therefore he excused from hunting 
up the name of the anatomist who recently published as new this old old 
method, or the pages of the journals which reproduced his paper without 
protest. 
The reader who requires more information concerning 
microscopical cements and varnishes than can be given in 
this chapter may consult with advantage the papers of CEMENTS AND VARNISHES. 
285 
Aubert, The Microscope, xi, 1891, p. 150, and Journ. Roy. 
Mic. Soc., 1891, p. 692; Beck, The Microscope, xi, 1891, 
pp. 338, 368, and Journ. Boy. Mic. Soc., 1892, p. 293; and 
the last edition of Behrens’ Tabellen zum Gebrauch bei mikro- 
skopischen Arbeiten (Bruhn, Braunschweig, 1892). 
452. Comparative Tenacity of Cements (see Behrens, Zeit. f. 
wiss. Mik., ii, 1885, p. 54, and Aubert, Amer. Mon. Mic. 
Journ., 1885, p. 227; Journ. Boy. Mic. Soc., 1886, p. 173).— 
Behrens gives the palm to amber varnish ; Aubert places 
Miller’s caoutchouc cement at the head of the list, Lovett’s 
cement coming halfway down, and zinc white cement at the 
bottom, with less than one quarter the tenacity of the caout- 
chouc cement. 
453. The Paper Cell Method.—According to my experience, 
the best way to make a fluid mount safe is the following :— 
By means of two punches I cut out rings of paper of about a 
millimetre in breadth, and of about a millimetre smaller in 
diameter than the cover-glass. Moisten the paper ring with 
mounting fluid, and centre it on the slide. Fill the cell thus 
formed with mounting fluid ; arrange the object in it; put 
the cover on; fill the annular space between the paper and 
the margin of the cover with glycerin jelly (a turn-table may 
be useful for this operation); and as soon as the gelatin has 
set turn a ring of Bell’s or other cement on it. 
For greater safety, the gelatin may of course be treated 
with bichromate according to Marsh’s plan, next §. 
454. Gelatin Cement (Marsh’s Section-cutting, 2nd ed., p. 
104).—Take half an ounce of Nelson’s opaque gelatin, soak 
well in water, melt in the usual way, stir in 3 drops of krea- 
sote, and put away in a small bottle. It is used warm. 
When the ring of gelatin has become quite set and dry, 
which will not take long, it may be painted over with a 
solution of bichromate of potash made by dissolving 10 grains 
of the salt in an ounce of water. This should be done in the 
daytime,” as the action of daylight is necessary to enable the 
bichromate to render the gelatin insoluble in water. The 
cover may then be finished with Bell’s cement. 286 
CHAPTER XXII. 
This process is particularly adapted for glycerin mounts. 
455. Bell’s Cement.—Composition unknown. May be ob- 
tained from the opticians or from J. Bell and Co., chemists, 
338, Oxford Street, London. 
This varnish flows easily from the brush, and sets quickly. 
For glycerin or other fluid mounts, the cover should be ringed 
as above described with glycerin jelly before applying the 
varnish. This precaution is especially necessary with glycerin. 
This is the best varnish for fluid mounts known to me. It is 
soluble in ether or chloroform. It is not attacked by oil of 
cedar. 
456. Miller’s Caoutchouc Cement.—Composition unknown. 
May be obtained from the opticians. A very tenacious and, 
which is frequently an important point, a quickly drying 
cement. It may be diluted by a mixture of equal parts of 
chloroform and strong alcohol (see Rousselet, Journ. Quek. 
Club, v, ii, 1895, p. 8). 
457. Clarke’s Spirit-proof Cement.—Mr. Ch. Rousselet has 
highly recommended this to me. It may be procured from 
Mr. J. Bolton, 25, Balshall Heath Road, Birmingham. 
458. Asphalt Varnish (Bitume de Judee).—Unquestionably 
one of the best of these media, either as a cement or a varnish 
'provided it be procured of good quality. It can be procured 
from the opticians or from the oil-shops. Kitton {Month. Mic. 
Journ., 1874, p. 34) recommends asphalt dissolved in benzol 
with the addition of a small quantity of gold size. 
459. Brunswick Black.—Best obtained from the opticians. 
A receipt for preparing it is given in Beale, How to Work, 
&c., p. 49. 
“ If a little solution of india-rubber in mineral naphtha be 
added to it, there is no danger of the cement cracking when 
dry.” Carpenter states that without this addition it is brittle 
when dry. Brunswick black is soluble in oil of turpentine. 
A most useful cement, works easily and dries quickly. It can 
be recommended for turning cells. 
460. Brunswick Black and Gold Size (Eulenstein, Beale, CEMENTS AND VARNISHES. 
287 
How to Work, &c., p. 49).—Equal parts of Brunswick black 
and gold size with a very little Canada balsam. 
461. Gold Size.—Receipts for preparing it may be found in 
the Micrographic Diet, or in Cooley’s Cyclopaedia; but it is 
certainly best to obtain it from the opticians or oil-shops. It 
is soluble in oil of turpentine. A good cement, when of good 
quality, and very useful for turning cells. 
462. Marine Glue.—Found in commerce. Carpenter says 
the best is that known as Gr K 4. 
It is soluble in ether, naphtha, or solution of potash. Its 
use is for attaching glass cells to slides, and for all cases in 
which it is desired to cement glass to glass. 
Receipts for preparing it may be found in Beale, p. 40, or 
in Cooley’s Cyclopaedia. 
463. Harting’s Gutta-percha Cement (see Beale’s How to 
Work, &c., p.49).—Marine glue serves the same purpose, viz. 
that of attaching cells to slides. 
464. India-rubber and Lime French Cement.—See Beale, p. 
58. 
465. Knotting [Journ. Roy.Mic. Soc., 1882,p. 745).—‘‘Patent 
knotting ” from oil and colour stores, exposed to the air 
until it has become of the proper consistency; — for mending 
cells and for preventing running-in of the finishing varnish 
(Northern Microscopist, ii, 1882). 
466. Turpentine, Venice Turpentine (Csokor, Arc/L mik. Anat., 
xxi, 1882, p. 353; Parker, Amer. Mon. Mic. Journ., ii, 1881, 
pp. 229-30; Journ. Roy. Mic. Soc. [N.S.], ii, 1882, p. 724).— 
Venice turpentine (Terebinthina veneta) is the liquid resinous 
exudation of Abies larix. It is seldom met with in a pure 
state. The following are the directions for preparing and 
using it given by Parker : 
Dissolve true Venice turpentine in enough alcohol, so that 
after solution it will pass readily through a filter, and, after 
filtering, place in an evaporating dish, and by means of a 288 
CHAPTER XXII. 
sand-bath evaporate down to about three quarters of the 
quantity originally used. (The best way to tell when the 
evaporation has gone far enough is to drop some of the 
melted turpentine, after it has evaporated down to about three 
quarters of its original volume, into cold water; if on being 
taken out of the water it is hard and breaks with a vitreous 
fracture on being struck with the point of a knife, cease 
evaporation and allow to cool.) 
Or (Csokor), common resinous turpentine of commerce is 
put in small pieces to melt over a water-bath, then poured 
into a suitable vessel and allowed to cool. It should form a 
brittle, dark brown mass, not yielding to the pressure of a 
finger. It is sometimes useful, in order to attain the right 
degree of hardness in the cold mass, to add a little resinous 
oil of turpentine to the melted mass, and then to evaporate 
for several hours over the water-bath. 
This cement is used for closing glycerin mounts ; it is 
applied in the following manner:—Square covers are used, 
and superfluous glycerin is cleaned away from the edges in 
the usual way. 
The cement is then put on with a piece of wire bent at 
right angles (No. 10 — 12 wire is taken, and copper is the 
best, as it gives to the turpentine a greenish tinge); the short 
arm of the wire should be just the length of the side of the 
cover-glass. The wire is heated in a spirit lamp, plunged 
into the cement, some of which adheres to it, and then 
brought down flat upon the slide at the margin of the cover. 
The turpentine distributes itself evenly along the side of the 
cover, and hardens immediately, so that the slide may be 
cleaned as soon as the four sides are finished. It is claimed 
for this cement that it is perfectly secure, very handy, and 
never runs in. Parker saw this cement, or a similar one 
known as Vendischer Damarlack, exclusively used for gly- 
cerin mounts in the Pathological Laboratory at Vienna. 
This is an extremely valuable method. It is very rapid and 
very safe. The cement sets hard in a few seconds. 
467. Colophonium and Wax (Kronig, Arch. f. mih. Anat., 
1886, p. 657; Journ. Boy. Mic. Soc., 1887, p. 344) .—Seven to 
nine parts of coloplionium are added piecemeal to two parts 
of melted wax, tlie whole filtered and left to cool. For use, CEMENTS AND VARNISHES. 
289 
the mass is melted by placing the containing vessel in hot 
water. The cement is not attacked by water, glycerin, or 
caustic potash. 
468. Apathy’s Cement for Glycerin Mounts (Zeit. f. wiss. 
Mik., vi, 2, 1889, p. 171).—Equal parts of hard (60° C. melt- 
ing-point) paraffin and Canada balsam. Heat together in a 
porcelain capsule until the mass takes on a golden tint, and 
no longer emits vapours of turpentine. On cooling, this forms 
a hard mass, which is used by warming and applying with a 
glass rod or brass spatula. One application is enough. The 
cement does not run in, and never cracks. 
469. Jtxlien’s Balsam-Paraffin, see Journ. New York Mic. Soc., ix, 
1893, p. 39, or Journ. Roy. Mic. Soc., 1893, p. 567. 
470. Paraffin.—Temporary mounts may be closed with pure 
paraffin, by applying it with a bent wire, as described, § 466. 
471. Canada Balsam, or Damar.— Cells are sometimes made with 
these. They are elegant, but in my experience are not reliable for per- 
manent mounts. 
472. Amber Varnish.—As above mentioned, Behrens finds 
this cement to possess an extreme tenacity. He does not 
give the composition of his varnish, which was procured from 
E. Pfannenschmidt at Dantzic. The following is from Cooley’s 
Cyclopsedia, art. “ Varnish:” 
“ Take of amber (clear and pale) 6 lbs., fuse it; add of hot 
clarified linseed oil 2 gallons, boil it until it “ strings ” well, 
then let it cool a little and add of oil of turpentine 4 gallons 
or q. s.” 
Other receipts, 1. c. 
473. Amber and Copal Varnish (Heydenreich, Zeit.f. wiss. 
Mih., 1885, p. 338).—An extremely complicated mode of 
preparation. The varnish may be obtained from Ludwig* 
Marx, at 110, Moskowskaja Sastawa, St. Petersburg; or 79, 
Gaden, Vienna; or 1, Romerthal, Mayence. 
474. Shellac Varnish (Beale, p. 28).—Shellac should he broken into 
small pieces, placed in a bottle with spirit of wine, and frequently shaken 
until a thick solution is obtained. The Micro. Dictionary says that the 
addition of 20 drops of castor oil to the ounce is an improvement. 290 
CHAPTER XXII. 
Untrustworthy, but useful for protecting balsam mounts from the action 
of oil of cedar. 
For a method of preparing chemically pure shellac (a somewhat important 
matter), see Witt, Zeit.f. wiss. Mile., 1886, p. 199. 
For Seaman’s shellac cement for attaching metal to glass, see Journ. 
Boy. Mic. Soc., 1888, p. 520. 
475. Sealing-Wax Varnish (Micro. Dict.,“ Cements ”).—Add enough 
spii-it of wine to cover coarsely powdered sealing-wax, and digest at a gentle 
heat. This should only be used as a varnish, never as a cement, as it is apt 
to become brittle and to lose its hold upon glass after a time. 
476. Tolu Balsam Cement (Carnoy’s Biol. Cell., p. 129). 
Tolu balsam . .... 2 parts. 
Canada balsam . . . . .1 part. 
Saturated solution of shellac in chlo- 
roform ...... 2 parts. 
Add enough chloroform to bring the mixture to a syrupy 
consistence. Carnoy finds this cement superior to all others. 
477. Stieda’s Zinc White or Red Cement (Arch. f. mile. Anat., 
1866, p. 435).—Rub up oxide of zinc with turpentine, and add, stirring con- 
tinually, for every drachm of the zinc oxide 1 ounce of a solution of damar 
in turpentine (of the consistency of thick syrup). This gives a white 
cement like Ziegler’s. For a red cement take, instead of zinc, cinnabar, 
and take 2 drms. of the metal for each ounce of the damar solution. If the 
cement has become too thick with age, dilute with turpentine, ether, or 
chloroform. Recent authors who have discussed this cement are agreed that 
it is quite untrustworthy as a cement, though it may he useful as a finish. 
478. Ziegler’s White Cement.—Composition unknown. Is very much 
used on the Continent. 
479. Kitton’s White Lead Cement (Month. Mic. Journ., 1876, p. 
221).—Equal parts of white-lead, red-lead, and litharge (all in powder), 
ground together with a little turpentine until thoroughly incorporated, then 
mixed with gold size. The mixture should be thin enough to work with a 
brush. No more of the cement should be made than is required for present 
nse, as it soon sets and becomes unworkable ; but a stock of the materials 
may he kept ready ground in a bottle. 
480. Lovett’s Cement (Journ. Boy. Mic. Soc., 1883, p. 786).—Two 
parts white-lead, two parts red oxide of lead (minium), three parts litharge. 
To be ground very fine, mixed dry, and kept so in a bottle. When required 
for use mix a little of the powder with gold size to the consistency of paint, 
taking care that no grit gets into it. 
481. Aspinall’s Enamel.—Stanley Kent (Journ. Boy. Mic. 
Soc., 1890, p. 821) finds this of great use, both for ringing 
slides and making cells. PABT II. 
SPECIAL METHODS AND EXAMPLES. CHAPTER XXIII. 
INJECTIONS —GELATIN MASSES. 
482. Introduction.—Injection masses are composed of a 
coloured substance, technically termed the colouring mass, 
and of a substance with which that is combined, technically 
termed the vehicle. 
The following formulae are grouped mainly according to 
the nature of the vehicle. 
For injections made for the study of the angiologyof verte- 
brates, the student will do well to follow the masterly 
practice of Robin and Ranvier, consulting also, if necessary, 
the excellent instructions given in the Lehrbuch cler vergleich- 
enden milcroshopischen Anatomie of Fob For injections of 
Invertebrates (and, indeed, for Vertebrates if it is desired to 
demonstrate the minute structure of environing tissues at the 
same time as the distribution of vessels) glycerin masses are 
frequently preferable to the gelatin masses chiefly employed 
by these authors; and I would recommend as particularly 
convenient the Prussian blue glycerin masses of Beale. 
Glycerin masses have the great advantage that they are used 
cold. 
483. Nitrite of Amyl as a Vaso-dilator.—As stated above, 
glycerin masses are certainly very convenient, and give very 
good results from the scientific—not from the aesthetic—point 
of view. They have a great defect for the injection of fresh 
specimens—that is, those in which rigor mortis has not set 
in : they stimulate the contraction of arteries. In these cases 
it may be advisable to use nitrite of amyl as a vaso-dilator. 
The animal may be anaesthetised with a mixture of ether and 
nitrite of amyl, and finally killed with pure niti’ite. Or, after 
killing by nitrite, a little nitrite of amyl in salt solution may 
be injected before the injection mass is thrown in. In any 
case it is advisable to add a little nitrite to the mass just 294 
CHAPTER XXIII. 
before using. The relaxing power is very great (see Oviatt 
and Sargent, in St. Louis Med. Journ., 1886, p. 207 ; and 
Journ. Boy. Mic. Soc., 1887, p. 341). 
Robin’s Masses. 
484. Robin’s Gelatin Vehicle (Traite', p. 30).—Take some 
gelatin, of the sort known as “ colle de Paris.” (This gelatin 
is found in commerce in the form of thin sheets, marked with 
lozenge-shaped impressions of the cords which supported 
them whilst drying.) Soak it in cold water, then heat in 
water over a water-bath. One part of gelatin should be taken 
for every 7, 8, 9, or even 10 parts of water; it is a common 
error to employ solutions containing too much gelatin. The 
solution is now to be combined with one of the colouring 
masses given below. 
This vehicle, like all gelatin masses, is liable to be attacked 
by mould if kept long; camphor and carbolic acid do not 
suffice to preserve it. 
Chloral hydrate added to the mass will preserve it (Hoyer). 
A sufficient dose, at least 2 per cent., should be employed 
(see below, §§ 493, 494). 
485. Robin’s Glycerin-gelatin Vehicle (Traite, p. 32).—Dis- 
solve in a water-bath 50 grms. of French gelatin (“colle de 
Paris ”) in 300 grms. of water, in which has been dissolved 
some arsenious acid; add of glycerin 150 grms., and of car- 
bolic acid a few drops. Unlike the pure gelatin vehicles, this 
mass does keep indefinitely. 
The colouring masses recommended for combination with 
the vehicles above described are made as follows : 
486. Carmine Colouring Mass (Traite, p. 33).—Rub up in a 
mortar 3 grins, of carmine with a little water and enough 
ammonia to dissolve tlie carmine. Add 50 grms. of glycerin, 
and filter. 
Prepare 50 grms. of acid glycerin (containing 5 grms. of 
acetic acid for every 50 grms. of glycerin), and add it by 
degrees to the carmine-glycerin, until a slightly acid reaction 
is obtained (as tested by very sensitive blue test-paper, moist- 
ened and held over the mixture). 295 
INJECTIONS—GELATIN MASSES. 
One part of this mixture is to be added to 3 or 4 parts of 
the gelatin injection vehicle (ante, Formula 484), or of the 
glycerin-gelatin (§ 485). 
487. Ferrocyanide of Copper Colouring Mass (ibid., p. 34). 
Take— 
(1) Ferrocyanide of potassium (concentrated 
solution) . . . . . . 20 c.c. 
Glycerin . . . . . . 50 ,, 
(2) Sulphate of copper (concentrated solu- 
tion) . . . . . . 35 ,, 
Glycerin . . . . . . 50 ,, 
Mix (1) and (2) slowly, with agitation; at the moment of 
injecting combine with 3 volumes of vehicle. 
488. Blue Colouring Mass (Prussian Blue) (Robin’s modifica- 
tion of Beale's formula, ibid., p. 35). 
Take— 
(a) Sulphocyanide of potassium (sol. sat.) 90 c.c. 
Glycerin . . . . . . 50 ,, 
(b) Liquid perchloride of iron at 30° . . 3 „ 
Glycerin . . . . . . 50 „ 
Mix slowly and combine the mixture with 3 parts of vehicle. 
It is well to add a few drops of HC1. 
489. Cadmium Colouring Mass [ibid., p. 36). 
Take— 
Sulphate of cadmium (sol. sat.) . . 40 c.c. 
Glycerin . . . . . . . 50 ,, 
and 
Sulphide of sodium (sol. sat.) . . . 30 ,, 
Glycerin . . . . . . 50 „ 
Mix with agitation and combine with 3 vols. of vehicle. 
O 
490. Green Colouring Mass (ibid., p. 37). 
Take— 
Arseniate of potasli (saturated solution) 80 c.c. 
Glycerin . . . . • • 30 ,, 
and 
Sulphate of copper (saturated solution) 40 „ 
Glycerin .• . . . . • 30 ,, 
Mix and combine with 3 vols. of vehicle. 296 
CHAPTER XXI11. 
491. Ranvier’s Carmine-Gelatin Mass (Traite technique, p. 
116).—Take 5 grins. Paris gelatin, soak it in water for half 
an hour, or until quite swollen and soft; wash it; drain it; 
put it into a test-tube and melt it, in the water it has absorbed, 
over a water-bath. When melted add slowly, and with con- 
tinual agitation, a solution of carmine in ammonia, prepared 
as follows:— grms. of carmine are rubbed up with a little 
water, and just enough ammonia, added drop by drop, to dis- 
solve the carmine into a transparent solution. 
When the carmine has been added to the gelatin, you will 
have about 15 c.c. of ammoniacal solution of carmine in 
gelatin, if the operations have been properly performed. This 
solution is to be kept warm on the water-bath, whilst you 
proceed to neutralise it by adding cautiously, drop by drop, 
with continual agitation, a solution of 1 part of glacial acetic 
acid in 2 parts of water. (When the mass is near neutrality, 
dilute the acetic acid still further.) The instant of saturation 
is determined by the smell of the solution, which gradually 
changes from ammoniacal to sour. As soon as the sour smell 
is perceived, the addition of acetic acid must cease, and the 
liquid be examined under the microscope. If it contains a 
granular precipitate of carmine, too much acid has been added, 
and the mass must be thrown away. 
Ranvier states that by practice the operator learns to attain 
to perfect neutralisation almost infallibly in this way, and 
that this is the only way to attain to it. Trust must not be 
pub in certain formulae that profess to indicate the propor- 
tions of ammonia and acetic acid necessary for neutralisation, 
on account of the variation in strength of the solutions of am- 
monia kept in laboratories. The method proposed by Frey 
of determining beforehand the quantity of a known acetic 
solution that is necessary for neutralisation of a given quantity 
of the ammonia employed, is not infallible because it often 
happens that commercial gelatin is acid; in which case the 
proposed method would cause the operator to overpass the 
point of saturation. 
Carmine-gelatin Masses. 
The mass having been perfectly neutralised is strained 
through new, flannel. 
492. How to Neutralise a Carmine Mass (Ville, Gaz. liebd. INJECTIONS—GELATIN MASSES. 
297 
d. Sci. med. de Montpellier, Fev., 1882 ; may be had separately 
from Delahaye et Lecrosnier, Paris).—Ville is of Ranvier’s 
opinion that the method of titration recommended by Frey is 
defective, but for a different reason. When carmine is treated 
with ammonia a certain proportion of the ammonia combines 
with the carmine to form a transparent purple compound, and 
the rest of the ammonia remains in excess. It is this excess 
that it is required to neutralise precisely. In Frey’s method 
a quantity of acid sufficient for the neutralisation of the whole 
of the ammonia employed is taken; hence, naturally, the point 
of neutralisation is overstepped, and a granular mass is the 
result. 
As to the acidity accidentally found in commercial gelatin, 
that source of error is easily eliminated. Instead of soaking 
the gelatin in water, it should be placed in a large funnel with 
a narrow neck, or better, in a stopcock funnel, and the whole 
should be placed under a tap, and a stream of water arranged 
in such a manner that the gelatin be constantly completely 
immersed. Washing for an hour or so in this way will 
remove all traces of acids mechanically retained in the gelatin. 
As to the neutralisation of the colouring mass, Ville is of 
opinion that the criterion of neutrality given by Ranvier—the 
sour smell that takes the place of the ammoniacal odour— 
cannot be safely relied on in practice. He considers it greatly 
preferable to employ dichroic litmus paper (litmus paper 
sensitized so as to be capable of being used equally for the 
demonstration of acids and bases). 
To prepare such a paper, the tincture obtained by decoction 
of cake litmus is slightly acidified by an excess of sulphuric 
acid. By this means the excess of alkali, or of alkaline 
carbonate, that is always present in litmus decoction, and 
which diminishes its sensibility as a reagent, is neutralised. 
The decoction is then heated and agitated with an excess of 
precipitated carbonate of baryta, and filtered. 
The solution of litmus thus obtained is exposed to the air 
in wide vessels until its intense blue colour has given place to 
a reddish tint. Strips of white unsized paper are then dipped 
in it, and dried in the shade on stretched threads, in a place 
free from vapour of ammonia. 
A shorter method consists in adding very dilute sulphuric 
acid, drop by drop, to the ordinary laboratory tincture of 298 
CHAPTER XXIII. 
litmus, until the colour changes to red. Then, by adding 
successively traces of alkali and very dilute sulphuric acid, 
the reddish dichroic tint may be obtained, and the paper pre- 
pared with the solution as before. 
The paper is used in the same way as ordinary litmus 
paper. A strip is moistened with distilled water and held as 
close as possible to the injection mass kept melted on a water- 
batli. It becomes blue at first, very rapidly and decidedly; 
but as fast as fresh quantities of acid are added this reaction 
becomes less evident, and at a certain moment the change of 
colour becomes very slow in making its appearance. It is 
then that the addition of acid should cease, and the operation 
is ended. 
Very delicate sensitized paper may also be prepared with 
other reagents than litmus—for instance, with Nessler’s 
reagent* or with alcoholic solution of liasmatoxylin, or with a 
solution made by adding a trace of dilute sulphuric acid to 
“ liqueur orange No. 3 ” (a liquid found in commerce, and 
used for detecting acids); the solution takes on a gooseberry 
red colour. 
The preparation of the injection mass is facilitated by em- 
ploying acetic acid and ammonia of known strength. For 
the acetic acid it is sufficient to keep the glacial acid in a 
well-stoppered bottle. But this will not suffice for the 
ammonia, which is notably lowered in strength through the 
mere pouring from one bottle into another. Ville has 
imagined an apparatus which allows of withdrawing a known 
quantity without permitting any access of air to the stock 
solution. Description and figures, 1. c. 
With the exception of the processes above described, Ville 
prepares the iujection mass exactly as Ranvier. 
493. Hoyer’s Carmine-Gelatin Mass (Biol. Centralb., 1882, 
p. 21).—Take a concentrated gelatin solution and add to it a 
corresponding quantity of neutral carmine staining solution 
(1. c., p. 17). Digest in a water-bath, until the dark violet-red 
colour begins to pass into a bright red tint. Then add 5—10 
* Nessler’s reagent may be prepared as follows:—Mercuric chloride, in 
powder, 35 grms. ; iodide of potassium, 90 grms.; water, 1750 c.c. Heat 
gently till dissolved in a large basin ; then add of stick caustic potash 320 
grms., and 50 c.c. of saturated solution of mercuric chloride (Wanklyn). 
From Cooley’s Cyclopsedia, s. v. “ Nessler’s Test.” INJECTIONS—GELATIN MASSES. 
299 
per cent, by volumes of glycerin, and at least 2 per cent, by 
weight of chloral, in a concentrated solution. After passing- 
through flannel it can be kept in an open vessel under a bell- 
glass. 
494. Fol’s Carmine-Gelatin Mass (Zeit. f. wiss. Zool., xxxviii, 
1883, p. 492). 
The following method of preparation has the advantage of 
producing masses that can be kept in the dry state for an 
indefinite length of time. (Fol finds that the addition of 
chloral hydrate to wet masses is not an efficient preservative.) 
One kilog. of Simeon’s photographic gelatin* is soaked for 
a couple of hours, until thoroughly soft, in a small quantity 
of water. The water is then poured off and the gelatin 
melted over a water-bath, and one litre of concentrated solu- 
tion of carmine in ammonia is poured in with continual 
stirring. (The carmine solution is prepared by diluting 
strong solution of ammonia with three or four parts of water 
and adding carmine to saturation; the undissolved excess of 
carmine is removed by filtration just before the solution is 
added to the gelatin.) 
To the mixture of gelatin and carmine, which should have 
a strong smell of ammonia, sufficient acetic acid is added to 
turn the dark purple colour of the mixture into the well-known 
blood-red hue. Exact neutralisation is not necessary. The 
mass is set aside until it has become firm, and is then cut up 
into pieces, which are tied up in a piece of tulle or fine netting. 
By means of energetic compression with the hand under water 
(it must be acidulated water, O'l per cent, acetic acid, other- 
wise the carmine will wash out : cf. Journ. Boy. Mic. Soc., 
iv, part 3, 1884, p. 474) the mass is driven out through the 
meshes of the stuff in the shape of fine strings, which are 
washed for several hours in a sieve placed in running water in 
order to free them from any excess of acid or ammonia. The 
strings are then again melted, and the molten mass is poured 
* This gelatin may be obtained either from the ordinary providers of 
articles used in photography, or direct from Simeon’s Gelatin-fabrik; 
Winterthur, Switzerland. Two sorts, a hard and a soft, are sold; the softer 
is to be preferred on account of its lower point of fusion. Probably the 
photographic gelatins of Hinrichs, of Frankfurt, and of Coignet, of Paris, 
would answer equally well; as also the best English preparations. 300 
CHAPTER XXIII. 
on to large sheets of parchment paper soaked with paraffin, 
and the sheets are hung up to dry in an airy place. When 
dry the gelatin can easily be separated from the sheets, and 
may be cut into long strips with scissors and put away, pro- 
tected from dust and damp, until wanted for use. In order 
to get the mass ready for use, all that is necessary is to soak 
the strips for a few minutes in water and melt them over a 
water-bath. 
The process may be simplified, without giving very greatly 
inferior results, as follows (Lehrb., p. 13). Gelatin in sheets 
is macerated for two days in the above-described carmine 
solution, then rinsed and put for a few hours into water acidu- 
lated with acetic acid. It is then washed on a sieve for several 
hours in running water, dried on parchment paper, and pre- 
served as above. 
This injection mass is very well spoken of. 
495. Other Carmine Gelatin Masses.—Gerlach’s Carmine-Gela- 
tin Mass (see Arch.f. mik. Anat., 1865, p. 148 ; and Ranvier’s Traite, p 
113). Thiersch’s Carmine-Gelatin Mass (see ibid.). Carter’s Car- 
mine-Gelatin Mass (see Beale, p. 113). Davies’ Carmine-Gelatin 
Mass (see his Prep, and Mounting of Mic. Objects, p. 138). 
Blue Gelatin Masses. 
496. Robin’s Prussian Blue Gelatin Mass (see above, § 488). 
497. Ranvier’s Prussian Blue Gelatin Mass (Traite, p. 119). 
—Twenty-five parts of a concentrated aqueous solution of 
soluble Prussian blue (prepared as directed below) mixed 
with 1 part of solid gelatin. 
The mixture of the Prussian blue with the vehicle is effected 
in the following manner : 
Weigh the gelatin, soak it in water for half an hour or an 
hour, wash it, and melt it in a test-tube, in the water it has 
absorbed, by heating over a water-bath. Put the solution 
of Prussian blue into another test-tube, and heat it on the 
same water-bath as the gelatin, so as to have the two at the 
same temperature. Pour the gelatin gradually into the 
Prussian blue solution, stirring continually with a glass rod. 
Continue stirring until the disappearance of the curdy pre- 
cipitate that forms at first. (Some gelatins produce a jper- INJECTIONS—GELATIN MASSES. 
301 
sistent precipitate ; these must be rejected ; but it must be 
borne in mind that the precipitate that invariably forms in 
even the best gelatins disappears if the heating be continued. 
It is essential to remember this when preparing Prussian blue 
and gelatin mass.) As soon as the glass rod has ceased to 
show blue granulations on its surface on being withdrawn 
from the liquid, it may be concluded that the Prussian blue 
is completely dissolved. Filter through new flannel, and 
keep the filtrate at 40° over a water-bath until injected. 
The soluble Prussian blue for the above mass is prepared 
as follows : 
498. Soluble Prussian Blue for Injection Masses (Ranvier, 
ibid.).—Make a concentrated solution of sulphate of peroxide 
of iron in distilled water, and pour it gradually into a con- 
centrated solution of yellow prussiate of potash. There is. 
produced a precipitate of insoluble Prussian blue. (An 
excess of prussiate of potash ought to remain in the liquid 
in order to ascertain whether this is the case take a small 
quantity of the liquid and observe whether a drop of sulphate 
of iron still precipitates it.) Filter the liquid through a felt 
strainer, underneath which is arranged a paper filter in a 
glass funnel. The liquid at first runs clear and yellowish 
into the lower funnel; distilled water is then poured little 
by little on to the strainer; gradually the liquid issuing from 
the strainer acquires a blue tinge, which, however, is not 
visible in that which issues from the lower filter. Distilled 
water is continually added to the strainer for some days until 
the liquid begins to run off blue from the second filter. The 
Prussian blue has now become soluble. The strainer is turned 
inside out and agitated in distilled water; the Prussian blue 
will dissolve if the quantity of water be sufficient. 
The solution may now be injected just as it is, or it may be 
kept in bottles till wanted, or the solution may be evaporated 
in a stove, and the solid residuum put away in bottle. 
For injections, if a simple aqueous solution be taken, it 
should be saturated. Such a mass never transudes through 
the walls of vessels. Or it may be combined with one fourth 
of glycerin, or with the gelatin vehicle above described. 
499. Soluble Prussian Blue (Guignet, see Journ. de Microgr 302 
CHAPTER XXIII. 
1889, p. 94 ; Journ. Roy. Mic. Soc., 1889, p. 463; or previous 
editions of this work). 
500. Brucke’s Soluble Berlin Blue (Arch. f. mik. Anat., 1865, 
p. 87).—Briicke first prepared it by taking a 10 per cent, 
solution of ferrocyanide of potassium, and precipitating by 
means of a dilute solution of sesquichloride of iron (taken in 
such a quantity as to contain just half as much chlorine as is 
necessary for the decomposition), and washing the precipitate 
on the filter until solubility is attained. 
Later on he employed a greater excess of ferrocyanide, and 
took just so much dilute solution of chloride of iron that the 
weight of the dry chloride employed came to or of that 
of the ferrocyanide. The precipitate was washed on a filter 
(using the filtrate to wash with) until nothing but a clear 
yellow liquid filtered off, then washed with water until the 
water began to run off blue, then dried, pressed between 
blotting-paper in a press, the resulting mass broken in pieces 
and dried by exposure to the air. 
A cheaper method is the following : 
Make a solution of ferrocyanide of potassium containing 
217 grammes of the salt to 1 litre of water. 
Make a solution of 1 part commercial chloride of iron in 
10 parts water. 
Take equal volumes of each, and add to each of them twice 
its volume of a cold saturated solution of sulphate of soda. 
Pour the chloride solution into the ferrocyanide solution, 
stirring continually. Wash the precipitate on a filter until 
soluble, and treat as above described. 
The concentrated solution of the colouring matter is to be 
gelatinised with just so much gelatin that the mass forms a 
jelly when cold. 
501. Thiersch’s Prussian Blue Gelatin Mass (Arch. f. mik. 
Anat., i, 1865, p. 148). 
Take— 
(1) A solution of 1 part gelatin in 2 parts water. 
(2) A saturated aqueous solution of sulphate of iron. 
(3) A saturated aqueous solution of red prussiate of 
potash. 
(4) A saturated aqueous solution of oxalic acid. INJECTIONS—GELATJN MASSES. 
303 
Now (a) mix 12 c.c. of the iron solution with one ounce of 
the gelatin solution at the temperature of 25° It. 
Then (b) mix, at the same temperature, 24 c.c. of the prus- 
siate solution with two ounces of the gelatin solution. 
(c) To the latter mixture add first 24 c.c. of the oxalic acid 
solution, stir well, and then add the gelatin and iron mixture 
(a). Stir continually, keeping the temperature at from 20° 
to 25° R. until the whole of the Prussian blue is precipitated. 
Finally, heat over a water-bath to about 70° R. and filter 
through flannel. 
502. F ol’s Berlin Blue Gelatin Mass (Zeit. f. wiss. Zool., 
xxxviii, 1883, p. 494).—A modification of Thiersch’s formula, 
last §. 120 c.c. of a cold saturated solution of sulphate of 
iron are mixed with 300 c.c. of the warm gelatin solution. 
In a separate vessel 600 c.c. of the gelatin solution are mixed 
with 240 c.c. of a saturated solution of oxalic acid, and 240 
c.c. of a cold saturated solution of red prussiate of potash 
are added to the mixture. The first mixture is now gradually 
poured into the second, with vigorous shaking, the whole is 
warmed for a quarter of an hour over a boiling water-bath, 
the mass is allowed to set, is pressed out into strings through 
tulle or netting, as described for the carmine mass, supra, 
§ 494, and the strings are washed and spread out to dry on 
the prepared paper. (It is necessai-y to dry the strings 
without re-melting in this case, because the mass does not 
readily melt without the addition of oxalic acid.) In order 
to prepare the mass for injection, the strings are put to swell 
up in cold water, and then warmed with the addition of 
enough oxalic acid to allow of complete solution. 
503. Hoyer’s Soluble Berlin Blue Gelatin Mass {Arch. f. mik. 
Anat., 1876, p. 649).—The filtered and not too much washed 
precipitate of soluble Berlin blue is brought in a little water 
on to a Graham’s dialyser, and the external water changed 
until the solution begins to pass through the parchment. 
Dilute the solution and filter through filter-paper, an opera- 
tion wThich becomes easy after dialysis. The solution may be 
injected pure (for lymphatics, for instance) or may be com- 
bined with gelatin. To do this, warm the solution almost to 
boiling-point, and add gradually a warm, thin solution of 304 
CHAPTER XXIII. 
gelatin until coagulation begins to set in. Strain through 
wetted flannel. 
Gelatin Masses of other Colours. 
504. Robin’s Cadmium Gelatin Mass (see § 489). 
505. Thiersch’s Lead Chromate Gelatin Mass {Arch. f. mik. 
Anat., 1865, p. 149). 
Make— 
(a) A solution of 1 part gelatin in 2 parts water. 
(b) A solution of 1 part neutral chromate of potash in 11 
parts water. 
(c) A solution of 1 part nitrate of lead in 11 parts water. 
Mix 4 parts of the gelatin solution with 2 parts of the lead 
solution, and in another vessel mix 4 parts gelatin solution 
with 1 part of the chromate solution. Heat both the mixtures 
to 25° R.; mix them together with continual stirring until all 
the chromate of lead is precipitated; heat over a water-bath 
to 70° R., and filter through flannel. 
Fol remarks that this is the most beautiful of yellow 
masses, but will not keep, as the chromate of potash causes 
the gelatin to pass over gradually into the insoluble state. 
506. Hoyer’s Lead Chromate Gelatin Mass {ibid., 1867, p. 
136). 
Take- 
One volume of a solution of gelatin containing 1 part of 
gelatin to 4 of water. 
One volume of cold satui’ated solution of bichromate of 
potash. 
And one volume cold saturated solution of sugar of lead 
(neutral plumbic acetate). 
Filter the gelatin solution through flannel, and mix in the 
bichromate solution. Then warm almost to boiling-point, and 
add gradually the (warmed) sugar of lead solution. Allow 
the mass to cool down to body temperature, and inject at 
once. Another mode of preparation is as follows :—Mix the 
sugar of lead solution with part of the gelatin solution, mix 
the bichromate solution with the remaining gelatin solution, 
heat the latter mixture, and pour into it the former mixture 
(gradually), stirring continually. INJECTIONS GELATIN MASSES. 
305 
If the solutions are mixed at a low temperature a lumpy 
granular precipitate is formed. Further, when solution of 
sugar of lead is added to a hot solution of bichromate of 
potash a rich orange-red precipitate is obtained; whilst if the 
solutions be mixed cold the precipitate is bright yellow. 
If the solutions of the two salts be kept ready prepared, 
the injection mass may be mixed in less than a quarter of an 
hour. Its advantages are that, on account of the extremely 
fine state of division of the precipitate, the mass is almost 
transparent, and runs so freely that even lymphatics may be 
perfectly injected with it, whilst its intensity of colour makes 
the vessels much more distinct than the very pale mass of 
Thiersch (last Section). It is also easier to manage than 
Thiersch’s mass, as it does not solidify so quickly. It shows 
well in the vessels by reflected, as well as by transmitted 
light. 
507. Fol’s Lead Chromate Gelatin Mass (Lehrb., p. 15). 
508. Hoyer’s Silver Nitrate Yellow Gelatin Mass (Biol. Cen- 
tralbl., ii, 1882, pp. 19, 22; Journ. Roy. Mic. Soc. [N.S.], iii, 
1883, p. 142).—“ A concentrated solution of gelatin is mixed 
with an equal volume of a 4 per cent, solution of nitrate of 
silver and warmed. To this is added a very small quantity 
of an aqueous solution of pyrogallic acid, which reduces the 
silver in a few seconds; chloral and glycerin are added as 
before ” (see ante, Hoyer’s formula for carmine-gelatin, 
No. 493). 
This mass is yellow in the capillaries and brown in the 
larger vessels. It does not change either in alcohol, chromic 
or acetic acid, or bichromate of potash, &c. 
509. Other Colours.—Hoyer’s Green Gelatin Masses (ibid.).— 
Made by mixing a blue mass and a yellow mass. Thieksch’s Green Gela- 
tin Mass (Arch. f. mik. Anat., 1865, p. 149).—Made by mixing the blue 
mass, § 501, and the yellow mass, § 505. Robin’s Scheele’s Green Gela- 
tin Mass (see §490). Hartig’s White Gelatin Mass (see Frey, Le 
Microscope, p. 190). Frey’s White Gelatin Mass (ibid.). Teichmann’s 
White Gelatin Mass (ibid., p. 191). Fob’s Brown Gelatin Mass 
(Zeit. f. wiss. Zool., xxxviii, 1883, p. 494). Miller’s Purple Silver 
Nitrate Gelatin Mass (see Amer. Mon. Mic. Journ., 1888, p. 50; 
Journ. Boy. Mic. Soc., 1888, p. 518; Zeit.f. wiss. Mik.,v, 3, 1888, p. 361). 
Robin’s Mahogany Gelatin Mass (see § 487). 306 
CHAPTER XXIII. 
510. Ranvier’s Gelatin Mass for Impregnation (Traits, p. 123). 
—Concentrated solution of gelatin, 2, 3, or 4 parts; 1 per 
cent, nitrate of silver solution, 1 part. 
511. Fol’s Metagelatin Vehicle (Lehrb., p. 17).—The opera- 
tion of injecting with the ordinary gelatin masses is greatly 
complicated by the necessity of injecting them warm. Fol 
proposes to employ metagelatin instead of gelatin. 
If a slight proportion of ammonia be added to a solution of 
gelatin, and the solution be heated for several hours, the 
solution passes into the state of metagelatin, that is, a state 
in which it no longer coagulates on cooling. Colouring masses 
may be added to this vehicle, which may also be thinned by 
the addition of weak alcohol. After injection, the prepara- 
tions are thrown into strong alcohol or chromic acid, which 
sets the mass. CHAPTER XXIV. 
INJECTIONS OTHER MASSES (COLD). 
512. Joseph’s White-of-Egg Injection Mass (Carmine) [Per. 
naturw. Sect. Schles. Ges. 1879, pp. 36—40; Journ. Roy. Mic. 
Soc. [N.S.J, ii, 1882, p. 274).—“ Filtered white of egg, diluted 
with 1 to 5 per cent, of carmine solution This mass 
remains liquid when cold; it coagulates when immersed 
in dilute nitric acid, chromic or osmic acid, remains trans- 
parent, and is sufficiently indifferent to reagents.” 
For Invertebrates. 
513. Bjeloussow’s Gum Arabic Mass {Arch. f. Anat. u. Phys., 
1885, p. 379).—Make a syrupy solution of gum arabic and a 
saturated solution of borax in water. Mix the solutions in 
such proportions as to have in the mixture 1 part of borax to 
2 of gum arabic. Rub up the transparent, almost insoluble 
mass with distilled water, added little by little, then force it 
through a fine-grained cloth. Repeat these operations until 
there is obtained a mass that is free from suspended gela- 
tinous clots. (If the operation has been successful, the mass 
should coagulate in the presence of alcohol, undergoing at the 
same time a dilatation to twice its original volume.) 
The vehicle thus prepared may be combined with any 
colouring mass except cadmium and cobalt. 
After injection the preparation is thrown into alcohol, and 
the mass sets immediately, swelling up as above described, 
and consequently showing vessels largely distended. 
Cold-blooded animals may be injected whilst alive with this 
mass. It does not flow out of cut vessels. Injections keep 
well in alcohol. Glycerin may be used for making them 
transparent. 
If it be desired to remove the mass from any part of a pre- 
paration, this is easily done with dilute acetic acid, which 
dissolves it. 308 
CHAPTER XXIV. 
Glycerin Masses.* 
514. Beale’s Carmine Glycerin Mass [How to Work, &c., 
p. 95).—Five grains of carmine are dissolved in a little water 
with the aid of about five drops of ammonia, and added to 
half an ounce of glycerin. Then add half an ounce of glycerin 
with eight or ten drops of acetic or hydrochloric acid, gradu- 
ally, with agitation. Test with blue litmus paper, and if 
necessary add more acid till the reaction is decidedly acid. 
Then add half an ounce of glycerin, two drachms of alcohol, 
and six drachms of water. I have found this useful, but not 
so good as the Prussian blue injections. 
515. Beale’s Prussian Blue Glycerin Mass (IIow to Work, &c., 
p. 93). 
Common glycerin ... 1 ounce. 
Spirits of wine .... 1 „ 
Ferrocyanide of potassium . . 12 grains. 
Tincture of perchloride of iron . 1 drachm. 
Water ..... 4 ounces. 
Dissolve the ferrocyanide in one ounce of the water and 
glycerin, and add the tincture of iron to another ounce. 
“ These solutions should he mixed together very gradually, 
and well shaken in a bottle, the iron being added to the solu- 
tion of the ferrocyanide of 'potassium. Next, the spirit and 
the water are to be added very gradually, the mixture being 
constantly shaken.” 
“ The water ” spoken of in the last sentence appears to 
mean the remaining three ouuces of water that were not 
mixed with the glycerin at first. 
Injected specimens should be preserved in acidulated gly- 
cerin, otherwise the colour may fade. 
516. Beale’s Acid Prussian Blue Glycerin Mass (ibid., p. 296). 
Price’s glycerin .... 2 fluid ounces. 
Tinct. of sesquicliloride of iron . 10 drops. 
Ferrocyanide of potassium . . 3 grains. 
Strong hydrochloric acid . . 3 drops. 
Water ..... 1 ounce. 
* See the remarks on Glycerin Masses, § 483. INJECTIONS—OTHER MASSES (COLD). 
309 
Proceed as directed above, dissolving the ferrocyanide in 
one half of the glycerin, the iron in the other, and adding the 
latter drop by drop to the former. Finally add the water and 
HC1. Two drachms of alcohol may be added to the whole if 
desired. 
I consider this a most admirable formula. I possess some 
of this mass prepared many years ago, in which not the 
smallest flocculus has made its appearance. The Prussian 
blue appears to be in a state of true solution. The mass runs 
well, and has not so much tendency to exude from cut capil- 
laries as might be supposed. Unfortunately it is a rather 
expensive preparation. 
517. Ranvier's Prussian Blue Glycerin Mass (Traite, p. 120).— 
Consists of the Prussian blue fluid, § 498, mixed with one fourth of gly- 
cerin. 
518. Other Colours.—Any of the colouring masses, §§ 486 to 490, or 
other suitable colouring masses, combined with glycerin, either dilute or 
pure. 
Aqueous Masses. 
519. Banvier’s Prussian Blue Aqueous Mass (Traite, p. 120). 
—The soluble Prussian blue, § 498, injected without any 
vehicle. It does not extravasate. 
520. Muller’s Berlin Blue (Arch.f.mih. Anat., 1865, p. 150). 
—Precipitate a concentrated solution of Berlin blue by means 
of 90 per cent, alcohol. 
The precipitate is very finely divided ; the fluid is perfectly 
neutral, and much easier to prepare than the formula of 
Beale. 
521. Mayer’s Berlin Blue (Mitth. zool. Stat. Neapel, 1888, 
p. 307).—A solution of 10 c.c. of tincture of perchloride of 
iron in 500 c.c. of water is added to a solution of 20 gr. of 
yellow prussiate of potash in 500 c.c. of water, allowed to 
stand for twelve hours, decanted, the deposit washed with 
distilled water on a filter until the washings come through 
dark blue (one to two days), and the blue dissolved in about 
a litre of water. 
522. Emery’s Aqueous Carmine (ibid., 1881, p. 21).—To a 10 per 310 
CHAPTEB XXIV. 
cent, ammoniacal solution of carmine is added acetic acid, with continual 
stirring, until the colour of the solution changes to blood-red through 
incipient precipitation of the carmine. The supernatant clear solution is 
poured off, and injected cold without further preparation. The injected 
organs are thrown at once into strong alcohol to fix the carmine. For injec- 
tion of fishes. 
523. Letellier's Vanadate of Ammonia and Tannin.—See Journ. 
Roy. Mic. Soc., 1889, p. 151, or previous editions. 
524. Taguchi's Indian Ink (Arch. f. mik. Anat., 1888, p. 
565; Zeit. f. wiss. Mik., 1888, p. 503).—Chinese or (better) 
Japanese ink well rubbed up on a hone until a fluid is obtained 
that does not run when dropped on thin blotting-paper, nor 
form a gre}r ring round the drop. Inject until the prepara- 
tion appears quite black, and throw it into some hardening 
liquid (not pure water). 
I believe this will be found useful for many purposes, 
especially for work amongst Invertebrates, as well as for 
lymphatics, juice-canals, and the like. 
Celloidin Masses. 
525. Schiefferdecker’s Celloidin Masses {Arcli. Anat. u. Phys., 
1882 [Anat. Abth.'j, p. 201). (For Corrosion preparations.) See previous 
editions of this work, or Whitman’s Methods in Microscopical Anatomy. 
526. Hochstetter’s Modification of Schiefferdecker’s Mass {Anat. 
Anz., 1886, p. 51; Journ. Roy. Mic. Soc., 1888, p. 159). 
Other Masses. 
527. Budge’s Asphaltum Mass.—See Arcli. f. mile. Anat., xiv, 1877, 
p. 70, or previous editions. 
528. Hover’s Shellac Mass {Arcli.f. mile. Anat., 1876, p. 645). See 
previous editions. 
This method, with some slight modifications of detail, has been recom- 
mended by Bellarminow {Anat. Anz., 1888, p. 650; see also Zeit.f. wiss. 
Mile., v, 4, 1888, p. 523, and Journ. Roy. Mic. Soc., 1889, p. 150). 
529. Hover’s Oil-eolour Masses (Interned. Monatschr.f. Anat., 1887, 
p. 341 ; see also Zeit.f. wiss. Mile., 1888, p. 80, and Journ. Roy. Mic. Soc., 
1888, p. 848). Pansch’s Starch Mass (see Arcli.f. Anat. u. Entw., 1877, 
p. 480; 1880, pp. 232, 371 ; 1881, p. 76; 1882, p. 60; 1883, p. 265 ; and a 
modification of the same by Gage, Amer. Mon. Mic. Journ., 1888, p. 195 ; 
and Journ. Roy. Mic. Soc., 1888, p. 1056). Teiebmann’s Linseed-Oil INJECTIONS—OTHER MASSES (COLD). 
311 
Masses (see S. B. Math. Kl. Krakau Akadvii, pp. 108, 158 ; Journ. 
Roy. Mic. Soc., 1882, pp. 125 and 716, and 1895, p. 704). 
530. Natural Injections (Robin, Traite, p. 6).—To preserve 
these throw the organs into a liquid composed of 10 parts of 
tincture of perchloride of iron and 100 parts of water. 
Retterer and Zellner use solution of Muller, see Journ. 
Roy. Mic. 8oc.} 1894, p. 641. CHAPTER XXY. 
MACERATION AND DIGESTION. 
Maceration. 
531. Methods of Dissociation.—It is sometimes necessary, 
in order to obtain a complete knowledge of the forms of the 
elements of a tissue, that the elements be artificially separated 
from their place in the tissue and separately studied after 
they have been isolated both from neighbouring elements and 
from any interstitial cement-substances that may be present 
in the tissue. Simple teasing with needles is often insufficient 
to effect the desired isolation, as the cement-substances are 
often tougher than the elements themselves, so that the latter 
are torn and destroyed in the process. In this case recourse 
must be had to maceration processes, by which is here meant 
treatment with media which have the property of dissolving 
or at least softening the cement-substances or the elements 
of the tissue that it is not wished to study, whilst preserving 
the forms of those it is desired to isolate. When this soften- 
ing has been effected the isolation is completed by teasing, 
or by agitation with liquid in a test-tube, or by the method 
of tapping, which last gives in many cases (many epithelia, 
for instance) admirable results which could not be attained 
in any other way. The macerated tissue is placed on a slide 
and covered with a thin glass cover supported at the corners 
on four little feet made of pellets of soft wax. By tapping 
the cover with a needle it is now gradually pressed down, 
whilst at the same time the cells of the tissue are segregated 
by the repeated shocks. When the segregation has pro* 
ceeded far enough, mounting medium may be added, and the 
mount closed. 
The student will do well not to neglect this simple method, 
which is one that it is most important to be acquainted with. 
A good material for making wax feet\ s obtained (Yosseler, 
Zeit.f. wiss. Mile., vii, 4, 1891, p. 461) by melting white wax MACERATION AND DIGESTION. 
313 
and stirring into it one half to two thirds of Venice turpen- 
tine. Care must be taken if the operation be performed over 
a naked flame, as the turpentine vapours are inflammable. 
532. Iodised Serum.—The preparation of this reagent has 
been given in Chap. XXI. The manner of employing it for 
maceration is as follows :—A piece of tissue smaller than a 
pea must be taken, and placed in 4 or 5 c.c. of weakly iodised 
serum in a well-closed vessel. After one day’s soaking the 
maceration is generally sufficient, and the preparation may be 
completed by teasing or pressing out, as indicated above; if 
not, the soaking must be continued, fresh iodine being added 
as often as the serum becomes pale by the absorption of the 
iodine by the tissues. By taking this precaution, the macera- 
tion may be prolonged for several weeks. 
It is obvious that these methods are intended to be applied 
to the preparation of fresh tissues, the iodine playing the part 
of a fixing agent with regard to protoplasm, which it slightly 
hardens. 
533. Artificial Iodised Serum (Frey, Le Microscope, p. 131; 
Ranvier, Traite, p. 77). 
The formula has been given in Chap. XXI. Ranvier states 
that he has been unable to obtain good results, for purposes 
of maceration, by this method. 
534. Alcohol.—Ranvier employs one-third alcohol (1 part of 
36° alcohol to 2 parts of water). Epithelia will macerate well 
in this in twenty-four hours. Ranvier finds that this mixture 
macerates more rapidly than iodised serum. 
Other strengths of alcohol may be used, either stronger 
(equal parts of alcohol and water) or weaker alcohol, for 
isolation of the nerve-fibres of the retina, for instance—Thin). 
All observers are agreed that one-third alcohol is a mace- 
rating medium of the highest order; List (Zeit.f. wiss. Mile., 
1885, p. 511) states that for glandular structures it should be 
used with precaution, on account of swellings that it produces 
in the cells, and that Muller’s solution, or osmic acid, should 
be preferred for such objects. 
535. Salt Solution.—10 per cent, solution of sodium chloride 
is a well-known and valuable macerating medium. 314 
CHAPTER XXV. 
536. Moleschott and Piso Borme’s Sodium Chloride and 
Alcohol (MoleschotFs Untersuchungen zur Naturlehre, xi, pp. 
99—107 ; Ranvier, Traite, p. 242).—10 per cent, solution of 
sodium chloride, 5 volumes; absolute alcohol, 1 volume. 
For vibratile epithelium, Ranvier finds the mixture inferior 
to one-third alcohol. 
537. Formaldehyde.—Gage has tested formaldehyde as a 
dissociating agent. He recommends the addition of two 
parts of formalin (40 per cent, solution of formaldehyde) to 
1000 parts of normal salt solution. He states that the results 
are highly satisfactory, the mixture acting quickly and yet 
retarding deterioration for some time (quoted from Fish, 
Proc. Am. Mic. Soc., xvii, 1895, p. 328). 
538. Chloral Hydrate.—In not too strong solution, from 2 to 
5 per cent., for instance, chloral hydrate is a mild macerating 
agent that admirably preserves delicate elements. Lavdowsky 
[Arch. f. mik. Anat., 1876, p. 359) recommends it greatly 
for salivary glands. Hickson [Quart. Journ. Mic. Sci., 1885, 
p. 244) recommends it for the study of the retina of 
Arthropods. 
539. Caustic Potash, Caustic Soda.—These solutions must be 
employed strong, 35 to 50 per cent. (Moleschott); so employed 
they do not greatly alter the forms of cells, whilst weak solu- 
tions destroy all the elements. (Weak solutions may, how- 
ever be employed for dissociating the cells of epidermis, 
hairs, and nails.) The strong solutions may be employed by 
simply treating the tissues with them on the slide. To make 
permanent preparations, the alkali should be neutralised by 
adding acetic acid, which forms with caustic potash acetate of 
potash, a well-known mounting medium (see Behrens, Kossel, 
and Schiepferdecker, Das Mikroskop, i, 1889, p. 156). It 
has been found by S. H. and S. P. Gage (Proc. Amer. Soc. of 
Microscopists, 1889, p. 35; Zeit. f. wiss. Mik., vii, 3, 1890, p. 
349) that instead of acetic acid, 60 per cent, acetate of potash 
solution, employed in considerable quantity and if desired 
with addition of 1 per cent, of acetic acid, may be used, the 
preparations either being mounted therein, or in glycerin 
or glycerin jelly. They may be stained if the acetate be first MACERATION AND DIGESTION. 
315 
washed out by treatment for twenty-four hours with alum 
solution. 
540. Sulphocyanides of Ammonium and Potassium (Stirling, 
Journ. Anat. and Phys., xvii, 1883, p. 208).—10 per cent, 
solution of either of these salts is, according to Stirling, an 
admirable dissociating medium for epithelium. Macerate 
small pieces for twenty-four to forty-eight hours. 
If a crystalline lens be macerated as above its fibres become 
beaded or moniliform. 
541. Soulier’s Sulphocyanide Mixtures (Travaux de VInst. 
zool. de Montpellier, Nouv. Ser., 2, 1891, p. 171). Soulier 
has found that Stirling’s solution greatly deteriorates cellular 
elements, but that good results are obtained by combining it 
with a fixing agent. He prepared the following series of 
mixtures : 
Sulphocyanide 
of ammonium 
or potassium 
of 10 percent., 
5 per cent., 
2’5 per cent., 
or l’2o per 
cent, strength ' 
Nos. 1. 2. 3. 4. 5. 6. 7- 8. 
20 c.c. 30 c.c. 35 c.c. 36 c.c. 37 c.c. 38 c.c. 39 c.c. 39-5 c.c. 
Solution of 
Ripart and 
Petit . . . 
20 c.c. lOc.c. 5 c.c. 4c.c. 3 c.c. 2c.c. 1 c.c. 0'5 c.c. 
The best results were obtained with series made with a 
2 per cent, solution of sulphocyanide. 
Soulier also obtained good results by combining liquid of 
Ripart and Petit with artificial serum of Kronecker instead 
of sulphocyanide, or with pepsin, eau de Javelle, 10 per cent, 
sulphate of soda, or 1*5 per cent, solution of caustic soda. 
And he further found that good results are obtained by 
combining solutions of chloride of sodium, or solutions of 
caustic potash or soda, with any of the usual fixing agents. 
542. Saliva, Artificial (for embryology of nerve and muscle) 
(Calberla’s formula, Arch. f. mik. Anat., xvi, 1879, p. 471, 
eb seq.).—After having made trial of various different mace- 
rating agents, with the object of obtaining isolation of tho 
developing muscle and nerve of embryos of Amphibia and 316 
CHAPTER XXV. 
Ophidia, Calberla found that the best results were obtained 
by means of Czerny’s mixtui’e of saliva and solutio Mulleri. 
This led him to imagine an artificial saliva, which on trial 
gave results as good as those obtained by natural saliva, or 
even better. 
Second formula (the first formula is suppressed, as being 
more complicated, and not giving better results) : 
Potassium chloride .... 0-4 
Sodium chloride ..... 0*3 
Phosphate of soda .... 02 
Calcium chloride . . . . . 02 
I 
PI 
This is dissolved in 100 parts of water, saturated with 
carbonic acid, and the solution combined with water and 
solutio Mulleri, one volume of the solution being combined 
with half a volume of Muller’s solution and a volume of 
water. 
In either case the Muller’s solution may be replaced by a 
21 per cent, solution of chromate of ammonia. The best 
results were obtained when the solutions were saturated with 
the C03 just before using. 
The tissues are isolated by teasing and shaking, and speci- 
mens mounted in concentrated acetate of potash. 
543. Landois’s Solution (Arch. f. mik. Anat., 1885, p. 445). 
Saturated sol. of neutral chromate of ammonia 5 parts. 
Saturated sol. of phosphate of potash . . 5 „ 
Saturated sol. of sulphate of soda . . . 5 „ 
Distilled water . . . . . . . 100 „ 
To be used in the same way as chromic acid :—Small pieces 
of tissue are macerated for one to three/or even four or five 
days, in the liquid, then brought for twenty-four hours into 
ammonia carmine diluted with one volume of the macerating 
liquid. 
Gierke particularly recommends this liquid for all sorts of 
macerations, but especially for the central nervous system, for 
which he finds it superior to all other agents. It is also re- 
commended for the same purpose by Nansen (v. Zeit.f. wiss. 
Mik., v, 2, 1888, p. 242). 
544. Permanganate of Potash.—Has an action similar to that MACERATION AND DIOxESTION. 
317 
of osmic acid, but more energetic. Is recommended, either 
alone or combined with alum, as the best dissociating agent 
for the fibres of the cornea (Rollett, Strieker’s Handbuch, 
p. 1108). 
545. Chromic Acid.—Generally employed of a strength of 
about 0'02 per cent. Specially useful for nerve tissues and 
smooth muscle. Twenty-four hours’ maceration will suffice 
for nerve-tissue. About 10 c.c. of the solution should be 
taken for a cube of 5 mm. of the tissue (Ranvier). 
546. Bichromate of Potash.—0‘2 per cent. 
547. Muller’s Solution.—Same strength. 
548. Muller’s Solution and Saliva (see above, § 542). 
549. Brock’s Medium (for nervous system of Mollusca, 
Intern. Monatsch. f. Anat., i, 1884, p. 349).—Equal parts of 
10 per cent, solution of bichromate of potash and visceral 
fluid of the animal. 
550. Mobius’s Media (quoted from Zeit. f. wiss. Mik., iii, 3, 
1886, p. 402). 
1. One part of seawater with 4 to 6 parts of 0’5 per cent, 
solution of bichromate of potash. 
2. 0‘25 per cent, chromic acid, 0'1 per cent, osmic acid, 
0’1 per cent, acetic acid, dissolved in sea water. For Lamelli- 
branchiata. Macerate for several days. 
551. Gage’s Picric Alcohol (Proc. Amer. Soc. of Microscopists, 
1890, p. 120; Zeit. f. wiss. Mik., ix, 1, 1892, pp. 87, 88).—95 
per cent, alcohol, 250 parts; water, 750; picric acid, 1. 
Recommended for most tissues, but especially for epithelia 
and smooth and striated muscle. This is also much recom- 
mended by Hopkins in the same place. A few hours’ macera- 
tion is generally sufficient. 
552. Osmic Acid.—O’l per cent., for from a few minutes to 
a fortnight (cortex of cerebrum—Rindfleisch) . May be fol- 
lowed by maceration in glycerin. 318 
CHAPTER XXV. 
553. Osmic and Acetic Acid (the Heetwjgs’ Liquid, Das Ner- 
vensystem u. die Sinnesorgane der Medusen, Leipzig, 1878, and 
Jen. Zeitschr., xiii, 1879, p. 457; Journ. Roy. Mic. Soc., iii, 
1880, p. 441, and [N.S.] iii, 1883, p. 732). 
0 05 per cent, ostnic acid . . .1 part. 
0‘2 ,, acetic acid . . 1 „ 
Medusae are to be treated with this mixture for two or 
three minutes, according to size, and then washed in repeated 
changes of 0-l per cent, acetic acid until all traces of free 
osmic acid are removed; they then remain for a day in 0’1 
per cent, acetic acid, are washed in water, stained in Beale’s 
carmine (in order to prevent the osmium from over-blacken- 
ing, and to assist the maceration), and are preserved in 
glycerin. 
For Actiniae the osmic acid is taken weaker, 0‘04 per cent.; 
both the solutions are made with sea water; and the washing 
out is done with O’2 per cent, acetic acid. If the maceration 
is complete, stain with picro-carmine; if not, with Beale’s 
carmine. 
554. Bela Hallee’s Mixture (.Morphol. Jahrb., xi, p. 321). 
—One part glacial acetic acid, 1 part glycerin, 2 parts water. 
Specially recommended for the central nervous system of 
Mollusca (Rhipidoglossa). A sufficient degree of maceration 
is obtained in thirty to forty minutes, the cells showing less 
shrinkage than with other liquids. 
555. Nitric Acid.—Most useful for the maceration of muscle. 
The strength used is 20 per cent. After twenty-four hours’ 
maceration in this, isolated muscle-fibres may generally be 
obtained by shaking the tissue with water in a test-tube. 
Preparations may afterwards be washed with water and put 
up in strong solution of alum, in which they may be pre- 
served for a long time (Hopkins, Proc. Amer. Soc., of Micro- 
scopists, 1890, p. 165; Zeit. f. wiss. Mih., ix, 1, 1892, p. 86). 
Maceration is greatly aided by heat, and at a temperature 
of 40° to 50° C. may be sufficiently complete in an hour 
(Gage). 
556. Nitric Acid and Chlorate of Potash (Kuhne’s method, 
Ueber die peripherischen Endorgane, &c., 1862; Ranviee, Traite, MACERATION AND DIGESTION. 
319 
p. 79).—Chlorate of potash is mixed, in a watch-glass, with 
four times its volume of nitric acid. A piece of muscle is 
buried in the mixture for half an hour, and then agitated 
with water in a test-tube, by which means it entirely breaks 
up into isolated fibres. 
557. Sulphuric Acid (Banvier, Traite, p. 78).—Sulphuric 
acid has been employed by Max Schultz for isolating the 
fibres of the crystalline. 
Macerate for twenty-four hours in 30 grms. of water, to 
which are added 4 to 5 drops of concentrated sulphuric acid. 
Agitate. 
Odenius found very dilute sulphuric acid to be the best 
reagent for the study of nerve-endings in tactile hairs. He 
macerated hair-follicles for from eight to fourteen days in a 
solution of from 3 to 4 grains of “ English sulphuric acid ” 
to the ounce of water. 
Hot concentrated sulphuric acid serves to dissociate horny 
epidermic structures (horn, hair, nails). 
558. Oxalie Acid.—Maceration for many days in concen- 
trated solution of oxalic acid has been found useful in the 
study of nerve-endings. 
559. Schiefferdecker’s Methyl Mixture (for the retina) 
{Arch. f. mik. Ancit., xxviii, 1886, p. 305).—Ten parts of gly- 
cerin, 1 part of methyl alcohol, and 20 pai-ts of distilled 
water. Macerate for several days (perfectly fresh tissue). 
560. Lysol (Beinke, Anat. Anz., viii, 1892, p. 582; Zeit. f. 
wiss. Alik., x, 2,1893, p. 224).—Lysol is an industrial product 
consisting in a solution of cresol in a neutral soap, and is 
used as an antiseptic. According to Beinke, it possesses a 
very rapid macerating action. He uses a 10 per cent, solu- 
tion in distilled water or in water containing alcohol and 
glycerin. Spermatozoa of the rat are said to be resolved 
into fibrils in a few minutes. The cortical cells of hairs are 
likewise resolved into fibrils in a few minutes. Epithelial 
cells of Salamandra are said to be dissociated instantaneously. 
Nuclear chromatin is destroyed, bringing into evidence the 
reticulated caryoplasm. 320 
CHAPTER XXV. 
For some conclusions founded on this reaction, see Arch. f. 
mik. Anat., xliii, 3, 1894, p. 398, et seg. 
Digestion. 
561. Beale’s Digestion Fluid (Archives of Medicine, i, 1858. 
pp. 296—316).—The mucus expressed from the stomach 
glands of the pig is rapidly dried on glass plates, powdered, 
and kept in stoppered bottles. It retains its properties for 
years. Eight tenths of a grain will dissolve 100 grains of 
coagulated white of egg. 
To prepare the digestion fluid, the powder is dissolved in 
distilled water, and the solution filtered. It filters readily. 
Or the powder may be dissolved in glycerin. The tissues to 
be digested may be kept for some hours in the liquid at a 
temperature of 100° F. (37° C.). 
562. Brucke’s Digestion Fluid (from Carnoy’s Biologie cellu- 
laire, p. 94). 
Glycerinated extract of pig’s stomach . . 1 vol. 
0’2 per cent, solution of HC1 . . .3 vols. 
Thymol, a few crystals. 
563. Bickfalvi’s Digestion Fluid (Gentrabl. f. d. med. Wiss., 
1883, p. 833).—One grm. of dried stomachal mucosa is mixed 
with 20 c.c. of 0-5 per cent, hydrochloric acid, and put into an 
incubator for three or four hours, then filtered. Macerate 
the tissue in the solution for not more than half an hour to an 
hour. 
564. Kuskow’s Digestion Fluid (Arch. f. mile. Anat., xxx, 
p. 32; cf. Zeit. f. wiss. Mile., iv, 3,1887, p. 384).—One part of 
pepsin dissolved in 200 parts of 3 per cent, solution of oxalic 
acid. The solution should be freshly prepared, and the 
objects (sections of hardened Ligamentum Nuchae) remain in 
it at the ordinary temperature for ten to forty minutes. 
565. Schiefferdecker’s Pancreatin Digestion Fluid (Zeit. f. 
wiss. Mile., iii_, 4, 1886_, p. 483).—Solution of pancreatin in 
water. Scliiefferdecker employs the “ Pankreatinum siccum” 
prepared by Dr. Witte, Eostock. A saturated solution is MACERATION AND DIGESTION. 
321 
made in distilled water, cold, and filtered. Pieces of tissue 
(epidermis) are macerated in it for three to four hours at 
about body temperature. Nuclei are preserved, and the 
forms of prickle-cells well shown. 
566. Kuhne’s Trypsin Methods (see Unters. a. cl. Phys. Inst. Univ. 
Heidelberg, i, 2, 1877, p. 219)."—Very complicated. 
567. Gedoelst’s Methods (see La Cellule, iii, 1887, p. 117, and v, 
1889, p. 126; also Zeit.f. wiss. Mik., vii, 1, 1890, p. 57). CHAPTER XXVI. 
CORROSION, DECALCIE1CATION, AND BLEACHING. 
Corrosion. 
568. Caustic Potash, Caustic Soda, Nitric Acid.—Boiling, or 
long soaking in a strong solution of either of these, is an effi- 
cient means of removing soft parts from skeletal structures 
(appendages of Arthropods, spicula of sponges, &c.). 
569. Eau de Javelle (Hypochlorite of Potash) (Noll’s method, 
Zool. Anzeig., 122, 1882, p. 528).—Noll remarks that the 
usual method of preparing the skeleton of siliceous sponges 
and similar structures by corroding away the soft parts by 
means of caustic potash has many disadvantages, of which a 
principal one is that the spicula are not preserved in their 
normal positions. He therefore proceeds as follows :—A piece 
of sponge is brought on to a slide and treated with a few drops 
of eau de Javelle, in which it remains until all soft parts are 
dissolved. (With thin pieces this happens in twenty to thirty 
minutes.) The preparation is then cautiously treated with 
acetic acid, which removes all precipitates that may have 
formed, and treated with successive alcohols and oil of cloves, 
and finally mounted in balsam. 
The same process is stated to be applicable to calcareous 
structures. I feel convinced, however, that if the structures 
are delicate, they will suffer, or be entirely destroyed. 
570. Eau de Labarraque (Hypochlorite of Soda) may be used 
in tlie same way as eau de Javelle. Looss (Zool. Anz., 1885, 
p. 333) finds that either of these solutions will completely 
dissolve chitin in a short time with the aid of heat. For this 
purpose the commercial solution should be taken concentrated 
and boiling. A formula for making it is given in § 597. 
If solutions diluted with 4 to 6 volumes of water be taken, 
and chitinous structures be macerated in them for twenty - CORROSION, DECALCIFICATION, AND BLEACHING. 
323 
four hours or more, according to size, the chitin is not dis- 
solved, but becomes transparent, soft, and permeable to stain- 
ing fluids, aqueous as well as alcoholic. The most delicate 
structures, such as nerve-endings, are stated not to be injured 
by the treatment. The method is applicable to Nematodes 
and their ova, an object well known for the resistance they 
oppose to ordinary reagents, and also to the removal of the 
albumen from ova of Amphibia, &c. 
This is undoubtedly a valuable method. 
571. Altmann’s Corrosion Method {Arch. f. mik. Anat., 1879, p. 471). 
—Whilst almost all animal tissues are very quickly destroyed by eau de 
Javelle, yet fats, and particularly fats hardened by osmic acid, withstand its 
action for a long time. If, then, you introduce some fat or other into a 
tissue, harden it with osmic acid and corrode the tissue with eau de Javelle, 
you will obtain a mould in osmium-blackened and hardened fat, of the 
spaces you had filled with the fat introduced. 
The method may be of much use in certain special researches, such as 
those on the choroid, iris, and pigmented organs. I recommend the 
reader to carefully study the article, which does not well bear abstracting. 
A good abstract will be found in Journ. Boy. Mic. Soc., 1879, p. 610, with 
plate. 
Decalcification and Desilicification. 
572. Decalcification.—In order to obtain the best results, it 
is important to employ only material that has been duly fixed 
and hardened secundum artem; and it is well not to put too 
much confidence in reagents that are said to have the pro- 
perty of hardening and decalcifying fresh material at the 
same time (Fish, Ref. Sandb. Med. Sci., Supp., p. 425.) 
573. Decalcification of Bone.—I take the following historical 
sketch from Busch’s article “ On the Technique of the Histo- 
logy of Bone” (Arch. f. mik. Anat., xiv, 1877, p. 481 ; see 
also the paper of Haug, in Zeit. f. iviss. Mik., viii, i, 1891, p. 1). 
The most widely used agent for decalcification is hydro- 
chloric acid. Its action is rapid, even when very dilute, but 
it has the disadvantage of causing serious swelling of the 
tissues. To remedy this, chromic acid may be combined with 
it, or alcohol may be added to it. Or a 3 per cent, solution 
of the acid may be taken and have dissolved in it 10 to 15 
per cent, of common salt. Or (Waldeyer) to a ToVo Per cent* 
solution of chloride of palladium may be added of its 
volume of HC1. 324 
CHAPTER, XXVI. 
Chromic acid is also much used, but has a very weak decal- 
cifying action and a strong shrinking action on tissues. For 
this latter reason it can never be used in solutions of more 
than 1 per cent, strength, and for delicate structures much 
lower strengths must be taken. 
Phosphoric acid has been recommended for young boues. 
Acetic, lactic, and pyroligneous acids have considerable 
decalcifying power, but cause great swelling. Picric acid 
has a very slow action, and is only suitable for very small 
structures. 
See further under the head of “ Bone.” 
574. Nitric Acid (Bcsch, 1. c.).—To all other agents Busch 
prefers nitric acid, which causes no swelling and acts most 
efficaciously, whilst at the same time it does not injuriously 
attack tissue-elements. 
One volume of chemically pure nitric acid of sp. gr. P25 is 
diluted with 10 vols. water. It may be used of this strength 
for very large and tough bones; for young bones it may be 
diluted down to 1 per cent. 
Fresh bones are first laid for three days in 95 per cent, 
alcohol; they are then placed in the nitric acid, which is 
changed daily, for eight or ten days. They must be removed 
as soon as the decalcification is complete, or else they will 
become stained yellow. When removed they are washed for 
one or two hours in running water and placed in 95 per cent, 
alcohol. This is changed after a few days for fresh alcohol. 
Young and foetal bones may be placed in the first instance 
in a mixture containing 1 per cent, bichromate of potash and 
ytj- per cent, chromic acid, and decalcified with nitric acid of 
1 to 2 per cent., to which may be added a small quantity of 
chromic acid per cent.) or chromate of potash (1 per cent.). 
By putting them afterwards into alcohol the well-known 
green stain is obtained. 
575. Nitric Acid and Alcohol.—3 per cent, of nitric acid in 
70 per cent, alcohol. Soak specimens for several days or 
weeks. I do not know who first recommended this admirable 
medium. Pure nitric acid, even if weak, readily exercises a 
gelatinising action on bone; whilst the addition of alcohol 
(or of alum) counteracts this action (Fish, Ref. Handb. Med. 
Sci., Supp., p. 425). CORROSION, DECALCIFICATION, AND BLEACHING. 
325 
Thoma (Zeit. /. wiss. Mik., viii, 2, 1891, p. 191) gives the 
following method.—Take 5 vols. of 95 per cent, alcohol and 1 
vol. pure concentrated nitric acid. Leave bones in this mix- 
ture, changing the liquid every two or three days, until 
thoroughly decalcified, which should happen even with large 
bones in two or three weeks. Wash out until every trace of 
acid is removed (t. e. for some days after no acid reaction is 
obtained with litmus paper) in 95 per cent, alcohol containing 
an excess of precipitated chalk. This may take eight to four- 
teen days, after which the tissues will stain well and may be 
treated as desired. 
576. Nitric Acid and Alum (Gage, quoted from Fish, ]. c.). 
—A saturated aqueous solution of alum is diluted with an 
equal volume of water, and to each 100 c c. of the dilute so- 
lution is added 5 c.c. of strong nitric acid. Change every 
two or three days, until the decalcification is complete. For 
teeth this is said to be, perhaps, a better decalcifier than the 
alcohol mixture. 
577. Hydrochloric Acid (see above, § 573).—Ranvieb says that it 
may be taken of 50 per cent, strength, and then has a very rapid action. 
To counteract the swelling action of the acid, sodium chloride maybe added. 
Two formulae of this sort have been given by von Ebnee (see Hatjg’s 
paper quoted in the last section). The first is 100 c.c. cold saturated solu- 
tion of sodium chloride in water, 100 c.c. water, and 4 c.c. hydrochloric 
acid. Preparations to be placed in this, and 1 to 2 c.c. hydrochloric acid 
added daily until they are soft. The second is, 25 parts of hydrochloric 
acid, 500 of alcohol, 100 of water, and 2'5 of sodium chloride. Haltg pre- 
fers the proportions of l'O to 5'0 of acid, 70 of alcohol, 30 of water, and 0 5 
of salt. 
578. Hydrochloric Acid and Chromic Acid (Bayekl, Arch.f. mile. 
Anat., 1885, p. 35).—Equal parts of 3 per cent, chromic acid and 1 per cent, 
hydrochloric acid. For ossifying cartilage. Haijg recommends equal parts 
of 1 per cent, hydrochloric acid and 1 per cent, chromic acid(l. c.). 
579. Hydrochloric Acid and Nitric Acid (Hopewell Smith, Journ. 
Roij. Mic. Soc., 1892, p. 433).—Place teeth in 12 parts of 10 percent. HC1, 
and after 15 hours add 1'5 parts of HN03, and after 48 hours add 1*5 parts 
more of HN03. After 75 to 80 hours remove and wash for half an hour in 
a solution of 5 grms. of lithium carbonate to an ounce of water. 
580. Hydrochloric Acid and Glycerin.—Glycerin, 95; hydrochloric 
acid, 5. Recommended for softening teeth in Squike’s Methods and For- 
mulae, p. 12. 326 
0HAPTEK XXVI. 
581. Picric Acid should be taken saturated. 
Piero-sulphuric acid should of course be avoided on account of the forma- 
tion of gypsum. 
Picro-nitric or Picro-hydrochloric Acid.—The reader will perhaps reflect 
that the last two fluids appear likely to be very useful for decalcifications. 
Mayer points out that the action is very rapid, and that the copiously 
evolved C02 often produces, mechanically, lesions in tissues; so that in 
many cases in which calcareous structures are concerned chromic acid is to 
be preferred, the more so as it more effectually hinders any collapsing of the 
structures that might result from the withdrawal of their supporting cal- 
careous elements. 
Picric acid fluids are good media, as though their action is slow they 
preserve tissues well, and leave them in a good state for subsequent staining. 
582. Phosphoric Acid.—10 to 15 per cent. (Haug, 1. c., in § 573). 
Somewhat slow, staining not good. 
583. Lactic Acid.—10 per cent, or more. Fairly rapid, preserves well, 
and may be recommended (Haug, 1. c.). 
584. Chromic Acid is employed in strengths of from 0T per cent, to 
2 per cent., the maceration lasting two or three weeks (in the case of bone). 
It is better to take the acid weak at first, and increase the strength gradu- 
ally. In any way the action is extremely slow, and it is therefore better to 
take one of the mixtures of chromic acid with a more energetic agent. 
585. Chromic and Nitric Acid.—Dissolve 15 grms. pure chromic 
acid in 7 oz. of distilled water, to which 30 minims of nitric acid are after- 
wards to he added. Macerate for three or four weeks, changing the fluid 
frequently (Marsh). 
Fol takes 70 volumes of 1 per cent, chromic acid, 3 of nitric acid, and 200 
of water (Lehrb., p. 112). 
Fish takes simply liquid of Perenyi. 
It remains to be added that even with the addition of nitric or hydro- 
chloric acid the action is excessively slow, frequently requiring months to be 
complete. 
586. Arsenic Acid.—4 per cent, aqueous solution, used at a tempera- 
ture of 30° to 40° C. (Squire’s Methods and Formulae, &c., p. 11). 
587. Phloroglucin (Andeer, Centralbl. f. d. med. Wiss., xii, 
xxxiii, pp. 193, 579; Intern. Monatschr., i, p. 350; Zeit.f. wiss. 
Mih’., 1885, pp. 375, 539 ; Journ. Roy. Mic. Soc., 1887, p. 504 ; 
Haug, Zeit. f. wiss. Mile., viii, 1, 1891, p. 8; Ferreri, ibid., ix, 
2, 1892, p. 236; Bull. R. Accad. Med. di Boma, 1892, p. 67). 
—This is the most recent of the decalcification methods. It 
has the advantages of being the most rapid of any, and of 
preserving the tissues very well (with the exception of 
blood). CORROSION, DECALCIFICATION, AND BLEACHING. 
327 
Phloroglucin by itself is not a solvent of lime salts; its 
function in the mixtures given below is so to protect the 
organic elements of tissues against the action of the miueral 
acids that these can be used in a much more concentrated 
form than would be otherwise advisable. 
Haug advises the following procedure :—Bring 1 grin, of 
phloroglucin into 10 c.c. of pure, not fuming nitric acid 
(1*4 sp. gr.), and warm very slowly and carefully with gentle 
agitation. There is formed a clear solution of (presumably) 
a nitrate of phloroglucin. Dilute the solution with 100 c.c- 
of distilled water, and add 10 c.c. of nitric acid. This gives 
a solution containing 20 per cent, of acid, which is the proper 
proportion. More water may be added to the solution, to 
make it up to 300 c.c., if nitric acid be also added in the 
proportion given. But the dilution must not be carried 
beyond this point, in order that the preservative action of 
the phloroglucin be not overmuch weakened. The process of 
decalcification in this solution is extremely rapid, and there- 
fore should be carefully watched. Foetal and young bones 
become quite soft in half an hour; small pieces of old and 
hard bones (femur, temporal bone) in a few hours. Teeth 
take longer, and may require, if time be an object, a solution 
made with 35 to 45 per cent, of nitric acid. Wash out for 
two days in running water. The tissues stain well. 
The solution may be made with hydrochloric acid instead 
of nitric acid, 30 per cent, of acid being taken, and 0’5 per 
cent, of sodium chloride added. 
For slow decalcification a 2 to 5 per cent, nitric acid solu- 
tion may be used, or a mixture containing of phloroglucin 1 
part, nitric acid 5, alcohol 70, and distilled water 30 parts. 
For the labyrinth, Ferreri advises a mixture containing 
1 grm. of phloroglucin, dissolved with the aid of heat in 10 
grms. of hydrochloric acid with 100 of water, 200 of 70 per- 
cent. alcohol being added after cooling. The mixture should 
be changed once a week during thirty to forty days. 
Desilicification. 
588. Hydrofluoric Acid (Mayer’s method, Zool. Anz., 1881, No. 97, 
p. 593).—The objects from which it is desired to remove siliceous parts are 
brought in alcohol into a glass vessel coated internally with paraffin (other- 
wise the glass would be corroded by the acid). Hydrofluoric acid is then 
added drop by drop (the operator taking great care to avoid the fumes, 328 
CHAPTER XXVI. 
which attack mucous membranes with great energy). A Wagnerella borealis 
may thus be completely desilieified in a few minutes. Small pieces of sili- 
ceous sponges will require a few hours, or at most a day. The tissues 
do not suffer; and if they have been previously stained with acetic acid 
carmine the stain does not suffer; at least, this was so in the case of 
Wagnerella. 
This dangerous method is best avoided. As regards sponges, I would 
point out that if well imbedded, good sections may be made from them 
without previous removal of the spicula. The spicula appear to he cut; 
probably they break very sharply when touched by the knife. Knives are 
of coui’sfe not improved by cutting such sections. 
Bleaching. 
589. Mayer’s Chlorine Method (Mitth. Zool. Stat. Neapel, ii, 
1881, p. 8).—This is a process imagined for the purpose of 
getting rid of the blackening that often occurs as a conse- 
quence of treatment by osmio acid. 
The specimens are put into alcohol (either 70 or 90 per 
cent.). Crystals of chlorate of potash are added until the 
bottom of the vessel is covered with them. A few drops of 
concentrated hydrochloric acid are then added by means of a 
pipette, and mixed in by shaking the vessel as soon as the 
green colour of the evolving chlorine has begun to show itself. 
Warm if necessary; but most objects, even large ones, may 
be bleached in half a day without the employment of heat. 
The tissues do not suffer. 
Instead of hydrochloric acid, nitric acid may be used; in 
which case the bleaching agent is the freed oxygen, instead of 
chlorine. 
The first method may be used for the purpose of removing 
pigment from the eyes of insects. 
590. Marsh’s Chlorine Method (Section Cutting, p. 89).— 
Marsh generates chlorine in a small bottle by treating crystals 
of chlorate of potash with strong HC1, and leads the gas (by 
means of a piece of glass tubing bent twice at right angles) 
to the bottom of a bottle containing the sections in water. 
(See a tig. of the apparatus in Journ, Hoy. Mic. Soc., iii, 1880, 
p. 854.) *" ' 
591. Chlorine Solution (Sargent's method).—Hydrochloric acid, 10 
drops ; chlorate of potash, h dr.; water, 1 oz, Soak for a day or two. Wash 
well. \ 
This method is intended for “ bleaching insects; ” it will be seen that it is CORROSION, DECALCIFICATION, AND BLEACHING. 
329 
only applicable to the preparation of hard parts, as soft tissues would be 
destroyed by the solution. 
592. Kreasote (Pouchet’s method, Journ. de VAnat., 1876, p. 8, 
et. seq.).—I gather from the paper here quoted that most of the granular 
animal pigments are soluble in kreasote. Other solvents are mentioned in 
this paper (“On the Change of Colouration through Nervous Influence”), 
but this appears to be the only one eapable of general histological appli- 
cation. 
593. Nitric Acid has a similar action. Parker [Bull. Mus. 
Comp. Zool., Cambridge, U.S.A., 1889, p. 173; see Zeit. f. 
wiss. Mile., viii, 1, 1891, p. 82) says that for eyes of scorpions 
the usual 5 to 10 per cent, solutions are not strong enough. 
He treats sections, fixed to the slide with Schallibaum’s 
medium, for about a minute with a solution of up to 50 per cent, 
of nitric acid in alcohol, or, still better, with a 35 per cent, 
solution of a mixture of equal parts of nitric and hydrochloric 
acid in alcohol. To make the solution, the acid should be 
poured slowly into the alcohol (not vice versa), and the mixture 
kept cool. 
594. Peroxide of Hydrogen (Oxygenated Water) (Pouchet’s 
method, M. Deval, Precis, &c., p. 234).—Macerate in glycerin 
to which has been added a. little oxygenated water (5 to 6 
drops to a watch-glass of glycerin). (Oxygenated water may 
be procured from perfumers or hair-dressers, by whom it is 
sold as a hair dye under the name of “Aureoline,” “ Golden 
hair-wash,” or the like.) 
The brownish-green colour communicated to tissues by 
chromic solutions may be changed to yellow by means of 
oxygenated water (see § 41). 
Osmium preparations may be bleached in the same way 
(see § 38). 
595. Peroxide of Sodium (Carazzi, Zool. Anz., 444, 1894, 
p. 135).—Peroxide of sodium, Na3 O2, is a yellowish powder, 
which in presence of water gives off oxygen, and communi- 
cates an alkaline reaction to the water. With acidified water 
this alkalinity is not produced. Carazzi advises that a IQ per 
cent, solution of tartaric or acetic acid be put into a suitable 
recipient, and a small quantity of the peroxide be put at the 
bottom of the recipient. Alcohol of 70 per cent, is then 
cautiously poured on to the surface of the liquid, and the CHAPTER XXYI. 
objects to be bleached (previously saturated with alcohol) are 
then put into the supernatant alcohol. Oxygen is given off 
from the bottom of the liquid, and rises and dissolves in the 
alcohol, bleaching the objects that have been placed there. 
Mineral acids must not be employed, and large quantities 
of peroxide must not be added to a small quantity of liquid, 
as the reaction under these conditions is very violent. 
596. Sulphurous Acid.—Prof. Gilson writes me that he finds 
alcoholic solution of sulphurous anhydride (SO2) very con- 
venient for the rapid decolouration of bichromate objects. 
A few drops suffice. 
597. Eau de Labarraque. Eau de Javelle (see §§ 569, 570).— 
These are bleaching agents. For the manner of preparing a similar solu- 
tion see Journ. de Microgr., 1887, p. 154, or Journ. Roy. Mic. Soc., 1887, 
p. 518. It is, shortly, as follows:—8 parts of caustic soda are dissolved in 
100 parts of distilled water, and chlorine is passed through to saturation. 
During the passage of the chlorine the solution must be surrounded with a 
mixture of salt and ice, otherwise the temperature rises, and chloride and 
chlorate of soda are produced. The resulting solution contains 7'45 per 
cent, of hypochlorite of soda. It is green ; and the more effectual the cold, 
the greener is the colour. The energy of the decolourising action is pro- 
portional to the greenness of the solution. Of course the method cannot be 
used for bleaching soft parts which it is desired to preserve. 
598. Chloroform helps to clear strongly pigmented chitin, and com- 
bined with nitric acid will decolourise it entirely (see below, in the chapter 
on Arthropods, Part II). 
599. Grenacher’s Mixture for Eyes of Arthropods and other 
Animals (Abh. nat. Ges. Halle-a.-S., xvi; Zeit. f. iviss. Mik., 
1885, p. 244). 
Glycerin ...... 1 part. 
80 per cent, alcohol . . . .2 parts. 
Mix and add 2 to 3 per cent, of hydrochloric acid. 
Pigments dissolve in this fluid, and so doing form a staiu 
which suffices in twelve to twenty-four horn's for staining the 
nuclei of the preparation. You may, if you like, first stain 
the objects with borax-carmine, and then put them into the 
liquid, the pigment being washed out more rapidly than the 
carmine. But the progress of the decolouration must be care- 
fully watched. CHAPTER XXVII. 
EMBRYOLOGICAL METHODS. 
600. Artificial Fecundation.—This practice, which affords 
the readiest means of obtaining the early stages of develop- 
ment of many animals, may be very easily carried out in the 
case of the Amphibia anura, Teleostea, Cyclostomata, Echino- 
dermata, and many Vermes and Coelenterata. 
In the case of the Amphibia, both the female and the male 
should be laid open, and the ova should be extracted from 
the uterus and placed in a watch-glass or dissecting-dish, and 
treated with water in which the testes, or, better, the vasa 
deferentia, of the male have been teased. 
Females of Teleostea are easily spawned by manipulating 
the belly with a gentle pressure; and the milt may be ob- 
tained from the males in the same way. (It may occasionally 
be necessary, as in the ease of the Stickleback, to kill the 
male, and dissect out the testes and tease them.) The sper- 
matozoa of fish, especially those of the Salmonidce, lose their 
vitality very rapidly in water; it is therefore advisable to add 
the milt immediately to the spawned ova, then add a little 
water, and after a few minutes put the whole into a suitable 
hatching apparatus with running water. 
Artificial fecundation of Invertebrates is easily performed 
in a similar way. It is sometimes possible to perform the 
operation under the microscope, and so observe the penetra- 
tion of the spermatozoon and some of the subsequent phe- 
nomena, as has been done by Fol, the Hertwigs, Selenka, and 
others for the Echinodermata and other forms. 
601. Superficial Examination.—The development of some 
animals, particularly some Invertebrates, may be to a certain 
extent followed by observations of the living ova under the 
microscope. This may usefully be done in the case of various 
Teleosteans, such as the Stickleback, the Perch, Macropodus, 
and several pelagic forms, and with Ghironomus, Asellus 332 
CHAPTER XXVII. 
aquaticus, Ascidians, Planorbis, many Coelenterata, &c. I 
advise the student to carefully draw the different stages so 
observed, for such drawings are most important aids to the 
study of the same stages by the section method. 
Some ova of Insecta and Arachnida which are completely 
opaque under normal conditions become transparent if they 
are placed in a drop of oil; if care be taken to let their 
surface be simply impregnated with the oil, the normal course 
of development is not interfered with (Balbiani). 
602. Preparation of Sections.—Osmic acid, employed either 
alone or in combination with other reagents, is an excellent 
fixing agent for small embryos, but not at all a good one for 
large ones. It causes cellular elements to shrink somewhat, 
and therefore brings out very clearly the slits that separate 
germinal layers, and any channels or other cavities that may 
be in course of formation. 
In virtue of its property of blackening fatty matters, 
myelin amongst them, it is of service in the study of the 
development of the nervous system. 
Chromic acid is indispensable for the study of the external 
forms of embryos; it brings out elevations and depressions 
clearly, and preserves admirably the mutual relations of the 
parts ; but it does not always preserve the forms of cells 
faithfully, and is a hindrance to staining in the mass. 
Picric liquids have an action which is the opposite of that 
of osmic acid ; they cause cellular elements to swell some- 
what, and thus have a tendency to obliterate spaces that 
may exist in the tissues. But notwithstanding this defect, 
the picric compounds, and especially Kleinenberg’s picro- 
sulphuric acid, are amongst the best of embryological fixing 
agents. 
Rabl (Zeit. f. wiss. Mik., xi, 2, 1894, p. 165) highly re- 
commends for embryos of Vertebrates, and also for other 
objects, the following pdatino-sublimate mixture. 
Platinic chloride, 1 per cent, solution 1 vol. 
Saturated aqueous sublimate solution 1 „ 
Distilled water . . . 1 „ 
This serves for a large number of blastoderms and young 
embryos (Pisces, Amphibia, Aves, Mammalia). Advanced 
embryos of Teleostea ought to be fixed in the warmed mix- EMBRYO LOG I CAL METHODS. 
333 
ture, in order to avoid rupture of the muscles aud shrinkage 
of the chorda. 
Rabl’s picro-sublimate mixture has been given § 61. It 
is recommended especially for somewhat advanced embryos, 
such as embryo chicks from the third or fourth day, and 
other embryos of a similar size. 
For imbedding, the celloidin-chloroform method of Viallanes gives excel- 
lent results, and so does paraffin. This latter is preferable in so far as it 
lends itself better to the rapid production of series of sections, and allows of 
the use of the Cambridge Rocking Microtome, the Minot, or the Reinhold- 
Giltay, which is perhaps the microtome par excellence of the embryologist. 
As to staining, my eminent fellow-worker, Dr. Henneguy, writing tbe 
chapter on embryological methods for the French edition of this work, 
advised staining in the mass with borax-carmine or alum-carmine (Henne- 
guy’s acetic acid formula) ; or, as an alternative, tbe staining of sections by 
Flemming’s method. The improvements that have in recent times been 
worked out in this method give still greater weight to the latter recom- 
mendation. 
603. Reconstruction of Embryos from Sections.—The study of 
a series of sections of any highly differentiated organism of 
unknown structure is so complicated that it is often necessary 
to have recourse to elaborate methods of geometrical or of 
plastic reconstruction in order to obtain an idea or a model 
of the whole. These methods have now been brought to so 
high a degree of complexity that a volume rather than a 
paragraph would be necessary to describe them. See Born, 
“ Die Plattenmodellirmethode,” in Arch. f. mile. Anat., 1883, 
p. 591, and Zeit. f. wiss. Mile., v, 4, 1888, p. 433; Strasser, in 
Zeit. f. wiss. Mile., iii, 2, 1886, p. 179, and iv, 2 and 3, pp. 168 
and 330; Kastschenko, in Zeit. f. wiss. Mile., iv, 2 and 3, 
1887, pp. 235-6 and 353, and v, 2, 1888, p. 173 (abstracts of 
all these papers may be found in Journ. Roy. Mic. Soc. of 
the years quoted). 
A simple, but in many cases quite efficient plan, has been described by 
For (Lehrb., p. 35) as follows:—Before cutting your sections, you make 
an outline drawing of your object, under the magnification that you intend 
to employ for the reconstructed drawing, and in a plane perpendicular to 
that of the intended sections. For instance, if you intend to make trans- 
verse sections of an embryo, begin by making a profile drawing of it, that 
is, a drawing of the outline of an ideal sagittal section of it. Then make 
your series of sections, and make drawings of them all under the same 
magnification as the sagittal drawing. Then trace over your sagittal draw- 334 
CHAPTER XXVII. 
ing a series of equidistant parallel lines in positions corresponding to the 
sections that have been made. If your sections are one hundredth of a 
millimetre thick, and your drawing be magnified one hundred times, the 
lines should he one millimetre apart (if you intend to reconstruct the whole 
of your sections, but the operation may frequently be abridged by only 
reconstructing say every fifth or every tenth section). 
You have now to fill in your outline drawing with details borrowed from 
the drawings of the sections. You may help yourself greatly in the follow, 
ing way:—A plate of glass, of a size suitable to the intended drawings, is 
covered with a layer of gelatin, and dried. On this is ruled a series of 
parallel lines, very close together, and ruled with differently coloured inks, 
the colours recurring in regular order. The plate is then cut into two 
unequal parts by a diamond, on a line perpendicular to the coloured lines. 
Lay one of the parts of the plate on the outline drawing so that the cut 
edge covers the line that corresponds to the first section you are going to 
fill in ; then lay the other part of the plate on the drawing of the section 
in such a position that the limits of the drawing correspond to the same 
coloured lines that cover the limits of the outline drawing on the other part 
of the plate already placed. 
Trace on the plate that covers the drawing of the section the outline of 
the internal organs. Lay it against its fellow-plate on the outline drawing, 
making the coloured lines correspond, and you will easily be able to mark 
off accurately on the outline drawing a series of dots that correspond in 
position to the outlines of the internal organs. This operation having been 
repeated for each of the sections that you desire to bring into your recon- 
struction, nothing remains but to join your dots by lines, and you will have 
filled up your outline drawing with a representation of the internal organs 
in the same plane. 
If any reader think this process complicated, he needs but to spend five 
minutes in trying it with a piece of tracing paper, and will find it to be in 
reality extremely simple. 
Another simple plan is to gum the drawings of the sections on cardboard 
of a thickness proportional to the thickness of the section and the magnifi- 
cation, cut out all the cavities of the drawing with a knife or fret-saw, and 
gum all the fretwork thus obtained together, This gives, of course, a 
model of the object. 
In simple cases it may be sufficient to adopt the plan 
described by Schaffer (Zeit. f. wiss. Mile., vii, 3,1890, p. 342). 
Careful outlines of the sections to be reconstructed are drawn 
on tracing-paper with the aid of the camera lucida, super- 
posed, and held up against the light for examination by 
transparence. Eyclesheimer’s method of producing orienta- 
tion lines in celloidin imbedding mass has been described in 
Chap. X, and some paraffin orientation methods in § 136. EMBRYOLOGICAL METHODS. 
335 
Mammalia. 
604. Rabbit—Dissection.—The rabbit may conveniently be 
taken as a type for this kind of work. 
For the study of the early stages the ova must be sought 
for in the tubse a certain number of hours after copulation. 
The dehiscence of the follicles takes place about ten hours 
after the first coitus. The tubse and cornua having been dis- 
sected out should be allowed to cool, and remain until the 
muscular contractions have ceased. Then, with the aid of 
fine scissors or a good scalpel, all the folds of the genital 
duct are carefully freed from their peritoneal investment. 
The tubas are then (if the ova are still within them, which 
is the case up to the end of the third day after coition) laid 
out on a long slip of glass, and slit up longitudinally by means 
of a pair of fine, sharp scissors. By means of needles and 
forceps the tubal mucosa is spead out so as to smooth out its 
folds as much as possible, and is carefully looked over with a 
strong lens or with a low power of the microscope. When 
the ova are found, a drop of some “ indifferent ” liquid is 
dropped on each, and it is carefully taken up with the point 
of a scalpel, a cataract needle, or a small pipette. They may 
be examined in the peritoneal fluid of the mother if the 
animal has been killed, or in its aqueous humour, or in 
amniotic liquid, or in blood-serum, or in Kronecker’s or other 
artificial serum. 
If you have not been able to find the ova with the lens or 
the microscope, scrape off the epithelium of the tubal mucosa 
with a small scalpel, mix it with a little indifferent liquid, 
and look for the ova under the microscope by transmitted 
light. 
Another method, employed by Kolliker, consists in in- 
jecting solution of Muller or weak osmic acid into the oviduct 
by means of a small syringe, and collecting the liquid that 
runs out in a series of watch-glasses, in which the ova can 
very easily be found by the microscope. 
The same doe may be made to serve for two observations, at some hours’ 
or days’ interval. A longitudinal incision of 8 to 10 centimetres length is 
made on the median or a lateral line of the abdomen; an assistant keeps 
the intestines in their place; a ligature is placed at the base of one of the 
uterine cornua, beneath the neck, and a second ligature around the mesome- 336 
CHAPTER XXVll. 
trium and mesovarium. The ovary, the tuba, and the cornu of that side 
are then detached with scissors. The abdomen is then closed by means of 
a few sutures passing through the muscle-layers and the skin. The animals 
support the operation perfectly well, and the development of the ova of the 
opposite side is not in the least interfered with. When it is desired to study 
these the animal may be killed, or may be subjected to a second laparotomy 
if it be desired to preserve it for ulterior observations. 
During the fourth, fifth, and sixth days after copulation the 
ova of the rabbit are free in the uterine cornua; they are 
easily visible to the naked eye, and may be extracted by the 
same manipulations as those of the tubes. After the sixth 
day they are at rest in the uterus, but have not yet contracted 
adhesions with the mucosa, so that they can still be extracted 
whole. At this stage the parts of the cornua where the ova 
are lodged are easily distinguishable by their peculiar aspect, 
the ova forming eminences of the size of a pea. The cornua 
should be cut up transversely into as many segments as there 
are eminences, care being taken to have the ova in the centre 
of the segments. You then fix each segment by means of 
two pins on the bottom of a dissecting dish, with the meso- 
metrial surface downwards and the ovular eminence upwards. 
The dissecting dish is then filled up with serum or liquid of 
Muller, or OT per cent, solution of osmic acid, or Kleinenberg’s 
picro-sulphuric acid, or nitric acid, or acetate of uranium 
solution. With a small scalpel a longitudinal incision is made 
on the surface of the ovular eminence, not passing deeper 
than the muscular layer; the underlying uterine mucosa is 
then gently dilacerated with two pairs of small forceps, and 
the ovum set free in the liquid. 
From the moment the ova have become adherent to the 
uterine mucosa they can no longer be extracted whole. The 
embryo being always situated on the mesometrial surface, the 
ovular eminence is opened by a crucial incision, and the strip 
of mucosa to -which the embryo remains adherent is fixed 
with pins on the bottom of the dish. Ed. v. Benbden (see 
Arch, de Biol., v, fasc. iii, 1885, p. 378) has been able by 
operating in this way in serum of Kronecker, and keeping the 
whole at blood temperature, to observe the circulation of the 
embryo for hours together. (If this be desired to be done, 
the crucial incision should not be too extended, so as to leave 
the terminal sinus intact.) EMBRYOLOGICAL METHODS. 
337 
Retterer (C. R. Soc. de Biol., 1887, p. 99) advises that for 
ova of the seventh day the segment of uterus containing 
them be opened on the mesometrial surface, for at that date 
no adhesion has yet been contracted with that side. By 
running in liquid of Kleinenberg by means of a pipette 
between the ovum and the free surface of the uterus, the 
ovum may be got away in the shape of a closed vesicle. 
605. Rabbit ; Microscopic Preparations.—In order to make 
permanent preparations of the different stages of fecundation 
and segmentation, v. Beneden [Arch, de Biol., i, 1, 1880, p. 
149) recommends the following process :—The living ovum 
is brought into a drop of 1 per cent, osmic acid on a slide, 
and thence into solution of Muller (or bichromate of am- 
monia or solution of Kleinenberg). After an hour the liquid 
is changed, and the whole is put into a moist chamber, where 
it remains for two or three days. It is then treated with 
glycerin of gradually increasing strength, and at last mounted 
in pure glycerin acidified with formic acid. Ova may be 
stained with Beale’s carmine or picro-carmine, after removal 
from the osmic acid and careful washing. 
In order to bring out the outlines of blastoderm-cells the 
living ovum may be brought into one third per cent, solution 
of nitrate of silver. After remaining there for half a minute 
to two minutes, according to the age of the vesicle, it is 
brought into pure water and exposed to the light. The pre- 
parations thus obtained are instructive, but blacken rapidly, 
and cannot be permanently preserved. 
After the end of the third day the blastodermic vesicle can 
be opened with fine needles, and the blastoderm washed, 
stained, and mounted in glycerin or balsam ; v. Beneden 
has also obtained good preparations by means of chloride of 
gold. 
For embryonic areas and more advanced embryos Kollikek 
recommends putting the ovum into 05 per cent, solution of 
osmic acid until it has taken on a somewhat dai’k tint, which 
happens in about an hour, and then treating it with successive 
alcohols for several hours. If the ovum be adherent to the 
uterine mucosa the portion of the membrane to which it is 
fixed should be left, stretched out with pins, in Ol per cent, 
solution of osmic acid for from four to six hours. The blasto- 338 
CHAPTEE XXVII. 
dermic vesicle can then easily be removed, and immersed for 
a few hours more in 0'5 per cent, solution of osmic acid, and 
finally be brought into alcohol. For sections Kolliker fixes 
with osmic acid. v. Beneden treats the ova for twenty-four 
hours with 1 per cent, solution of chromic acid, then washes 
well, and brings them through successive alcohols. Chromic 
acid has the advantage of hardening thoroughly the vesicle, 
and maintaining at the same time the epiblast-cells perfectly 
adherent to the zona pellucida. v. Beneden also recommends 
the liquid of Kleinenberg. Henneguy writes that he fre- 
quently employs it for embryonic areas and embryos of various 
ages, always with excellent results. Fol’s modification of the 
liquid of Flemming, and Ranvier and Yignal’s osmic acid and 
alcohol mixture (§ 37), also give excellent results. For stain- 
ing, Henneguy recommends borax-carmine or Delafield’s 
heematoxylin for small embryos ; for large ones he found 
that his acetic acid alum-carmine was the only reagent that 
would give a good stain in the mass. I think Carmalum is 
now indicated. 
For sections, pure paraffin. Cut in series and mount in 
balsam. 
Piersol (Zeit. f. wiss. Zool., xlvii, 2, 1888, p. 155) has been 
lately using for fixation either Kleinenberg’s solution or, for 
young stages, Altmann’s 3 per cent, nitric acid. Staining 
and cutting as above. 
See also Weysse, Proc. Amer. Acad. Arts and Sci., 1894,. 
p. 285 (blastodermic vesicle of Sus scrofa) and Sobotta 
Arch. f. mih. Anat., xlv, 1895, p. 15 (fecundation and 
segmentation of the ovum of the mouse; fixation in Flem- 
ming’s weak mixture, sections stained with Benda’s iron 
hmmatoxylin). 
Aves. 
606. Superficial Examination.—Excellent instructions on this 
head are given in Foster and Balfour’s Elements of Embryo- 
logy, to which, as it is certain to be in the student’s hands, he 
may be referred. What follows here is given merely as being 
of more recent publication. 
If it be desired to observe a living embryo by transmitted 
light, the egg should be opened under salt solution, as de- EMBEYOLOGICAL METHODS. 
339 
scribed below. A little of the white is then removed through 
the window, the egg is lifted out of the liquid, and a ring of 
gummed paper is placed on the yolk so as to surround the 
embryonic area. As soon as the paper adheres to the vitel- 
line membrane, which will be in a few minutes, a circular 
incision is made in the blastoderm outside the paper ring. 
The egg is put back into the salt solution, and the paper 
ring removed, carrying with it the vitelline membrane and 
the blastoderm, which may then be brought into a watch- 
glass or on to a slide and examined under the microscope 
(Dcval). 
607. Gerlach’s Window Method (Nature, 1886, p. 497; Journ. Boy. 
Mic. Soc., 1886, p. 359).—Remove with scissors the shell from the small 
end of the egg; take out a little white by means of a pipette; the blasto- 
derm will become placed underneath the window thus made, and the white 
that has been taken out may he replaced on it. Paint the margins of the 
window with gum mucilage, and build up on the gum a little circular wall 
of cotton wool; place on it a small watch-glass (or circular cover-glass), and 
ring it with gum. When the gum is dry, the cover is further fixed in its 
place by means of collodion and amber varnish, and the egg is put hack in 
its normal position in the incubator. The progress of the development may 
be followed up to the fifth day through the window. 
A description of further developments of this method, with figures of 
special apparatus, will he found in Anat. Anz., ii, 1887, pp. 583, 609; see 
also Zeit.f. wiss. Mih., iv, 3, 1887, p. 369. 
608. Preparation.—During the first twenty-four hours of 
incubation it is extremely difficult to separate the blastoderm 
from the yolk, and they should be fixed and hardened together. 
In later stages, when the embryo is conspicuous, the blasto- 
derm can easily be separated from the yolk, which is very 
advantageous. To open the egg, lay it on its side and break 
the shell at the broad end by means of a sharp rap; then 
carefully remove the shell bit by bit by breaking it away 
with forceps, working away from the broad end until the 
blastoderm is exposed. The egg should be opened in salt 
solution, then lifted up a little, so as to have the blastoderm 
above the surface of the liquid; the blastoderm is then treated 
with some fixing solution dropped on it from a pipette (1 per 
cent, solution of osmic acid, or Ranvier and Vignal’s osmic 
acid and alcohol mixture, iodised serum, solution of Kleinen- 
berg, 10 per cent, nitric acid, &c.). By keeping the upper 
end of the pipette closed, and the lower end in contact with 340 
CHAPTER XXVII. 
the liquid on the blastoderm, the blastoderm may be kept well 
immersed for a few minutes, and should then be found to be 
sufficiently fixed to be excised. (Of course, if you prefer it, 
you can open the egg in a bath of any fixing liquid [10 per 
cent, nitric acid being convenient for this purpose] of such a 
depth as to cover the yolk; and having exposed the blasto- 
derm, leave it till fixed [fifteen to twenty minutes] ; but I 
think the procedure above described will generally be found 
more convenient.) 
The egg is put back into the salt solution, and a circular 
incision made round the embryonic area. The blastoderm 
may then be floated out and got into a watch-glass, in which 
it may be examined, or may be brought into a hardening 
liquid. 
Before putting it into the hardening fluid, the portion of 
vitelline membrane that covers the blastoderm should be 
removed with forceps and shaking. 
Fixation in 10 per cent, nitric acid has the advantage of 
greatly facilitating the separation of the blastoderm. The 
acid should be allowed to act for ten minutes, after which it 
is well to bring the preparation into 2 per cent, solution of 
alum (cf. Hofmann, Ziet. f. wiss. Mik., x, 4, 1893, p. 485). 
In order to counteract the turning up of the edges of the 
blastoderm that generally happens during the process of 
hardening, it is well to get the blastoderm spread out on the 
convex surface of a watch-glass, and leave it so during the 
hardening. 
For hardening, Foster and Balfour recommend solution of 
Kleinenberg for five hours, followed by alcohol. Or chromic 
acid, a solution of 0'1 percent, for twenty-four houi’S, followed 
by a solution of 03 per cent, for twenty-four hours more, then 
by 70 per cent, alcohol for a day, 90 per cent, alcohol for two 
days, and lastly absolute alcohol. They also recommend a 0-5 
per cent, solution of osmic acid, in which the embryo remains 
for two hours and a half in the dark, and after washing is 
brought into absolute alcohol. 
Henneguy prefers the osmic acid and alcohol mixture of 
Ranvier and Yignal, or Flemming’s mixture followed by suc- 
cessive alcohols. 
Staining and imbedding may be performed by the usual 
methods. EMBRYOLOGIOAL METHODS. 
341 
Up to about the fiftieth hour embryos may be mounted 
entire, in glycerin or balsam. 
609. M. Duval’s Orientation Method (Ann. d. Sc. nat. Zool.r 
1885).—In the early stages of the development of the ova of 
Aves, before the appearance of the primitive streak, it is 
difficult to obtain a correct orientation of the hardened cica- 
tricula, so as to be able to make sections in any desired direc- 
tion. Duval, starting from the fact that during incubation 
the embryo is almost always found to be lying on the yolk in 
such a position that the big end of the egg is to the left, and 
the little end to the right of it, marks the position of the 
blastoderm in the following way. 
With a strip of paper 5 millimetres wide and 50 millimetres 
long you construct a sort of triangular bottomless box. You 
lay this on the yolk, enclosing the cicatricula in such a posi- 
tion that the base of the triangle corresponds to what will be 
the anterior region of the embryo, and its apex to the posterior 
region; that is to say, if the big end of the egg is to your 
left, the apex of the triangle will point towards you. You 
now, by means of a pipette, fill the paper triangle with O'3 
solution of osmic acid. As soon as the preparation begins to 
darken you put the whole egg into weak chromic acid, remove 
the white, and put the rest into clean chromic acid solution 
for several days. After hardening you will find on the sur- 
face of the yolk a black triangular area, which encloses the 
cicatricula and marks its position; you cut out this area with 
scissors and a scalpel, and complete the hardening with 
chromic acid and alcohol. 
See also the critique and modification of this method by 
Kionka, Anat. Sefte, x, p. 391; Zeit. f. wiss. Mik., xi, 2, 
1894, p. 250 (stain with borax carmine, adding to the acid 
alcohol used for washing out a few drops of aqueous solution 
of Orange G, which stains the vitellus yellow). 
See also the method of Hirota, Journ. Roy. Mic. Soc.} 1895, 
p. 118. 
610. Vialleton’s Method (Anat. Anzeig., vii, 1892, pp. 624—627; 
Journ. Roy. Mic. Soc., 1892, p. 889).—Egg opened in salt solution, blasto- 
derm excised and removed to a glass plate, then treated with 1 per cent, 
nitrate of silver solution, washed with water, and put into 70 per cent, 
alcohol for six to twelve hours in the dark. Borax-carmine, alcohol, 
dammar1. 342 
CHAPTER XXVII. 
Reptilia. 
611 General Directions.—The methods described above for 
the embryology of birds are applicable to the embryology of 
reptiles. During the early stages the blastoderm should be 
hardened in situ on the yolk ; later the embryo can be iso- 
lated, and treated separately with Kleinenberg’s solution and 
alcohol (Strahl, Arch.f. Anat. u. Pliys., 1881, p. 123). 
612. Perenyi’s Method (Zool. Anzeig., 274, 1888, p. 139, and 276, p. 
196).—Fix in the following special mixture : 
3 parts 20 per cent, nitric acid. 
3 parts 1 per cent, chromic acid. 
4 parts absolute alcohol. 
For embryos of Lacerta. Fix for twenty minutes. Wash out for an hour 
with 70 per cent, alcohol, and then with strong alcohol. Stain with Dela- 
field’s haematoxylin, and treat the stained material for three to five minutes 
with 1 per cent, chromic acid. 
613. See also Kupffer’s Method (Arch.f. Anat. und Entwick., 1882, 
p. 4). Sarasin’s Method (Semper’s Arbeiten, 1883, p. 159), and Mitstj- 
kuri (Journ. Boy. Mic. Soc., 1894, p. 750). 
Amphibia. 
614. Preliminary.—In order to prepare the ova of Amphibia 
for section cutting, it is essential to begin by removing their 
thick coats of albumen. This may be done by putting them 
for two or three days into 1 per cent, solution of chromic 
acid, and shaking well; but ova thus treated are very brittle, 
and do not afford good sections. A better method is that 
described by Whitman (Amer. Natural., xxii, 1888, p. 857), 
and by Blochmann (Zool. Anz., 1889, p. 269). Whitman puts 
the fixed eggs into a 10 per cent, solution of sodium hypo- 
chlorite diluted with 5 to 6 volumes of water, and leaves them 
there till they can be shaken free, which happens (for Necturus) 
in a few minutes. Blochmann takes eau de Javelle (potassium 
hypochhmte), and dilutes it with 3 to 4 volumes of water, 
and agitates the eggs previously fixed with solution of 
Flemming, for fifteen to thirty minutes in it. The ova are 
afterwards preserved in alcohol in the usual way. Some 
other means of attaining the same end are given in the 
following paragraphs. 343 
EMBRYOLOGICAL METHODS. 
615. Axolotl.—The ova are easier to prepare than those of 
the Anura, because the yoke is separated from the albuminous 
layer by a wide space filled with a liquid that is not coagulated 
by reagents. Put the eggs for a few hours into picro-sulphuric 
acid, then pierce the inner chorion with fine scissors or needles, 
and gently press out the ovum. Harden in alcohol. 
616. Triton (Scott and Osborn, Quart. Journ. Mic. Soc., 
1879, p. 449).—The albumen is here present in the form of 
several concentric coats, which are very delicate. Incise each 
of them separately with fine scissors, turn out the ovum, and 
fix it. Solution of Kleinenberg is the reagent that gives the 
best results. 
617. Triton (Hertwig, Jew. Zeit.f. Naturw., 1881-2, p. 291).— 
Put the eggs into a mixture of equal parts of 2 per cent, 
acetic acid and 05 per cent, chromic acid. After ten hours 
incise the membranes, opening one end of the inner chorion, 
and turn out the embryos and bring them into successive 
alcohols. 
618. Salamandra (Babl, Morphol. Jahrb., xii, 2, 1886, p. 
252).—For his more recent methods see § 602. 
619. Rana (0. Hertwig, Jen. Zeit. f. Naturw., xvi, 1883, 
p. 249).—The ova are thrown into nearly boiling water (90° 
to 96° C.) for five to ten minutes. The albuminous envelope 
of the ovum is then cut open, and the ovum extracted under 
water. The ova are then brought into 0*5 per cent, osmic 
acid, or into alcohol of 70, 80, and 90 per cent. Chromic 
acid makes ova brittle ; they ought not to remain in it for 
more than twelve hours. Chromic acid destroys or attacks 
the pigment of the ova, whilst alcohol preserves it, which is 
frequently important for the study of the germinal layers. 
Morgan (Amer. Nat., xxv, 1891, p. 759; Journ. Boy. Mic. 
Soc., 1892, p. 284) has the following. During the periods in 
which it is difficult or impossible to remove the inner jelly- 
membrane the eggs can be freed as follows :—Each egg is cut 
out with scissors from the general jelly-mass, and put for 
from one to twelve hours into saturated solution of picric 
acid in 35 per cent, alcohol containing “the same amount of 
sulphuric acid as in Kleinenberg’s solution.” Wash for 344 
CHAPTER XXVII. 
several hours in several changes of alcohol, beginning with 
35 per cent., and increasing the strength gradually up to 70 
per cent. About the second day in the 70 per cent, alcohol 
the inner membrane begins to swell, and on the third or 
fourth day may be pierced by a needle, and the egg removed 
and placed in 80 per cent, alcohol (see also Whitman, Meth. 
of Research, p. 156; and Schultze, Zeit. f. wiss. Zool., v, 
1887, p. 177). 
620. Sulphate of Copper Hardening Liquid. — I take from the 
Lehrhuch of Pol (p. 106) the following formula, which was first published 
by Remak, then modified by G-oette, and is said to be useful for hardening 
the ova of Amphibia : 
2 per cent, solution of sulphate of copper . . 50 c.c. 
Alcohol of 25 per cent. 50 „ 
Rectified wood vinegar . . . . .35 drops. 
621. Other Methods.—Roux, Study of Cleavage Cells (Cytro- 
pism of the Ovum), see Arch. f. Entwickelungs-mechanik, i, p. 44 ; 
Amer. Natural., xxix, 1895, p. 511; Journ. Roy. Mic. Soc., 1895, p. 598. 
Production of Hemiembryos of the Prog, &c., Avat. Anz., ix, 8, 
1894, p. 248, and 9, p. 265. Born, Arch. f. mik. Anat., xliii, 1894, p. 1; 
Rabl, Zeit. f. wiss. Mik., xi, 2, 1894, p. 165 (his fixing methods have been 
given). 
Pisces. 
622. Teleostea in General.—The ova of many of the bony 
fishes can be studied by transmitted light in the living state; 
but those of the Salmonidas must be hardened and removed 
from their envelopes for the study of the external forms of 
the embryo. 
To this end the ova may be put for a few minutes into 
water containing 1 to 2 per cent, of acetic acid, and thence 
into 1 per cent, chromic acid. After three days the capsule 
of the ovum may be opened at the side opposite to the em- 
bryo, and be removed with line forceps. The ovum is put for 
twenty-four hours into distilled water, and then into succes- 
sive alcohols. Embryos thus prepared show no deformation, 
and their histological elements are fairly well preserved. But 
the vitellus rapidly becomes excessively hard and brittle, so 
as greatly to interfere with section cutting. 
The following processes give good results as regards sec- 
tion cutting. EMBRYOLOGICAL METHODS. 
345 
Put the ova for a few minutes into 1 per cent, osmic acid ; 
as soon as they have taken on a light brown colour bring 
them into Muller’s solution. Open them therein with fine 
scissors—the vitellus, which immediately coagulates on con- 
tact with air, dissolves, on the contrary, in Muller’s solution 
—and the germ and cortical layer can be extracted from the 
capsule of the ovum. They should be left in clean Muller’s 
solution for a few days, then washed with water for twenty - 
four hours, and brought through successive alcohols. 
Another method (Henneguy) is as follows :—The ova are 
fixed in solution of Kleinenberg containing 10 per cent, of 
acetic acid. After ten minutes they are opened in water con- 
taining 10 per cent, of acetic acid, which dissolves the vitellus. 
The embryos are put for a few hours into pure solution of 
Kleinenberg, and are then brought through alcohol of 
gradually increasing- strength. 
623. Kollmann’s Fixative (Kollmann, Arch.f. Anat. u. Phys., 1885, 
p. 296). 
Bichromate of potash . . . . .5 per 100. 
Chromic acid. . . . . . . 2 ,, 
Concentrated nitric acid . . . . 2 ,, 
For ova of Teleostea. Fix for twelve hours, wash with water for twelve 
hours, then remove the chorion, and put the ova into 70 per cent, alcohol. 
624. Rabl’s Method (see § 602). 
625. Pekenyi’s Method (see § 50). 
626. Kowalewsky’s Method (see Zeit. f. iviss. Zool., xliii, 1886, p. 
434). 
627. Boyer’s Methods.—See Bull. Mus. Comp. Zool., Harvard, xxiii, 
1892, p. 93; Journ. Boy. Mic. Soc., 1892, p. 699. 
628. Rabl-Ruckhard’s Method (Arch. f. Anat. u. JEntw., 1882, p. 67). 
—Fix in 10 per cent, nitric acid for fifteen minutes. Remove the mem- 
branes to avoid deformation of the embryos, and put the ova back into the 
acid for an hour. Wash out in 1 to 2 per cent, solution of alum for an hour, 
and harden in alcohol. 
Modification of this method by Goronowitsch (see Morph. Jahrb., x, 
1884, p. 381). 
629. Pelagic Pish Ova (Whitman’s method; Amer. Natural., xvii, 
1883, pp. 1204-5 ; Journ. Boy. Mic. Soc. [N.S.], iii, 1883, p. 912, and 
Methods of Research, &c., p. 152).—Fix bj treatment first for five to ten 
minutes with a mixture of equal parts of sea water and | per cent, osmic 
acid solution, and then for one or two days with a modified Merkel’s solu- 
tion (due to Eisig), consisting of equal parts of 0‘25 per cent, platinum 346 
CHAPTER XXVII. 
chloride and 1 per cent, chromic acid. Prick the membrane before trans- 
ferring to alcohol. Whitman found that the usual Merkel’s fluid caused 
maceration of the embryonic portion of the egg. Picro-sulphuric acid 
causes the embryonic cells to swell, and in many cases to become com- 
pletely disorganised. The osmic acid treatment is necessary in the case of 
segmenting ova because the Merkel’s fluid does not kill rapidly enough, so 
that eggs placed in it may even pass through one or two stages of cleavage 
before dying. This fluid arrests the process of blackening by the osmium, 
or will even bleach the objects if blackening has set in. See also Agassiz 
and Whitman, in Proc. Amer. Acad. Arts and Sciences, xx, 1884. For 
later stages the authors recommend the method of Perenyi. And see the 
experiments detailed by Collinge, Ann. and Mag. Nat. Hist., x, 1892, p. 
228; Journ. Boy. Mic. Soc., 1892, p. 883. 
Tunicata. 
630. Distaplia.—Davidoff (Mitth. Zool. 8tat. Neapel, ix, 1, 
1889, p. 118) has some important observations on the fixation 
of the ova of D. magnilarva. The best reagent is a mixture 
of 3 parts of saturated solution of corrosive sublimate and 1 
of glacial acetic acid. The ova to remain in it for from half 
an hour to an hour, and be then washed for a few minutes in 
water and brought through successive alcohols. Another 
reagent, almost as good, consists of 3 parts of saturated solu- 
tion of picric acid and 1 of glacial acetic acid, the objects to 
remain in it for three to four hours, and then be brought into 
70 per cent, alcohol. 
631. Amarcecium (Maurice and Schulgin, Ann. Sci. Nat. 
Zool., xvii, 1884).—Stain in borax-carmine, wash out, and 
stain for fifteen to twenty hours in very weak solution of bleu 
de Lyon in 70 per cent, alcohol with a few drops of acetic 
acid. In sections the epiblast and hypoblast appear chiefly 
blue, the mesoblast-cells, on the contrary, appearing almost 
entirely red. 
632. Test-Cells of Ascidians (Morgan, Journ. of Morphol., iv, 1890, 
p. 195).—Tease fresh ovaries in very weak osmic acid, wash in distilled 
water, treat for half an hour with 1 per cent, silver nitrate, wash for half 
an hour in 2 per cent, acetic acid, and reduce in sunlight. Imbed in paraffin. 
By this process the limits of the follicle cells are demonstrated. 
Mollusca. 
633. Cephalopoda (Ussow, Arch, de Biol., ii, 1881, p. 582). 
—Segmenting ova are placed, without removal of the mem- EMBRYOLOGICAL METHODS. 
347 
branes, in 2 per cent, solution of chromic acid for two minutes, 
and then in distilled water, to which a little acetic acid (one drop 
to a watch-glassful) has been added, for two minutes. If an 
incision be now made into the egg-membrane the yolk flows 
away and the blastoderm remains; if any yolk still cling to 
it, it may be removed by pouring away the water and adding 
more. 
Watase {Journ. of Morphol., iv, 1891, p. 249; Journ. Boy. 
Mic. Soc., 1892, p. 152) kills the ova in the macerating mix- 
ture of the Hertwigs (§ 553), and as soon as the blastoderm 
turns white and opaque removes it under dilute glycerin. 
Treatment with liquid of Perenyi is recommended for surface 
views of cleavage. 
634. Gastropoda (Henneguy). — Ova of Helix may be fixed 
for from four to six hours in Mayer’s picro-nitric solution 
(§ 76). The carbonate of lime that encrusts the external 
membrane is thus dissolved, and the albuminous coat of the 
egg is coagulated. The egg is opened with needles, the 
albumen comes away in bits, and the embryo can be removed. 
Treat with successive alcohols, and imbed in paraffin. 
635. Limax (early stages) (Mark, Bull. Mus. Comp. Zool., Harvard 
Coll., vi, 1881).—The ova are treated with acetic acid of 1 to 2 per cent, for 
four or more hours. The two external membranes are incised with fine 
scissors, and the egg squeezed out in its albumen membrane. This is dis- 
sected off on a slide, the egg is separated from the albumen, stained, and 
mounted in glycerin. 
For later stages, or for making sections, osmic acid is used instead of 
acetic acid, and the egg is hardened with its albuminous coats. 
Miss A. Henchman {Bull. Mus. Comp. Zool., Harvard, xx, 
1890, p. 171; Journ. Roy. Mic. Soc., 1891, p. 274; Zeit. f. 
wiss. Mile., viii, 2, 1891, p. 216) finds that the best way of 
obtaining embryos is to keep some twenty-five or thirty adults 
(of L. maximus) in a large tin pail with a cover perforated 
with small holes. They should be fed on cabbage, and the 
vessel kept very clean. Eggs are generally found in the 
morning in bunches of thirty to forty. As they are more 
abundant in the early stages of confinement it is better to 
obtain a few slugs often than many at once. In a moderately 
warm room hatching occurs between the twenty-second and 
twenty-seventh day. The eggs must be carefully protected 
from desiccation. 348 
CHAPTER XXVII. 
Kill with 033 per cent, chromic acid, or with liquid of 
Perenyi. It is best to remove only the outer envelope before 
putting into the chromic acid, the inner membrane being 
removed after two or three minutes therein. Where Perenyi 
is used the membranes must be removed first, as the albumen 
will else coagulate in such a way as to prevent the removal of 
the embryos. 
See also Schmidt, Studien zur Entwickelungsgesch. d. Pul- 
monaten, Dorpat, 1891; and Washburn, Amer. Natural., xxviii, 
1894, p. 528; or Journ. Roy. Mic. Soc., 1894, p. 531. The 
paper of Kofoid in Bull. Mus. Comp. Zool., Harvard Coll., 
xxvii, 2, p. 35, contains a detailed account of the treatment 
appropriate to the ova of Agriolimax agrestis, which are 
smaller than those of Limax maximus, studied by Miss 
Henchman. See also the abstract of these methods in Journ. 
Roy. Mic. Soc., 1895, p. 701. 
636. Chiton, see Metcalf, Stud. Biol. Lab. Johns Hopkins TJniv., v, 
1893, p. 251, or Journ. Boy. Mic. Soc., 1894, p. 531. 
Arthropoda. 
637. Fixation of Ova.—In most cases the ova of Arthropods 
are fixed by heat in a more satisfactory way than by any other 
means. This may be followed either by alcohol or some 
watery liardeuing agent. If it be desired to avoid heating, 
picro-sulphuric acid or liquid of Perenyi may be tried. 
638. Removal of Membranes.—This is frequently very difficult, 
and it may often be advisable not to attempt to remove them, 
but to soften them with eau de Javelle or eau de Labarraque 
(see the methods of Looss and List). 
Morgan (Aimer. Natural., xxii, 1888, p. 357; Zeit. f. wiss. 
Mik., vi, 1, 1889, p. 69) recommends (for the ova of Peri- 
planeta) eau de Labarraque diluted with five to eight volumes 
of water, and slightly warmed. Thus used, it will soften the 
chitin membranes sufficiently in thirty to sixty minutes if 
employed before fixing. Fixed ova take longer. The fluid 
must, of course, not be allowed to penetrate into the interior 
of the ovum. 
639. Henking’s Methods.—For the whole subject of the 
technique of the embryology of Iusecta see an elaborate EMBRYOLOGICAL METHODS. 
349 
paper by this specialist in Zeit. f. wiss. Mik., viii, 2, 1891, 
p. 156. Henking agrees with other workers at this subject, 
that in the majority of cases heat is the only available fixing 
agent that will give fair results. He kills ova by plunging- 
them into hot water, or by pouring hot water on to them in 
a watch-glass, and then removing into 70 per cent, alcohol. 
But, as might be expected, he finds that the preservation of 
structures by this method is far from being perfectly satisfac- 
tory, cell-contours being not at all sharply brought out by it, 
and achromatic cell-structures being but imperfectly pre- 
served. He finds that in some cases ova may be fixed with 
liquid of Flemming, which, as may be supposed, gives incom- 
parably better results in these respects. Suitable ova may 
be put into liquid of Flemming (Henking does not say which 
formula) for half an hour, then for two hours into the same 
diluted with three volumes of water, then treated with alcohol 
as usual. Boverbs picro-acetic acid was found not to pene- 
trate the membranes. 
Henking thinks that eau de Javelle for softening mem- 
branes is best avoided. Membranes should either be dissected 
away or left in situ, and cut with the rest of the egg, accord- 
ing to the nature of the case. The great obstacle to section 
cutting is the brittleness of the yolk. This difficulty may be 
overcome as follows :—After fixing and treating with alcohol, 
prick the chorion and stain with borax-carmine. Put the 
stained ova for twelve hours into a mixture containing 20 c.c. 
of 70 per cent, alcohol, one drop of concentrated hydrochloric 
acid, and a knife-pointful of pepsin (it is not necessary that 
all the pepsin should be dissolved). The ova may then be 
treated with alcohol, oil of bergamot, and paraffin, and (with 
some exceptions, amongst which is Bombyx mori) will be found 
to cut without crumbling. 
The contents of fresh ova may conveniently be studied by 
means of the following fluid : 
Distilled water . . . .80 c.c. 
Glycerin . . . . . 16 „ 
Formic acid . . . . . 3 ,, 
1 per cent, osmic acid . . . 1 ,, 
Dahlia ...... 004 grm. 
The eggs are simply teased in a drop of the liquid, and a 350 
CHAPTER XXVII. 
cover-glass put on. If it be desired to preserve the prepara- 
tion, nothing more is necessary than to lute the cover-glass. 
640. Lepidoptera (Bobeetzky, Zeit. f. iviss. Zool., 1879, p. 
198).—Ova (of Pieris cratsegi and Porthesia chrysorrhoea) are 
slightly warmed in water and put for sixteen to twenty hours 
into 0'5 per cent, chromic acid. The membranes can then be 
removed, and the ova brought for a few hours into absolute 
alcohol, stained with carmine, and cut. 
641. Blattida (Patten, Quart. Journ. Mic. Sci., 1884, p. 549). 
—The ova or larva) are placed in cold water, which is gra- 
dually raised to 80° C. You leave off heating as soon as the 
ova have become hard and white. Pass very gradually 
through successive alcohols, beginning with 20 per cent.; 
stain with Kleinenberg’s hasmatoxylin or Mayer’s cochineal 
(only alcoholic stains will traverse the chorion). The ova 
may remain in the haematoxylin for five or six days, and be 
washed out in alcohol containing one drop of HC1 per 20 
grins., in which they should remain for several days, and then 
be soaked in pure alcohol until they have regained their 
violet colour. Penetrate with benzol and imbed in paraffin. 
Wheeler (Journ. of Alorph., iii, 1889, p. 292; Journ. Boy. 
Mic. Soc., 1890, p. 250) dissects out ovarian ova in salt solu- 
tion and fixes in liquid of Perenyi (fifteen minutes), then 
treats with alcohol, and stains with borax-carmine. Laid 
eggs may be killed by Patten’s method. After heating, the 
two lips of the crista of the capsule may be separated with 
fine forceps and pieces of the walls torn away, and the eggs 
pushed out of the compartments formed by their choria and 
hardened as desired. Good results are also obtained by 
heating to 80° C. for ten minutes in liquid of Kleinenberg, 
and preserving in 70 per cent, alcohol. This causes the en- 
velopes to dilate and stand off from the surface of the egg, 
so that they can easily be dissected away. 
Cholodkowsky (Mem. Acad. Imp. St. Petersburg, xxxviii, 
1891; Zeit. f. wiss. Alik., ix, 1, 1892, p. 80) recommends 
cutting off the ends of the cocoons and fixing for twelve hours 
in liquid of Perenyi, or for a few minutes in a solution of 
1 part iodine, 1 part iodine of potassium, and 300 parts 
water, heated to boiling-point. EMBRYO LOGICAL METHODS. 
351 
Heymons (Zeit.f. iviss. Zool., liii, 1892, p. 434; Zeit.f. wiss. 
mik., ix, 3, 1893, p. 343) finds that Cholodkowsky's methods 
are good for the study of general relations of parts, but not 
satisfactory for the preservation of delicate detail. For 
young embryos it is better to incise the cocoon at the end by 
which it inheres in the body of the mother, bring it for two 
minutes into water heated to 90° C., and open in Flemming, in 
which the embryo should be dissected out. 
642. Diptera (Henking, Zeit.f. wiss. Zool., xlvi, 1888, p. 289; 
Zeit.f. iviss. Mik., 1889, p. 59).—Ova still contained within 
the fly may be fixed by plunging the animal for some time 
into boiling water, then dissecting out and bringing them into 
70 per cent, alcohol. Laid eggs may have boiling’ water 
poured over them, or be put into solution of Flemming in a 
test-tube which is plunged into boiling water until the eggs 
begin to darken (about a minute). Cold solution of Flemming 
easily causes a certain vacuolisation of the contents of the 
ova. Open the ova at the larger end, stain with borax- 
carmine for fifteen to thirty hours, and cut in paraffin. 
See also (for Chironomus) Ritter, Zeit. f. wiss. Zool., i, 
1890, p. 408 ; Zeit.f. wiss. Mik., viii, 1, 1891, p. 87 (strings 
of ova fixed with hot 30 per cent, alcohol containing a little 
sublimate, and stained in the mass by immersion for several 
days in picro-carmine). 
643. Phalangida (Balbiani).—The ova of Phalangium opilio are 
enclosed in a chorion covered with yellow corpuscles which renders them 
quite opaque. They may be cleai-ed by treating them with water contain- 
ing a little solution of caustic potash and raised to boiling point. The ova 
are then laid on blotting-paper, and the chorion is removed by rubbing 
them gently with a small brush. The vitelline membrane remains intact 
and transparent, and the embryo may be studied through it. 
644. Phalangida (Henkihg, Zeit. f. wiss. Mik., iii, 4, 1886, pp. 470 
et. seq.).—Fix with boiling water or “ Flemming.” Preserve the ova in 90 
per cent, alcohol. To open the chorion, bring them back into 70 per cent, 
alcohol, which causes them to swell up so that the chorion can easily be 
pierced with needles, and the ovum turned out. 
645. Araneina.—See Balfouk {Quart. Journ. Mic. Sci., 1880, p. 167); 
Kishinotjye {Journ. Coll. Sci. Imp. Univ. Japan, iv, 1891, p. 55; Zeit.f. 
wiss. Mik., ix, 2, 1892, p. 215): Loot {Bull. Mus. Comp. Zool., Harvard, 
xii, 3, 1886 ; Zeit. f. wiss. Mik., iii, 2, 1886, p. 242). 
646. Aphides (Will, Semper’s Arbeiten, 1883, p. 223).—Sections to be 352 
CHAPTER XX VII. 
made through the entire animals containing the ova and embryos. The 
animals are killed in water of 70° C., and brought into alcohol. 
647. Astacus (Reichenbach, from Zeit.f. wiss. Mik., 1886, p. 400).— 
Fix in water gradually warmed to 60° or 70° C. (if the chorion should burst, 
that is no evil), harden for twenty-four hours in 1 to 2 per cent, bichromate 
of potash or 05 per cent, chi'omic acid, wash out for the same time in 
running water, and bring into alcohol. Remove the chorion, remove the 
embryo from the yoke by means of a sharp knife, and stain with picro- 
carmine and mount in balsam. 
648. Amphipoda (Orchestia) Uljanin, Zeit.f. wiss. Zool., xxxv, 1881, 
p. 441).—Ova in the earliest stages o£ development were treated for two 
hours with picro-sulphuric acid (Kleinenberg’s formula). This causes the 
chorion to swell and burst. Ova in later stages, in which the embryo is 
surrounded by a cuticular membrane, which encloses an albuminous liquid, 
must have this membrane torn with needles and the albuminous liquid 
allowed to ooze out before placing in the picro-sulphuric acid. 
Vermes. 
649. Taenia (v. Beneden, Arch, de Biol., ii, 1881, p. 187).— 
Ova in which a chitinous membrane has formed around the 
embryo are impervious to reagents. They may be put on a 
slide with a drop of some liquid and covered. Then, by with- 
drawing the liquid by means of blotting-paper, the cover may 
be made to gradually press on them so as to burst the mem- 
branes, and the embryo may then be treated with the usual 
reagents. 
650. Planaria (Iijima, Zeit. f. iviss. Zool., xl, 1884, p. 359).— 
The capsule containing the ova (of fresh-water Planaria) is 
opened with needles on a slide, in a drop of 2 per cent, nitric 
acid. The ova are extracted and covered (the cover being- 
supported by paper, or by wax feet). After half an hour 
they are treated with successive alcohols under the cover, 
and finally mounted in glycerin. For sections, the whole of 
the contents of a capsule is hardened in the mass in 1 per- 
cent. chromic acid and cut together. 
651. Lmnbricus (Kleinenberg, Quart. Journ. Mic. ScL, 1879, 
p. 207).—Fix with Kleinenberg’s picro-sulphuric acid, or, 
which is not quite so good, with vapours of osmium, pass 
through successive alcohols, stain with Kleinenberg’s liseina- 
toxylin, and cut in paraffin. 
Wilson [Journ. of Morph., iii, 1889, p. 445; Journ. Roy. EMBRYOLOGICAL METHODS. 
353 
Mic. Soc., 1890, p. 402) finds that liquid of Perenyi is by far 
the best fixing reagent, being in most respects superior even 
to Flemming’s. Fix for fifteen to sixty minutes ; wash out in 
70 per cent, alcohol. 
For Nereis, see von Wistinghausen, Mitth. Zool. Stat. 
Neajpel, x, 1891, p. 41; or Zeit.f. iviss. Mik., x, 4, 1893, p. 479. 
652. Ascaris.— Seethe chapter on “ Cytological Methods.” 
Echinodermcita, Coelenterata, and Parifera. 
See the paragraphs treating of these groups in the chapter 
on “ Zoological Methods.” 
For the maturation and fecundation of the ova of the Echi- 
nodermata, see also the chapter on “ Cytological Methods.” CHAPTER XXVIII. 
CYTOLOGICAL METHODS. 
653. Subjects for Study.—One of the best objects for this 
purpose is the tail of young larvae of Amphibia, both Anura 
and Urodela. 
In the living animal the epithelial cells and nuclei (in the 
state of repose) are so transparent as to be invisible in the 
natural state. They may, however, be brought out by cura- 
rising the larva; or, still better, by placing the curai’ised 
larva for half an hour in 1 per cent, chloride of sodium solu- 
tion. Normal larvae may be used for the study of the active 
state of the nucleus, but much time is saved by using curare. 
Curare.—Dissolve 1 part of curare in 100 parts water, and 
add 100 parts of glycerin. Of this mixture add from 5 to 10 
drops (according- to the size of the larva), or even more for 
large larvae, to a watch-glassful of water. From half to one 
hour of immersion is necessary for curarisation. The larvae 
need not be left in the solution until they become quite 
motionless; as soon as their movements have become slow 
they may be taken out and placed on a slide with blotting- 
paper. If they be replaced in water they return to the 
normal state in eight or ten hours, and may be re-curarised 
several times. 
Etherisation.—Three per cent, alcohol, or 3 per cent, 
ether maybe used in a similar way. These reagents cause no 
obstruction to the processes of cell-division, and are useful, 
but their action as anesthetics is inconstant. 
Indifferent Media.—One per cent, salt solution, iodised 
serum, syrup, cold water (+ 1° C.), and warm water (35°— 
40° 0.). The tail may be excised from the living animal and 
studied for a long time in these media (Peremeschko, Arch. 
/. mik. Anat., xvi, 1879, p. 437). 
Perhaps (Flemming, ibid., pp. 304 et seg.) the very best 
subject for these studies is Salamandra. The adult offers 
for study the thin transparent bladder; in the larva the gills 
and caudal “fin” may be studied in the living state. The CYTOLOGICAL METHODS. 
355 
gills are difficult to fix in position for observation, and are 
obscured by pigment. In the fin there is always a spot, near 
to the hind limbs, that is free from pigment; and on lightly 
coloured larvae other such spots may be found on the ventral 
half of the fin and on the lateral line. On a flat-finned larva 
it is possible to study these spots with high-power glasses. 
The larva may be fixed in a suitable cell, or wrapped in 
moist blotting-paper, or maybe curarised; or the tail maybe 
excised. (It is preferable to cut through the larva close in 
front of the hind limbs.) 
Objects for Preparation.—A favorable object for prepara- 
tion is found in the gill -plates, delicate laminaa that are to be 
found attached to the gill-cartilages on the mouth side. 
The lungs, parietal peritoneum, and mesentery of the 
larvae are also very favorable objects for preparations (see 
Flemming, Arch. f. mik. Anat., xxxv, 1890, p. 275; and xxxvii, 
1891, pp. 249 and 685). To prepare the lungs the larva, 
which should be of not more than 4 cm. length, should be 
killed by immersion in chromo-aceto-osmic acid, the body- 
cavity cut into, and the viscera gently drawn out and exposed 
to the action of the liquid, care being taken not to let the 
lungs get into folds. After fixation they should be carefully 
got on to a slide, and a small strip removed from their mar- 
gins on either side by means of a scalpel, after which the two 
walls may be separated from each other, and utilised as thin, 
flat preparations. 
Another excellent object is the intestine of the adult, of 
which sections may be made by the paraffin method, as re- 
commended in the important paper of M. Heidenhain, a Tiber 
Kern und Protoplasma,” in Festsclir. z. 50 jdhr. Jubil. d. H. 
Prof. Geheimr. v. Kolliker (also in separate reprint), Engel- 
mann, Leipzig, 1892, p. 111. This organ offers for study, 
besides the large epithelium cells of the intestinal crypts, 
numberless examples of leucocytes, an extremely favorable 
object. 
Larvae may be bred from adults kept in confinement, and 
supplied with a vessel of water, in which they will place the 
larvae of their own accord. In May gravid females may be 
killed and the larvae extracted. The larvae must be kept in 
frequently changed water and fed every day or two. Aquatic 
worms may be used for feeding them, e. g. Tubifex rivulorum. 356 
CHAPTER XXVIII. 
It is extremely important that they should be fed regularly 
and abundantly, for, if not, cell-divisions in the tissues become 
rare, and may even cease altogether. 
Adults may easily be kept in a vivarium. The bottom 
should be covered to a depth of about four inches with mould; 
a shallow dish (a photographic developing dish is as good as 
anything) filled with water should be sunk in the mould, and 
the mould should be covered with moss. The animals should 
be fed with worms, which they are sure to take during the 
night (Henneguy, Lemons sur la Cellule, p. 68). 
Lonneberg (see Zeit.f. wiss. Mik., x, 3, 1893, p. 377) recom- 
mends the intestinal epithelium of hibernating snails as an 
object that furnishes an abundance of karyokinetic figures, 
ihe snails should be kept for some days in a warm room, their 
epiphragm should then be removed, and they should be fed 
for three days with cabbage leaves before being dissected. 
Other classical subjects of study will be found mentioned 
in the following paragraphs. 
654. Stains for living Cells.—It is sometimes of the very 
gieatest importance to be able to stain a cell in the living 
state, even though it be but feebly and imperfectly. See 
§ 213. 
655. Study of Fresh and lightly fixed Cells.—It lias been 
rightly pointed out by Flemming that so-called “ indifferent ” 
liquids must not be believed to be without action on nuclei. 
Iodised serum, salt solution, serum, aqueous humour, lymph, 
better deserve the name of weak hardening agents. Between 
these, and such energetic hardening agents as Flemming’s 
mixture, come such light fixing agents as picric acid or very 
dilute acetic acid. These it is whose employment is indicated 
for the study of fresh isolated cells. 
A typical example of this kind of work is as follows:— 
Tease out a piece of living tissue in a drop of acidulated solu- 
tion of methyl green (0-75 per cent, of acetic acid). This is a 
delicate fixing agent, killing cells instantly without change 
of form. Complete the fixation by exposing the preparation 
for a quarter of an hour to vapour of osmium, and add a drop 
of solution of Bipart and Petit and a cover. 
Or you may fix the preparation, after teasing, with vapour of CYTOLOGICAL METHODS. 
357 
osmium for half a minute to two minutes, then add a drop of 
methyl green, and after five minutes wash out with 1 per cent, 
acetic acid, and add solution of Ripart and Petit and cover. 
Or you may kill and fix the cells by teasing in solution of 
Ripart and Petit (to which you may add a trace of osmium if 
you like), and afterwards stain with methyl green. 
I have found Pictet’s chloride of manganese (§ 386) very 
useful as an examination medium. A little solution of dahlia 
may be added to it. 
Henking’s mixture, which has been given above (§ 639), may also be 
found useful. 
For Flemming’s methods for the study of the division of the ova of 
Echinodermata, see Arch. f. mik. Anat., xx, 1881, p. 3, or previous editions 
of this work. 
Other fixing agents, such as picric acid or weak sublimate 
solution, may of course be used, and in some cases doubtless 
should be preferred. Other stains, too, such as Bismarck 
brown, may be used as occasion dictates ; and of course other 
examination media than solution of Ripart may be employed. 
But, for general purposes, the methyl-green-osmium-and- 
Ripart’s-medium method gives such good results, and is so 
very convenient, that it may well be called the classical 
method for the study of fresh cells. I think great credit is 
due to Carnoy for his frequent insistence on the excellence 
and handiness of this method. 
656. Some Microchemical Reactions.—Methyl green is a test 
for chromatin, in so far as it colours nothing- but the chro- 
matin in the nucleus. It is, however, not a perfect test, for 
the intensity of the coloration it produces varies greatly in 
different nuclei, and may in certain nuclei be extremely weak, 
or (apparently) even altogether wanting. In these cases 
other tests must be applied in order to establish with certainty 
the presence or absence of that element. The following sug- 
gestions are taken from Carnoy, who is, I believe, the only 
writer—on the zoological side, at all events—who has insisted 
on the necessity of applying microchemical methods in a 
systematic manner to the study of cells. 
Chromatin is distinguished from the lecithins and from 
albuminoids by not being soluble, as these are, in water and 
in weak mineral acids, such as OT per cent, hydrochloric 358 
CHAPTER XXVIII. 
acid. It is easily soluble in concentrated mineral acids, in 
alkalies, even when very dilute, and in some alkaline salts, 
such as carbonate of potash and biphosphate of soda. In 
the presence of 10 per cent, solution of sodium chloride it 
swells up into a gelatinous mass, or even, as frequently 
happens, dissolves entirely {Biol. Cell., pp. 208-9). It is only 
partially digestible (when in situ in the nucleus) in the usual 
laboratory digestion fluids. 
-The solvents of chromatin that are the most useful in 
practice are 1 per cent, caustic potash, fuming hydrochloric 
acid, or cyanide of potassium, or carbonate of potash. 
These last generally give better results than dilute alkalies. 
I hey may be employed in solutions of 40 to 50 per cent, 
strength. If it be desired to remove all the chromatin from 
a nucleus the reaction must be prolonged, sometimes to as 
much as two or three days, especially if the operation be con- 
ducted on a slide and under a cover-glass, which is the safer 
plan. 
It must bo remembered that these operations must be per- 
formed on fresh cells, for hardening agents bring about very 
considerable modifications in the nature of chromatin, render- 
ing it almost insoluble in ammonia, potash, or sodic phos- 
phate, &c. Hydrochloric acid, however, still swells and 
dissolves it, though with difficulty. 
Partial digestion may render service in the study of the 
chromatic elements of nuclei. Chromatin resists the action 
of digestive fluids much longer than the albumins do; so that 
a moderate digestion serves to free the chromosomes from 
any caryoplasmic granulations that may obscure them, whilst 
at the same time it clears up the cytoplasm. 
The term ‘ chromatin ’ has been used in the above paragraphs in a sense 
in which the term “nuclein” is employed by many writers. It is now 
known that there exists a whole series of nucleins, differing chiefly in respect 
of their richness in phosphorus and proteids. At one end of the chain is 
nucleic acid, with 9 to 11 per cent, of phosphorus, and without any proteids 
(this compound occurs in nature in the heads of spermatozoa) ; in the middle 
are what are generally termed the nucleins, consisting of proteid with vary- 
ing amounts of nucleic acid; and at the other extreme are nucleins which 
are nearly all proteid, containing only 0'5 to 1 per cent, of phosphorus, and 
are in fact the same substances which have received the name of “nucleo- 
albumin they may also be termed the artificial plastins. 
These substances have both been isolated from the most diverse tissues of CYTOLOGICAL METHODS. 
359 
the animal body, and have been prepared artificially. A corresponding 
series of nucleins exists within the nucleus itself. There are those that 
contain most nucleic acid; these are readily soluble in alkalies, and precipi- 
table with difficulty by acid : chromatin is one of these. There are others 
more insoluble in alkalies and poorer in nucleic acid; these are the plastins, 
the pyrenin of nucleoli being one of them. And there are others even 
poorer in nucleic acid ; these are the nucleo-albumins (which exist also in 
the cytoplasm) : the paralinin, or “ nuclear sap,” appears to be in part com- 
posed of these, in part of phosphorus-free compounds. 
There appears to be some doubt whether chromatin is or is not nucleic 
acid itself. The principal reactions in which it resembles nucleic acid are 
given by Halliburton (Goulstonian Lectures on the Chemical Physiology 
of the Animal Cell, 1893, p. 574 of the Report in the British Medical 
Journal, No. 1681, March 18th, 1893, from which place also I have con- 
densed the above remarks on the chemistry of the nucleins) as follows:— 
“ 1. It does not give Millon’s nor the xantho-proteic reactions. 2. It is easily 
soluble in alkalies, soluble with difficulty in acids. 3. It is soluble in an 
acetic acid solution of potassium ferrocyanide. 4. After treatment with 
concentrated copper sulphate solution for twenty-four hours it loses its 
affinity for stains. It is not, however, dissolved by the copper sulphate as 
Schwarz stated. 5. It has a great affinity for anilin dyes, especially for 
basic dyes like methyl green. If a mixture of methyl green and acid 
fuchsin is employed, nucleic acid is stained green. The nucleins next richest 
in phosphorus are stained a blue-violet tint, whereas the phosphorus-poorest 
are coloured red. Now in the dividing nucleus, when the amount of chro- 
matin is at its maximum, the nucleus stains green ; whereas in the resting 
nucleus, where there is more pyrenin, a blue colour is observed.” 
It results from experiments of the nature of that mentioned under No. 5 
(which, by the way, does not appear to me to be quite correctly stated), that 
chromatin is a basopliilous body in Ehrlich’s sense (explained in § 268), 
and that the albumins, and consequently cytoplasm in general are acido- 
philous. 
These considerations appear to justify the employment of the term 
“ chromatin ” for the element of the nucleus that stains with methyl green, 
the term “ nuclein ” having obtained a wider extension. 
See also Kossel, in Behrens, Kossel, und Schiefferdecker’s Das 
MiJcrosJcop, &c., ii, p. 47; the same, in Verh. d. physiol. Ges., Berlin, Oct. 
21st, 1892; Malfatti, Per. d. naturw. med. Vereines in Innsbruck, 1891-2 ; 
Schwarz, Cohn’s Beitr. z. Biol. d. Pflanzen, v, 1, 1887, p. 1, or Zeit.f. 
iviss. Mik., iv, 4, 1887, p. 530; Zacharias, Ber. d. deutschen botan. Ges. 
x, 1893, p. 188, and xi, 1893, p. 293, or Zeit. f. wiss. Mik., x, 1, 1893, p. 
80, and x, 3, 1893, p. 373; Lilienfeld, Verh. d. phys. Ges. Berlin, 1892-3, 
No. 11, or Zeit. f. wiss. Mik., x, 1, 1893, p. 80; Zimmehmann, Zeit. f. wiss. 
Mik., xii, 4, 1896, p. 458. 
Eor microchemical methods for the study of the distribution of assimilated 
iron compounds in cells, see the masterly paper of Macallum, in Quart. 
Journ. Mic. Sci., No. 150, 1895, p. 175. 
Eor the microchemical detection of phosphorus in tissue-elements see the 
paper of Lilienfeld and Monti, in Zeit. f. physiol. Cliemie, xvii, 1892, 360 
CHAPTER XXVIII. 
p. 410 (Report in Zeit.f. wiss. Mil., ix, 3, 1893, p. 332) ; also a short notice 
in Halliburton’s Goulstonian Lectures, 1893 (see Brit. Med Journ 
March 11th, 1893, p. 505). 
657. Cytological Fixing Agents.—The following is in great 
part taken from the numerous papers of Flemming in the 
Arch. f. mile. Anat. from the year 1879 onwards, and from his 
Zellsubstanz Kern- und Zelltheilung. 
Osmic acid (y to 2 per cent.) preserves the form of the 
entire cell, but swells the nuclei and rounds off nucleoli. It 
renders the nuclear “ reticulum ” undiscernible. Picric acid, 
either concentrated or dilute, and chromic acid, OT to 0'5 per 
cent., are to be preferred to alcohol and other agents for the 
study of the cells of Vertebrates. Shrinking and distortion of 
the nuclear figures (and, with picric acid, swellings of them) 
are to be expected, but other agents have the same defect to 
a much greater degree; alcohol especially causes entanglement 
of the threads. Acetic acid does the same, and causes 
swelling besides. Stronger chromic acid solutions cause 
shrinking. Neither of these reagents is harmless as regards 
the nuclei of red blood-corpuscles. The salts of picric acid 
(potash-, soda-, and baryta-salts) are most harmful. Weak 
(i. e. not more than 1 per cent.) acetic, hydrochloric, or nitric 
acid, combined with clearing in glycerin and staining, may be 
useful for bringing out chromatin and nucleoli. Chloride of 
gold preserves the form well, but generally leaves the nuclear 
structures unstained. Nitrate of silver is hopelessly uncon- 
trollable in its action. Alcohol has much the effect of chromic 
acid, but often causes a much greater shrinking of the nuclei. 
Bichromate of potash and chromate of ammonia bring out very 
sharply the appearance of a reticulum, but these appearances 
cannot be accepted as true (1. c., p. 334, et seg.). 
“ Those who seek to study cell-division by means of bichromate 
of potash or other chromic salts are hopelessly in the wrong 
road.” And this because of the injurious action of the 
bichromate, not on the body of the cell, which it preserves 
well, but on the chromatin structures. Chromic salts are 
excellent reagents for general histological work, but not for 
nuclear structures. They dissolve nucleoli, destroy nuclear 
“networks,” and swell up and distort karyokinetic figures to 
such a degree that the appearances obtained from them are 
merely unnatural caricatures of the true structure. CYTOLOGICAL METHODS. 
361 
Altmann’s nitric acid method is excellent for the purpose 
of hunting for cell-divisions in tissues; but the minute struc- 
ture of the figures is not so well preserved as it is by means 
of chromic or picric acid. The same must be said of Kleinen- 
berg’s picro-sulphuric acid method. (I am not alone in hold- 
ing that this is a most untrustworthy cytological reagent; see, 
for instance, Holl [Sitzb. k. Akad. Wiss. Wien, xcix, 1890, 
p. 311; Zeit. f. wiss. Mik., ix, 1, 1892, p. 89], who found that 
it frequently reduced chromosomes to the state of mere lumps, 
“ Krumeln.”) 
There are two fixing mixtures which may be said to be 
classical for cytological studies, Flemming’s chromo-aceto-osmic 
acid mixture, §§ 46, 47, and Hermann’s platino-aceto-osmic acid 
mixture, § 65. As to the former of these, Flemming has the 
following explanations:—Attempts to omit the chromio acid 
from the formula did not give good results. The omission of 
acetic acid (as in Max Flesch’s formula, § 45) causes the 
figures to be far less sharply brought out. The presence of 
acetic or formic acid in all osmium solutions is favorable to 
the precision of subsequent staining with heematoxylin, picro- 
carmine, or gentian violet. But mixtures of osmic and acetic 
acid without chromic acid (Eimer) do not give such good 
results as the chromo-aceto-osmic acid mixture. Mixtures 
of picric acid with osmic acid or with osmic and acetic acid 
(proportions of the latter as in the chromo-aceto-osmic mix- 
ture [§ 46], but of picric acid about 50 per cent.) fix quite as 
well as the chromic mixtures, but precise staining is even 
more difficult than with pure osmic acid preparations. Flem- 
ming concludes that the beneficial effects of the osmium in 
all these mixtures are to be ascribed to the instantaneous 
rapidity with which it kills, the function of the other acids of 
the mixture being to render the structures distinctly visible. 
Mixtures containing osmic acid should therefore be em- 
ployed whenever it is desired to fix the chromatic figures as 
faithfully as possible; whilst pure chromic acid should be 
taken whenever very sharp staining is the more important 
point. 
For the study of achromatic figures he recommends the 
chromo-acetic acid mixture (§ 43), followed by staining in 
haematoxylin (anilins, he states, do not give so good results 
for this purpose). 362 
CHAPTER XXVIII. 
For the study of polar corpuscles he recommends the 
osmium mixtures, or pure chromic acid followed by Staining 
with gentian violet. 
The above account stands nearly as it stood in the first 
edition. The state of things at present is as follows :—It is 
admitted by all competent observers that the chromo-aceto- 
osmic mixture is, with at most one or two possible exceptions, 
by far the best fixing agent for nuclei. But some observers 
have stated that it does not always preserve the cell-body 
well. This is a question that has been already discussed in 
§§ 46 and 47. I will only add here that after considerable 
experience I see no reason to distrust Flemming’s mixture as 
a preservative of any kind of protoplasm, provided it be used 
in the proper way. It must be taken of the proper strength, 
it must be used with very small objects, so that it may act on 
all parts of them with its full strength, and not be filtered and 
diluted through thick walls of tissue before coming into con- 
tact with the object of study; and it must be allowed to act 
for the proper time. 
This brings us to another point. There are two Chromo- 
aceto-osmic mixtures—the old weaker one, and the new stronger 
one. Flemming recommended the strong one primarily as 
affording a means of differentiating kinetic chromatin from 
resting chromatin. He did not recommend it as a reagent for 
general work. Whether of these two solutions should be 
used for general work ? According to my experience, the 
strong solution does preserve both nuclear structures and 
caryoplasmic structures quite as faithfully at least as the old 
formula, and some structures most decidedly much better. 
Of course the one and the other should be taken according 
to the nature of the object you are dealing with ; but I think 
it may safely be stated as a general rule that if you take the 
strong mixture, and fix thoroughly in it, you are not likely to 
go far wrong. And what is meant by a thorough fixation ? 
Half an hour may be taken to be generally enough; but for 
very delicate things, such as the Nebenkern and the achro- 
matic figure, eighteen hours or more may be desirable. 
As to the platino-aceto-osmic mixture of Hermann, it has 
already been explained in § 65 that the point of superiority 
over Flemming’s mixture that is claimed for it lies in a more 
faithful preservation of cytoplasm and achromatic structures. CYTOLOGIOAL METHODS. 
363 
That the alleged superiority really exists appears to be the 
general opinion of those who have worked with this reagent. 
Flemming (Arch. f. mik. Anat., xxxvii, 1891, p. 685; Zeit. f. 
wiss. Mik., viii, 3, p. 343) agrees that it gives a peculiarly 
sharp demonstration of spindle fibres, centrosomes, and polar 
corpuscles ; but thinks that the chromo-aceto-osmic mixtures 
give a somewhat more faithful preservation of the chromatic 
elements. According to my experience there are slight 
differences in the behaviour of these two reagents which may 
be taken advantage of (see § 65). 
Niessing (Arch. f. mik. Ancit., xlvi, 1895, p. 147) lias tlie 
following- two modifications of Hermann’s mixture : 
(1) Platinic chloride, 10 per cent, solution . 25 
Osmio acid, 2 per cent. . . . .20 
Glacial acetic acid . . . . 5 
Distilled water ...... 50 
(2) The same with saturated aqueous solution of corrosive 
sublimate instead of the water. 
0. vom Rath’s picro-platinic mixtures have beengiven(§80). 
Liquid of Merkel, or the modification of it by Brass, which 
will be given in the chapter on Protozoa, may be found useful. 
For Lindsay Johnson’s platinic mixture see § 97. 
Some observers have had good results with liquid of 
Perenyi, especially for achromatic structures. 
Two or three of the fixing agents proposed by other writers 
may also rank as first-class reagents for this kind of work. 
There is Rabl’s chromo-formic acid (§ 44). Fix in this for 
twelve to twenty-four hours, wash out well with water, and 
pass into alcohol. And there is the same observer’s platinum 
chloride solution (§ 64). In Rabl’s paper in Ancit. Anz., iv, 
1889, p. 21, he recommends that Salamanclra larvae be fixed 
(for twenty-four hours) in a solution of from one tenth to one 
eighth per cent, strength. In his earlier work he used solu- 
tions of 1 : 300 strength. Platinum chloride has the pecu- 
liarity of causing a slight shrinkage of the chromatin, which 
helps to bring into evidence the granules of Pfitzner and the 
longitudinal division of the chromosomes. 
There remain to be mentioned several fixing agents with which very 
important work has been done. These, however, are, I think, not quite first- 
class reagents for the purpose, the brilliant results that have been obtained 
with them having been obtained rather in spite of their defects than on 364 
CHAPTER XXVIII. 
account of their good qualities. For instance, acetic alcohol is a reagent 
with which some of the most important work in recent cytology has been 
done—namely, mueh of that on the maturation and fecundation of the ovum 
of Ascaris. It is evident that for such an extraordinarily impenetrable 
object as the ovum of Ascaris, the employment of some such highly pene- 
trating fluid as acetic acid is imperatively indicated, notwithstanding the 
serious defects that it may have. • 
Cabnoy (La Cellule, iii, 1, 1886, p. 6) used at first a mixture of three 
parts of absolute alcohol with 1 of glacial acetic acid; later (ibid., iii, 2, 
1887) the chloroform mixture (§ 69). From five to fifteen minutes is enough 
for even the most resistent ova. 
Yan Beneden and Nett (Nouvelles Bech. sur la Fee. et la Division 
mitosique, 1887) employed a mixture of equal parts of absolute alcohol and 
glacial acetic acid, or even pure acetic acid. 
Acetic alcohol may be washed out with either pure alcohol, or with dilute 
glycerin (Calberla’s formula would be a good one in many cases). For 
further details see ante, § 69. 
M. Heidenhain (Ueb. Kern u. Protoplasma, 1892, p. 113) has been 
using corrosive sublimate on account of its convenience, and, above all, on 
account of the great facility it affords for the employment of certain stains. 
Ihe beautiful figures of “attraction spheres” and other cytoplasmic struc- 
tures given in this paper show that the most brilliant results may be 
obtained by this means. But I would remark that the figures of nuclear 
structures appear to me less convincing. The author figures and describes, 
under the name of “ Lanthanin,” an acidophilous earyoplasmic substance 
exhibiting a minutely reticular arrangement. The figures remind me of 
appearances which I frequently obtained in certain nuclei when working 
with sublimate some years ago, and which I regarded as artefacts, and in 
consequence was led to the abandonment of sublimate as a cytological fixa- 
tive. I do not find in Heidenhain’s paper that he has instituted control 
experiments to show that the reticular arrangement of his “ lanthanin ” is 
preformed in the nucleus, and would point out the need of such experiments 
before either the existence of the lanthanin reticulum or the fidelity of the 
reagent can be deemed established. It should further be noted that with 
sublimate material it is impossible to obtain with safranin, gentian violet, and 
some other stains, the delicate differentiations that they give with chrom- 
osmium material. 
Altmann (Arch. f. Anat. u. Entwickel., 1892, p. 223; Zeit. f.wiss. Mik., 
ix, 3, 1893, p. 331) has a new fixative for resting nuclei, viz. a 2'5 per cent, 
solution of ammonium molybdate to which is added about 025 per cent, of 
chromic acid. He also mentions (Die Elementarorganismen, 1890; cf. 
Zeit.f.wiss. Mile., vii, 2, 1890, p. 201) a fixation with nitrate or picrate of 
meicuiy, foi the demonstration of his “ bioblasts.” I have no personal 
knowledge or other information concerning either of these methods. 
Lemon juice (fresh, filtered) has been warmly recommended as a fixative 
for nuclei by van Gehuchten (Anat. Anzeig., iv, 1889, p. 52). Fix for 
five minutes, wash well with water and stain with methyl green, and exa- 
mine in liquid of Ripart and Petit. CYTOLOGICAL METHODS. 
365 
Heat has been recommended, but I believe it to be altogether objection- 
able. Henking (Zeit. f. iviss. Zool., xcix, 3, 1890, p. 503 ; Zeit.f. iviss. 
Mik., vii, 2, 1890, p. 211) has found that it totally destroys achromatic 
structures. 
658. Chromatin Stains.—Stains appropriate for fresh or 
lightly fixed tissues have been mentioned in § 655. 
For sections of hardened tissues we have the choice between 
the finer hsematein stains (or carmalum), and those obtained 
by means of safranin, gentian violet, and some other anilins, 
used according to the regressive method. 
Of the hsematein stains, hsemalum gives very good results 
with sublimate material; but for chrom-osmium material I 
decidedly prefer in general an iron lake, M. Heidenhain’s or 
Benda’s. 
Thionin has been warmly recommended by M. Heidenhain 
as giving a stain even superior to that of safranin. After 
trial, I think the recommendation is justified. Methyl green 
is, in my experience, inferior for preparations destined to be 
mounted in balsam. Carmalum is a very precise stain, and 
useful for sublimate material, but is decidedly too weak for 
chrom-osmium material. 
Babes’s supersaturated safranin stain (Arch. f. mile. Anat., xxii, 1883, 
p. 361) may also occasionally be useful. It is as follows :—A supersaturated 
solution of safranin in water is warmed to 60° C. and filtered warm. On 
cooling, it becomes turbid through the formation of small crystals. Sections 
are placed in a watch-glass with some of this turbid solution, and the whole 
is warmed for a few seconds (till the liquid becomes clear) over a spirit 
lamp. Allow the whole to remain for one minute, and wash out with water, 
and treat with alcohol and turpentine in the usual way. Tissues which do 
not take on the stain at once must be warmed over and over again. Clove 
oil must be avoided for clearing. 
Bor some remarks of Bataillon and Koehler on the stain of borax- 
methylen-blue, see Comptes Benclus, cxvii, 1893, p. 521, or Journ. Boy. 
Mic. Soc., 1894, p. 41. 
659. Plasma Stains.—I have been unable to discover a single 
thoroughly satisfactory one. All of those known to me are 
of an imperfect electivity, in so far as it is difficult if not 
impossible to limit their action with the desired precision and 
certitude to the element that it is desired to bring into promi- 
nence by staining. Almost all of them colour too readily the 
enchylema or hyaloplasm at the same time as the plasmatic 
reticulum. And, on the other hand, there are many im- 366 
CHAPTER XXVIII. 
portant elements of the cell which refuse to stain sufficiently. 
Spindles, for instance, can be made to stain, but only weakly 
at the best : it is impossible to get them to stain vigorously 
and at the same time distinctively. 
The best, or least defective plasma stain for hardened tissues 
that I know of is Kernschwarz ; see § 334, and also my paper 
“ Sur le Nebenkern et sur la formation du Fuseau,” in La 
Cellule, xi, 2, 1896, p. 257. 
After Kernschwarz, perhaps the best results I have had 
have been obtained by means of Flemming’s orange method. 
It is a very good stain for normal cytoplasmic structures, but 
a very poor one for the Nebenkern (“ Spheres ” of some 
authors) and other spindle relics. 
Benda’s Safranin and Lightgriin or Sdureviolett gives 
sometimes splendid results, but is capricious. It is very good 
for the Nebenkern, less so for equatorial spindle relics. 
For Sdurefuchsin and Orange G, see §§ 305 and 300. 
Ehrlich-Biondi mixture is a celebrated plasma stain. It 
will work with chrom-osmium material. It is of no use 
whatever for polar corpuscles or spindle relics. 
For Rawitz’ Inversion Stain, see § 308. I have obtained 
some vigorous images of polar corpuscles by this process, but 
I certainly do not think that it is a recommendable one. 
The Osmic Acid and Pyrogallol Process, § 377, gives a very 
fair and frequently useful plasma stain; but I do not con- 
sider it to be a method of quite the first class. 
The Iron-LLsematein Lakes of Benda and M. Heidenhain 
give good plasma stains, according to the degree of extrac- 
tion. The iron lake of M. Heidenhain is said to be a specific 
stain for “ centrosomes.” I am not concerned to dispute the 
alleged fact, but must be allowed to hold that the status of 
the corpuscles stained by this process has not been placed on 
a perfectly satisfactory footing. It is said by Heidenhain, 
and by other observers who have repeated his observations, 
that the reaction in question is obtained in a sharper form 
by combining the hgematein stain with a foregoing stain with 
Bordeaux B. It is held by Heidenhain that the foregoing 
stain with Bordeaux, which is a general stain taking effect 
on both chromatin and plasma, but having no affinity for cen- 
trosomes, so satisfies the chemical affinities of the first-named 
elements that they no longer hold the iron lake with the CYTOLOGICAL METHODS. 
367 
usual tenacity, but give it up freely in the extraction process, 
whilst the centrosomes, not having taken up so much Bor- 
deaux, are free to hold the hsematein strongly; which, it is 
stated, they do. This hypothesis is put forward as a theory 
under the name of Subtractive Staining by Heidenhain, 
and under the name of Tinctorial Preoccupation by Unna 
(Zeit.f. wiss. Mik., xii, 4, 1896, p. 454). 
The instructions given by Heidenhain for the employment 
of the Bordeaux are not so detailed as might be desired. He 
states (Arch. f. mik. Anat., xlii, 1894, p. 665) that the sec- 
tions (sublimate sections wei’e used by him) are to be stained 
for twenty-four hours or more in “ a weak ” solution of Bor- 
deaux, until they have attained such an intensity of colour as 
that “ they would just be fit for microscopic examination 
with high powers” (1. c., p. 440, note), and that they be then 
brought inlo the ferric alum. After mordanting and staining, 
the haematein is to be extracted in the iron alum until the 
chromatin has become entirely or almost entirely colourless. 
The Bordeaux is not supposed in this process to act as a plasma 
stain ; it goes away in the subsequent processes. Instead of 
Bordeaux, “ anilin blue ” may be used in the same way. I 
have repeatedly tried the Bordeaux process, and with my 
objects (I have not tried Heidenhain’s) have observed not 
the slightest difference in the preparations obtained in this 
way, and those obtained by the iron haematein without the 
Bordeaux. The solution of Bordeaux used by me was of 1 
per cent, strength. 
I have obtained some good plasma stains both with 
Ehrlich’s triacid and with his acidophilous mixture, and for 
some purposes should prefer these to the Ehrlich-Biondi 
mixture. 
Hermann (Arch. f. mile. Anat., xxxvii, 4, 1891, p. 583) recommends a 
modification of the hcematoxylin impregnation method of Pal :—Testes 
of Proteus put for twelve to eighteen hours into hematoxylin, 1 grin.; 
absolute alcohol, 70 c.c.; water, 30 c.c., in the dark, and washed out for the 
same time in 70 per cent, alcohol, also in the dark. Sections having been 
made are treated with dilute pale rose-coloured solution of permanganate of 
potash till they turn ochre-coloured, rinsed in water, and washed out for 
removal of the brown peroxide of manganese in Pal’s oxalic acid mixture 
diluted with 5 to 10 volumes of water. They are finally stained for three 
to five minutes in safranin. 
See also on this subject the paper of Hermann, •“ Methoden zum Studium 368 
CHAPTER XXVIII. 
des Archoplasmas und der Centrosomen tierischer und pflanzlicher Zellen,” 
in Ergebnisse der Anatomie, Band ii, 1892 (1893), p. 23. 
For Heidenhain's Vanadium hssmatoxylin, see Cohn in Anat. 
Hefte, 1895, p. 302, or Zeit. f. wiss. Mik., xii, 3, 1896, p. 359. 
The achromatic structures of the ovum of Ascaris are best 
demonstrated, according to van Beneden et Neyt (Nouv. 
Rech.) by fixing with acetic alcohol {ante, § 657), and bringing 
the ova into one-third glycerin in which is dissolved a little 
malachite green. The “ spheres attractives ” stain green. 
See also Boveri’s Zellen-Studien (in Jen. Zeitschr. f. Naturw., 
xxi, 1887, p. 423, or sold separately). Herla {Arch, de Biol., 
xiii, 1893, p. 423) advises a mixture of vesuvin 0-25, mala- 
chite green 0’25, distilled water 100, and glycerin 10; the 
stain to be washed out with dilute glycerin. 
660. Nucleus of Balbiani (“Noyau Vitellin,” “ Cellule Embryo- 
gene”) (Zool. Anz., 1883, p. 659).—This may be observed in the fresh 
state, without the addition of any reagent, in the ova of some animals, 
amongst others a great number of Arachnida and Myriapoda. It may be 
brought out more distinctly by treating the ova with a mixture of equal 
parts of acetic acid and 1 per cent, osmic acid, to which is added a little 
sodium chloride. This mixture does not render ova so granular as pure 
dilute acetic acid. For sections, stain by Henxeguy's permanganate 
safranin process. 
661. Cell-granules.—These for the most part undoubtedly 
metabolic products are best studied in gland-cells and blood- 
and lymph-corpuscles, and in certain elements belonging to 
the group of connective tissues, and the reader is therefore 
referred to the sections on “ Connective Tissues ” for the 
appropriate methods. It will suffice here to state that the 
most generally employed stains for cell-granules are the mix- 
tures of Ehrlich. 
For Altmaxn’s “ Bioblasts,” see, besides that author’s Studien iiber die 
Zelle, 1886, his Die Elementarorganismen, Leipzig, 1890, and a paper in 
Arch.f. Anat. u. Entwickel., 1892, p. 223; also Zeit. f. wiss. Mik., vii, 2, 
1890, p. 199 ; ix, 3, 1893, p. 331; and L. and It. Zoja, in Mem. R. 1st. 
Lombardo di Sci. e Lettere, xvi, 3, vii, p. 237. Omitting minutiae and 
variations, it may be said that these granules may be demonstrated by fixing 
for twenty-four hours in a mixture of equal parts of 5 per cent, bichromate 
of potash and 2 per cent osmic acid, imbedding in paraffin, staining sections 
for a minute on the slide held over a flame with a solution of 20 grms. of 
acid fuchsin in 100 c.c. of anilin water (p. 190), and washing out with CYTOLOGICAL METHODS. 
369 
saturated alcoholic solution of picric acid diluted with 2 volumes of water, 
heat being used as before to aid the differentiation, and finally clearing 
with xylol and mounting in balsam. Fischer (Ancit. Anz., ix, 1894, No. 
22, p. 678) has given reasons for believing that these granules may consist, 
in part at all events, of precipitates of albuminates (principally peptones) 
thrown down by the fixing agents (see also § 657). 
662. More Special Methods.—For accounts of the methods that have 
been employed in the study of tissue-cells, and more especially of sexual 
products, now suppressed for want of space, see previous editions, or the 
Traite des Metliodes techniques de VAnat. microscopique, Lee et 
Henneguy. CHAPTER XXIX. 
TEGUMENTARY ORGANS. 
663. Epithelium.—One of the chief methods of obtaining 
preparations giving instructive surface views of epithelia is 
the nitra te of silver method. For this see ante, § 358, et seq., 
in the chapter on “ Impregnation Methods.” The reader 
may also consult with advantage the admirable instructions 
given by Ranvier in his Traite technique, p. 246, et seq., and 
the memoir of Tourneux and Hermann in the Journ. de VAnai., 
1876, p. 200. 
The per chloride of iron and pyrogallic acid method of the 
Hoggans, § 379, and the osmic acid and pyrogallic acid pro- 
cess, § 377, may also be found useful here. But in many 
cases impregnation with methylen blue will doubtless be found 
preferable. 
Sections are easily made by the usual methods. The best 
hardening agent for skin appears to be Muller’s solution. 
This was the conclusion of F. E. Schultze in 1867 (Arch. f. 
mik. Anat., p. 145); and it is that of Tizzoni, the author of 
important researches on this organ (Bull, delle Sc. med. di 
Bologna, 1884, p. 259), and of Behn (Arch. f. mik. Anat., 
xxxix, 1892, p. 581). Simple bichromate of potash solution 
will do about as well. 
For glandular epithelium it is frequently better to employ 
a chromic acid liquid, or osmic acid (see, for example, Ranvier, 
loc. cit., p. 258, et seq.), or absolute alcohol (Blaue, Arch. f. 
Anat. u. Phys., 1884, p. 231) ; “ Kleinenberg ” is not so good. 
Macerating Media.—For soft epithelia, mild macerating 
agents, such as iodised serum, one-third alcohol, saliva, or 
Schultze’s mixture of saliva and solution of Muller, or a 
mixture of saliva with three to four volumes of physiological 
salt s-olution (Bizzozero, Intern. Monatsschr. f. Anat., 1885, 
p.278);—for hard epithelia, energetic dissociating agents, 
such as 40 per cent, solution of caustic potash. TEGUMENTARY ORGANS. 
371 
Minot (Amer. Natural., xx, 1886, p. 575; cf. Journ. Boy. 
Mic. Soc., 1886, p. 872) recommends maceration for seveival 
days in 06 per cent, solution of sodium chloride containing 
Ol per cent, of thymol, which allows the isolation of the 
epidermis of and is useful for the study of the 
development of hairs. 
Another method, given by Mitrophanow (see Zeit. f. wiss. 
Mik., v, 4, 1888, p. 513), is as follows :—An embryo of axo- 
lotl is fixed for a quarter of an hour in 3 per cent, nitric acid, 
and then brought into one-third alcohol. After an hour the 
epidermis begins to come away in places; and if the embryo 
be put for twenty-four hours into stronger spirit, it will come 
away almost entirely. 
For the purpose of separating the epidermis from the 
corium, Loewy (Arch. f. mik. Anat., xxxvii, 1891, p. 159 ; 
Zeit. f. wiss. Mik., viii, 2, 1891, p. 222) recommends macerat- 
ing for twenty-four to forty-eight hours, at a temperature of 
about 40° C., in 6 per cent, pyroligneous acid. Acetic acid 
of -g- per cent. (Philippson) is also good. 
For ciliated epithelium, see the methods of Englemann 
under “ Mollusca.” 
664. Prickle-Cells and Intercellular Canals.—Besides macera- 
tion, which is one of the most important of the methods for 
the study of these objects, impregnation may be useful. 
Mitrophanow (Zeit. f. wiss. Zool., 1884, p. 302, and Arch. f. 
Anat. u. Phys., 1884, p. 191) recommends the following pro- 
cess :—Wash with distilled water the tail of an axolotl larva; 
put it for an hour into 025 per cent, solution of gold chloride 
with one drop of hydrochloric acid to a watch-glassful of the 
solution ; wash, and reduce in a mixture of one part of formic 
acid with six parts of water. 
On this subject see the important memoirs of Ide, in La 
Cellule, iv, 2, 1888, p. 409, and v, 2, 1889, p. 321. 
665. Plasma-fibrils of Epithelium.—Kromayer’s process for 
demonstrating his intra- and intercellular fibrils of epithelia 
(Arch. f. mik. Anat., xxxix, 1892, p. 141; Zeit. f. wiss. Mik., 
ix, 1, 1892, p. 84, and ix, 3, 1893, p. 355) is as follows:— 
Sections are stained for five minutes in a mixture of equal 
volumes of anilin water (p. 190) and concentrated aqueous 372 
CHAPTER XXIX. 
solution of methyl violet 6 b. They are well washed in water 
and treated with solution of iodine in iodide of potassium until 
they become blue-black (one to thirty seconds). They are again 
washed with water, dried with blotting-paper, and treated 
with a mixture of 1 vol. of anilin to 2 vols. of xylol until 
sufficiently differentiated, when they are brought into pure 
xylol. Very thin sections will require more xylol in propor- 
tion to the anilin, viz. 1 : 3 or 1 :4: thicker ones may require 
more anilin, viz. 3:5 or 3:3. Gentian or Krystallviolett 
will do instead of methyl violet, but not quite so well. For 
a discussion of the points of difference between the fibrils of 
Kromayer and the fibres of Herxheimer, see Ehrmann, and 
Jadassohn, Arch. f. Dermatol, u. Syphilis, 1892, 1, p. 303; 
Zeit. f. iviss Mih., ix, 1893, p. 356. 
For the same object Unna (Monatsh.f. prakt. Dermatol., xix, 
1894, p. 1 and p. 277, et seq. ; Zeit.f. wiss. Mih., xii, 1, 1895, 
pp. 61, 63) has given a whole series of minutely detailed 
methods, from which the following are some extracts : 
1. Wasserblau-Orcein.—Stain sections for ten minutes 
in a neutral aqueous 1 per cent, solution of Wasserblau, rinse 
with water and stain for five or ten minutes in a neutral 
alcoholic 1 per cent, solution of Griibler’s orcein. Dehydrate, 
clear, and mount in balsam. This method may be varied as 
follows: 
(a) Ten minutes in the Wasserblau, and thirty minutes or 
more in the orcein. 
(b) Take for the second stain an acid solution of orcein. 
(a) Stain for only one minute in the Wasserblau, but for 
thirty or more in the neutral orcein. 
2. Stain for half an hour or more in a strong solution of 
hsemalum, rinse with water, stain for half a minute in a satu- 
rated aqueous solution of picric acid, and dehydrate for thirty 
seconds in alcohol containing 0-5 per cent, of picric acid. 
3. Hsemalum for two hours, neutral orcein as above for ten 
to twenty minutes. 
Besides these methods, Unna gives half a dozen others 
which can hardly be usefully detached from the theoretical 
part of his papers. 
666. Horn, Hair, and Nails.—The elements of hairs and nails 
may be isolated by prolonged maceration in 40 per cent, potash TEGUMENTARY ORGANS. 
373 
solution, or by heating with concentrated sulphuric acid. 
See also von Nathusius, Zool. Anz.,siv, 1892, p. 395. 
Horny tissues stain well in safranin or gentian violet 
(Reinke, Arch. f. mik. Anat., xxx, 1887, p. 183; Zei.t. f. vn'ss. 
Mile., iv, 3, 1887, p. 383). 
,667. Tactile Hairs.—Kanvier (Traite, p. 914) recommends 
for the study of the nerve-endings the boiled formic acid and 
gold-chloride method, § 371. A tactile hair having been 
isolated with its bulb, and its capsule incised, is put for about 
an hour into the formic acid and gold-chloride mixture, the 
gold is reduced in slightly acidulated water, hardening is 
completed in alcohol, and longitudinal and transverse sections 
are made. For Heasch’s method see Zeit. f. wiss. Mik., iv, 4, 
1887, p. 492. 
668. Skin Nerves.—Wolters (Arch.f. Dermatol, u. Syphilis, 
1892 ; Zeit. f. wiss. Mik., ix, 3, 1893, p. 360) employs the 
chloride of vanadium and hsematoxylin method, which will 
be given in the chapter on “ Nerve-cell and Cell-process 
Stains.” 
669. Intra-epidermic Nerve-fibres.—May be studied by the 
gold method. Ranvier (Traite, p. 900) recommends the 
boiled formic acid and gold-chloride method, § 371. 
He also (p. 910) recommends this method for the study of 
the tactile menisci of the pig’s or mole’s snout. 
Pieces of skin are impregnated as directed § 371, and 
after reduction are brought into alcohol, which completes the 
hardening, and stays the further reduction of the gold. 
Sections are then made. 
For the study of the tactile menisci of the snout, Ranvier 
also recommends the lemon juice and gold-chloride method, 
§ 372. 
The metliylen blue impregnation method should also be 
employed in the study of these nerve-endings. 
670. Tactile Corpuscles (Fischer, Arch. f. mik. Anat., 1875, 
p. 366).—Fischer employed the gold method of Lowit—see 
§ 370. Kanvier (Traite, p. 918) also recommends this method, 374 
CHAPTER XXIX. 
as well as his two gold methods, §§ 371, 372. Pieces of skin 
are first impregnated whole, then hardened by alcohol and 
sectioned. He finds (as do other authors) that osmic acid 
and picro-carmine are invaluable aids to the study of these 
structures, and to that of the corpuscles of Pacini. Purpurin 
and hgematoxylin may also be used for after-staining. See 
Raxvier, Traite, p. 919; and see also Laxgerhaxs, Arch. f. 
mik. Anat., 1873, p. 730; Kultschizky, ibid., 1884, p. 358; 
and Smirnow, Intern. Monatschr. f. Anat., &c., x, 1893, 6, 
p. 241 ; Zeit. f. wiss. Alik., x, 2, 1893, p. 254 (this observer 
recommends, besides the gold method of Lowit, the rapid 
bichromate of silver method of Golgi). 
671. Corpuscles of Herbst and Corpuscles of Grandry.— 
Dogiel {Arch. f. Anat. u. Entwickel., 1891, p. 182; Zeit. f. 
iviss. Mik., viii, 4, 1892, p. 520) prefers the methylen blue 
method (Chap. XVII). Four per cent, solution of methylen 
blue, warmed to 40° C., is injected into blood-vessels of the 
heads of ducks or geese; pieces of skin are removed from 
the beaks, sectioned in pith, and the sections brought on to 
slides and moistened with aqueous or vitreous humour from 
the animal, and left for a few minutes exposed to the air (it 
is well to add to the aqueous or vitreous humour a few 
drops of y per cent, methyl blue solution). After about ten 
to thirty minutes the nerve-endings are seen to be stained, 
and the sections are then brought into picrate of ammonia, 
and treated as described in the chapter on “Methylen Blue.” 
Geberg {ibid., x, 2, 1893, p. 244) has also employed this 
method. He has also made use of simple osrnic acid, and of 
the gold method of Arxstein, which is as follows : 
Pieces of skin are macerated for twenty-four hours in lime 
water, after which the horny layer may easily be removed. 
This being done, the skin is cut up into small pieces which 
are put for five minutes into a 025 per cent, solution of 
chloride of gold. Reduction sets in very rapidly, and the 
preparations become brown in a few minutes. The reduction 
is completed by putting them for twenty-four hours into dis- 
tilled water. During this time there forms a granular pre- 
cipitate which is removed by putting the pieces into a 025 
per cent, solution of cyanide of potassium and brushing them 
vigorously with a camel’s-liair brush; after which they are TEGUMENTARY ORGANS. 
375 
mounted in damar. Geberg used the gold chloride of 0\5 per 
cent, strength, and allowed it to act for thirty minutes. 
See also the method of Carriere, Arch. f. mik. Anat., 1882, 
p. 146, or previous editions of this work. 
672. Corpuscles of Meissner and of Krause (Cornea and Con- 
junctiva Bulbi and Palpebrarum) (Dogiel, Arch. f. mik. Anat., 
xxxvii, 1891, p. 602, and xliv, i, 1894, p. 15).—A fresh bulb 
should be enucleated in toto, and incised along a line running 
parallel to the equator and 5 to 8 mm. behind the corneal 
margin. The anterior segment thus obtained is freed from 
the ciliary body, lens, &c., the conjunctiva being left in situ 
on the cornea. It is then cut into pieces which are stained 
for an hour to an hour and a half in methylen blue + 
aqueous humour, and further treated for examination and 
preservation as described in the chapter on “ Methylen Blue.” 
See also Longworth’s methods, Arch. f. mik. Anat., 1875, 
p. 655. 
673. Similar Objects.—Papillae Foliatse of the Rabbit (Hermann). 
—See Zeit.f. wiss. Mile., v, 4, 1888, p. 524. Olfactive Organs of Verte- 
brates (Dogiel, Arch. f. mik. Anat., 1887, p. 74). Organs of a “Sixth 
Sense ” in Amphibia (Mitrophaxow).—See Zeit. f. wiss. Mik., v, 4, 
1888, p. 513. (This paper contains some details as to staining with 
“ Wasserblau,” for which see also Biol. Centralb., vii, 1887, p. 175.) 
Nerve-endings in Tongue of Frog (Fajerstain [Fetjerstein], Arch, 
de Zool. exper. et gen., vii, 1889, p. 705 ; Zeit.f. wiss. Mik., vii, 3, 1889, 
p. 357).—(Amongst other methods for the study of the terminal discs, the 
methylen blue method is recommended; see previous editions). Tongue 
of Rabbit, vox Lenhossek, Zeit.f. iviss. Alik., xi, 3, 1894, p. 377 (Ramon 
y Cajal’s double Golgi-method). 
674. Cornea.—There are three chief methods for the study 
of the corneal tissue—the methylen blue method, the silver 
method, and the gold method. 
For the methylen blue method see Chap. XVII, particularly 
§§ 295 and 296. 
Negative images of the corneal cells are easily obtained by 
the dry silver method (Klein). The conjunctival epithelium 
should be removed by brushing from a living cornea, and the 
corneal surface well rubbed with a piece of lunar caustic. 
After half an hour the cornea may be detached and examined 
in distilled water. 376 
CHAPTER XXIX. 
In order to obtain 'positive images of the fixed cells the 
simplest plan (Ranvier) is to macerate a cornea that has been 
prepared as above for two or three days in distilled water. 
There takes place a secondary impregnation, by which the 
cells are brought out with admirable precision. 
The same result may be obtained by cauterising the cornea 
of a living animal as above, but allowing it to remain on the 
living animal for two or three days before dissecting it out, 
or by treating a negatively impregnated cornea with weak 
salt solution or weak solution of hydrochloric acid (His). 
But the best positive images are those furnished by gold 
chloride. Ranvier prefers his lemon-juice method to all 
others for this purpose (see § 372). It is important that the 
cornea should not remain too long in the gold solution, or the 
nerves alone will be well impregnated. 
Ranvier also recommends this method as being the best for 
the study of the nerves. 
Rollett (Strieker’s Handbuch, p. 1115) recommends a 
double impregnation with silver followed by gold for obtaining 
gold-stained negative images. A cornea having been treated 
for a short time only with 0-5 per cent, silver nitrate solution, 
and the silver reduced, is treated with 0'5 per cent, gold 
chloride solution. The brown stain of the silver disappears 
immediately the preparation is placed in the gold solution ; 
after a few minutes the preparation is exposed to the light in 
acidulated water. Reduction of the gold rapidly takes place, 
and in the place of the former brown stain of the silver the 
ground-substance shows the well-known blue of reduced gold. 
The cells are, however, visible, being recognisable by their 
granular appearance and pale yellow tint. 
Renaut (Gomptes Rend., 1880, ler sem., p. 137) gives the 
following process for corneal corpuscles :—Cornea of frog : 
formic acid, 20 per cent., ten minutes; gold chloride, 1 per 
cent., twenty-four hours; formic acid, 33'3per cent., twenty- 
four hours. 
Hoyer’s method has been given, § 374. 
675. Cornea, other Methods (Rollett, Strieker’s Eandb., 
p. 1102).—Rollett strongly recommends the following plan :— 
A fresh cornea is placed (in humor aqueus) in a moist chamber, 
and exposed to the action of iodine vapour. As soon as it has TEGTTMENTARY ORGANS. 
377 
become brown the epithelium may easily be peeled off. If the 
reaction is not complete the cornea may be put back into the 
iodine chamber. When sufficient iodine has been absorbed 
the preparation may be examined, and it will be found that 
the network of corneal cells is brought out with an evidence 
hardly inferior to that of gold preparations. The method 
never fails, which is not the case with the gold method. It 
is admirable as a fixing method. 
For dissociation of the fibres Rollett recommends macera- 
tion in a solution of permanganate of potash or a mixture of 
this solution with alum. As soon as the tissue has become 
brown it is shaken in a test-tube with water, and breaks up 
into fibres and bundles of fibres. 
Taktuferi (Anat. Anz., v, 1890, p. 524; Zeit. f. iciss. Mih., vii, 3, 1890, 
p. 365) has the following method for demonstrating corneal cells and the 
ramifications of their processes :—A cornea is placed for three or more days 
in a solution of 15 grammes of hyposulphite of soda to 100 c.c. of distilled 
water, kept at a temperature of about 26°, then removed for two days into 
water containing very finely powdered chloride of silver (if left longer the 
corneal cells will not be stained, but innumerable elastic fibres will be 
demonstrated). The preparations are said to be permanent. Some further 
details are mentioned in Zeit.f.wiss. Mik., xi, 3, 1894, p. 346. 
676. Crystalline (Hardening of) (Lowe, Arch.f. mik. Anat., 
1878, p. 557).—A fresh bulb is placed in a vessel containing 
several litres of 1 per cent, bichromate of potash solution, 
which is frequently changed for stronger solutions until the 
strength of a cold saturated solution is attained. The bulb 
must remain in this for at least a year and a half, in order 
that the crystalline may attain the right degree of hardness. 
For Maceration, use Max Scliultze’s sulphuric acid solution, 
supra, § 557. 
Formaldehyde may perhaps be found useful for hardening. CHAPTER XXX. 
MUSCLE AND TENDON (NERVE-ENDINGS). 
Striated Muscle. 
677. Muscle-cells.—For the study of these and allied sub- 
jects see, inter alia, Behrens, Kossel, und Schiefferdecker, 
Das Mikroskop, &c., vol. ii, pp. 154—161; also, for the ap- 
plication of the gold method to the study of muscle-cells, 
Schafer, Proc. Roy. Soc., xlix, 1891, p. 280 ; or Journ. Roy. 
Mic. Soc., 1891, p. 683. 
678. Sarcolemma.—Besides the places quoted in last section, 
see Solger, Zeit. f.wiss. Mik., vi, 2, 1889, p. 189 (small pieces 
of fresh muscle teased and examined in cold saturated solu- 
tion of cai’bonate of ammonia). 
679. Sections (Rollktt , Denkschr. math.naturw. Kl.~k.Akad. 
Wiss. Wien, 1885; Zeit.f. wiss. Mik., 1886, p. 92).—Besides 
the usual section methods, the following methods of Rollett 
should be noted :—(1) The method mentioned (§ 183) of freez- 
ing living tissue in white of egg. (2) The same method 
applied to recently fixed muscle. 
680. Dissociation.—See Chap. XXV. 
Langebhaxs’ methods for Amjphioxus (Arch. f. mih. Anat., 1875, p. 
291).—For isolation of the muscle-plates macerate the fresh animal in 20 
per cent, nitric acid. 
For isolation of the nervous system macerate an animal for three days in 
20 per cent, nitric acid, then place it for twenty-four hours in water, and 
shake forcibly. The whole of the nervous system may thus be sepai'ated, 
almost down to the finest peripheral terminations of nerves. 
681. Nerve-endings.—For the study of nerve-endings in 
muscle, both motor and sensory, the four chief methods are 
the methylen-blue method, the gold method, the silver method, 
and the bichromate of silver method of Golgi. MUSCLE AND TENDON NERVE-ENDINGS. 
379 
682. Nerve-endings—the Methylen-blue Method.—The prin- 
ciples of the impregnation of nerve-tissue with methylen blue 
have been explained in Chap. XVII. 
Biedermann’s procedure for the muscles of Astacus has been 
indicated in § 291 (see also Zeit. f. iviss. Mik., vi, 1, 1889, p. 
65). After impregnating- as there directed the carapace 
should be opened, and the muscles exposed to the air in a 
roomy moist chamber for from two to six hours, in order that 
the stain may differentiate. The abdominal and caudal 
muscles are those which give the best results. 
For Hydrophilus piceus, Biederinann proceeded by inject- 
ing 0'5 c.c. of methylen-blue solution between the ultimate 
and penultimate abdominal rings, in the ventral furrow, and 
keeping the animals alive in water for three to four hours. 
After this time the thorax should be opened by two lateral 
incisions, and the muscles of the first pair of legs (which are 
the most suitable) removed and exposed to the air for three or 
four hours in a moist chamber, and finally examined in salt 
solution. 
Gerlach (Sitzb. k. math.-phys. Cl. k. bayer. Akad. Wiss. 
Miinchen, 1889, ii, p. 125 ; Zeit. f. miss. Mik., vii, 2, 1890, 
p. 220) injected frogs, either through the abdominal vein or 
through the aorta, with 4 to 5 c.c. of a 1 : 400 solution in 1 
per cent, salt solution, and examined pieces of muscle (pre- 
ferably the head and eye muscles) in serum of the animal, 
afterwards fixing the preparations with pier ate of ammonia 
and mounting in glycerin jelly. 
The procedure of Dogiel has been given in § 291. 
683. Nerve-endings—the Gold Method.—Fischer {Arch. f. 
mik. Anat., 1876, p. 365) used the gold method proposed by 
Lowit (Wien. Sitzgsber., Bd. lxxi, Abth. 3, 1875, p. 1), and 
employed by himself in his researches on the tactile corpuscles 
(Arch. f. mik. Anat., xii, p. 366). See ante, § 370. 
Biedermann, in the paper quoted in the last section, recom- 
mends for Astacus a similar procedure, the preliminary treat- 
ment with formic acid being omitted, and the muscles being 
put for a couple of days into glycerin after reduction in the acid. 
The procedure of Trinchese (Mem. R. Accad. 1st. Bologna, 
5, ii, p. 279; Zeit. f. iviss. Mik., ix, 2, 1892, p. 238) is also 
practically identical. 380 
CHAPTER XXX. 
Ranvier (Traite, p. 813) finds that for the study of the 
motor terminations of Batracliia the best method is his lemon- 
juice and gold-cliloride process (§ 372). The delicate ele- 
ments of the arborescence of Kiihne are better preserved by 
this method than by the simple method of Lowit. 
For the study of the motor plates of reptiles, fishes, birds, 
and mammals, he finds {ibid., p. 826) that his formic acid and 
gold-chloride method (§ 371) gives preparations infinitely 
superior to those obtainable by the method of Lowit ; but the 
lemon-juice method is still better, especially for lizards and 
mammals. The branches of the terminal arborescence are 
more regular than in preparations obtained by the formic 
acid process. 
684. Nerve-endings—the Silver Method.—Ranvier finds that 
the silver nitrate method of Cohnheim is also useful. He 
employs it as follows {ibid., p. 810) :—Portions of muscle 
(gastrocnemius of frog) having been very carefully teased out 
in fresh serum are treated for ten to twenty seconds with 
nitrate of silver solution of 2 to 3 per 1000, and exposed to 
bright light (direct sunlight is best) in distilled water. As 
soon as they have become black or brown they are brought 
into 1 per cent, acetic acid, where they remain until they have 
swelled up to their normal dimensions (the swelling induced 
by the acid serving to make up for the shrinkage caused by 
the nitrate of silver). They are then examined in a mixture 
of equal parts of glycerin and water. 
This process gives negative images, the muscular substance 
is stained brown, except in the parts where it is protected by 
the nervous arborescence, which itself remains unstained. 
The gold process gives 'positive images, the nervous structures 
being stained dark violet. 
685. Nerve-endings—the Bichromate of Silver Method.—The 
osmium bichromate and silver method of Golgi has been suc- 
cessfully applied by Ramon y Cajal to the study of the ter- 
minations of nerves and of tracheae in the muscles of insects, 
and is doubtless susceptible of still wider applications. The 
process used by him is the rapid one. 
In Zeit. f. wiss. Mik., vii, 3, 1890, p. 332, he gives it as 
follows:—Fresh muscle of insects put into a mixture of 20 MUSCLE AND TENDON (NERVE-ENDINGs). 
381 
parts 3 per cent, bichromate solution and 5 parts 1 per cent, 
osmium solution for twelve to twenty-four hours, then into 
0*75 per cent, nitrate of silver for one day, then alcohol of 40 
degrees (presumably Baume, i. e. about 90 per cent.), then 
clove oil followed by resinified turpentine and (if I understand 
rightly) balsam. 
686. Nerve -endings—other Methods.—For the following see pre- 
vious editions :—Bremer (Arch. f. mile. Anat., 1882, p. 195) ; Ciaccio 
(Journ. cle Microyrciphie, 1883, p. 38); Wolff (Arch. f. mih. Anat., 1881, 
p. 355); Carl Sachs (ibicl.); Krause (Intern. Monatschr.f. Anat. u. Hist.; 
Zeit.f. iviss. Mih., 1885, p. 547) ; Negro (Zeit.f. wiss. Mih., v, 2, 1888, p. 
240); Sihler, Verhancl. d. Physiol. Ges. Berlin, 1894-5 ; Zeit.f. wiss. 
Mih., xii, 3, 1896, p. 389 (these two are hsematoxylin stains); Boccardi 
(Zeit.f. iviss. Mih., iv, 4, 1887, p. 492); Kuhne (Zeit.f. Biol., xxiii, v, 1887, 
p. 1; Zeit.f. wiss. Mih., iv, 4, 1887, p. 495—this paper contains a critical 
review of the different gold methods); Golgi (Mem. delle R. Accad. cli Sci. 
di Torino, ii, 32); Marshall (Quart. Journ. Mic. Sci., 1890, p. 73; 
Journ. Roy. Mic. Soc., 1890, p. 404); Mays (Zeit.f. Biol., 1884, p. 449; 
Zeit. f. uiss. Mih., 1885, p. 242). 
Ten don. 
687. Corpuscles of Golgi (Ranvier, Traite, p. 929).—Take 
the tendon of the anterior and superior insertion of the gemini 
muscles of the rabbit. Free it as far as possible from adherent 
muscle-fibres. Treat it according to the formic acid and gold 
method (§ 371), and after reduction of the gold scrape the 
tendon with a fine scalpel, in order to remove the muscle-fibres 
that mask the “ musculo-tendinous organs.” 
688. Corpuscles of Golgi (in the tendons of the motores 
bulbi oculi) (von Maechi’s methods, Archivio per le Scienze 
Mediche, vol. v, No. 15).—The enucleated eyes, together with 
their muscles, were put for not less than three days into 
2 per cent, bichromate of potash. The muscles and tendons 
were then carefully dissected out, stained with gold chloride 
and osmic acid (Golgi’s method, supra, § 686), and by the 
methods of Manfeem, given in § 374. 
Mount all these preparations in glycerin (balsam clears too 
greatly). The methods only succeed completely during fine 
sunny weather. 
689. Corpuscles of Golgi (Cattaneo, Arcli. ital. cle Biol., x, 1888, 
p. 337).—The method here recommended is the ai*senic acid gold method of 
Golgi quoted above, § 686, 382 
CHAPTER XXX. 
See also Ruffixi (Atti R. Acc. Lincei Roma, 1892, p. 442 ; Zeit. f. wiss. 
Mik., ix, 2, 1892, p. 237), who recommends the method of Fischer. 
690. Corpuscles of Golgi (Ciaccio, Mem. R. Acc. Sci. Bologna 
[4], t. x, 1890, p. 301; Zeit. f. wiss. Mik., vii, 4, 1891, p. 507). 
—For Amphibia the usual gold methods are not satisfactory, 
because the ground-substance of the tendon takes the stain 
at the same time as the nerve-endings. Pieces of tendon 
should be put into 0T per cent, hydrochloric acid or 02 per 
cent, acetic acid until quite transparent. They should then 
be put for five minutes into a mixture of 0T per cent, gold 
chloride and 0T per cent, potassium chloride. After that 
they are put back into the acetic acid, and remain there for 
a day in the dark, and for two or three hours more in the 
sunlight. When they have become somewhat violet they a re 
put for a day into 0T per cent, osmic acid, and finally mounted 
in Price’s glycerin acidulated with 05 per cent, of acetic or 
formic acid. 
691. The Methylen-blue Method.—I find no mention of the 
application of this method to the study of the relations of 
nerve and tendon, for which it would seem a priori to be 
peculiarly suitable. 
Smooth Muscle. 
692. Test for Smooth Muscle (Retterer, Comptes Rend. Soc. 
Biol., iv, 1887, p. 645 ; Journ. Roy. Mic. Soc., 1888, p. 843).— 
If a specimen of tissue be fixed in a mixture of ten volumes 
of 90 per cent, alcohol and one volume of formic acid, well 
washed, and stained for twenty-four to thirty-six hours with 
alum-carmine, the cytoplasm of smooth muscle will be found 
to be stained red, whilst connective-tissue cells remain un- 
stained, and are swollen. 
693. Smooth Muscle—Isolation of Fibres (Schwalbe, Arch.f. 
mik. Anat., 1868, p. 394).—Maceration in weak chromic acid 
solution (002 per cent, proved a generally useful strength). 
This is a better reagent than osmic acid, 1 per cent, acetic 
acid (Molescliott), weak sulphuric acid, pyroligneous acid 
(Meissner), 20 per cent, nitric acid (Reichert), 32 to 35 per MUSCLE AND TENDON (NERVE-ENDINGS). 
383 
cent, potash solution (Moleschott), as it preserves better than 
any of these the finer structure of the cells. 
G-age’s methods.—See Journ. Roy. Mic. Soc., 1887, p. 327 ; 
and §§ 539, 551, and 555, ante. 
Mobius, liquid for maceration of the muscle of Cardium 
(see above, § 550). 
Ballowitz, muscle of Cephalopoda, see Arch. f. mik. Anat., 
xxxix, 1892, p. 291 ; Zeit.f. wiss. Mik., ix, 3, 1893, p. 344. 
694. Smooth Muscle, Specific Stain for (Unna, Monatsh. f. 
prakt. Dermatol., xix, 1894, p. 533; Zeit. f. wiss. Mik., xii, 2, 
1895, p. 243).—Sections stained for ten minutes in poly- 
chromatic methylen blue solution, rinsed in water, and brought 
for ten minutes into 1 per cent, solution of red prussiate of 
potash. This fixes the colour, so that the sections will now 
bear differentiating with acid alcohol. They are treated ac- 
cordingly with alcohol acidified with 1 per cent, of hydro- 
chloric acid for about ten minutes (until the collagen ground 
comes out white). Absolute alcohol, essence, balsam. 
In the same place see also another stain with acid orcein, 
haematein, Saurefuclisin, and picric acid. 
695. Iris (Dogiel, Arch. f. mik. Anat., 1886, p. 403).—An 
enucleated eye is divided into halves, and the anterior one 
with the iris brought for some days into a mixture of two 
parts one-third alcohol and one part 0’5 per cent, acetic acid. 
The iris can then be isolated, and split from the edge into an 
anterior and posterior plate, and these stained according.,'to 
the usual methods. 
696. Iris (Koganei, Arch. f. mik. Anat., 1885, p. 1).—The 
pigmented epithelium can be removed by brushing with a 
small brush after prolonged maceration in solution of Muller- 
The pigment may also be bleached by chlorine water, which, 
however, should only be allowed to act for a few hours, until 
the pigment has become of a light brown; complete de- 
coloration may be obtained by prolonging the reaction for 
twenty-four hours, but then the tissues suffer. (See Journ. 
Roy. Mic* Soc., 1886, p. 874.) 
See also Canfield, in Arch. f. mik. Anat., 1886, p. 121; and 
Dostoiewsky, ibicl., p. 91 (sections stained with htema- 384 
CHAPTER XXX. 
toxylin and eosin, or [List, Zeit. f. wiss. Mile., iii, 4, 1886, p. 
514] with Renaut’s haematoxylic glycerin). 
697. Stomach of Triton (see Stilling and Pfitzner, in Arch, 
mile. Anat., 1886, p. 396). 
698. Bladder of Frog, Innervation of (Wolff, Arch. f. mik. 
Anat., 1881, p. 362).—A frog is killed and a solution of gold 
chloride of 1 : 20,000 injected into the bladder through the 
anus. (If the injection flows out on removal of the syringe, 
tie the frog’s thighs together.) Now open the frog, dissect 
away the attachments of the bladder, ligature the intestine 
above the bladder, and cut away the abdomen of the frog so 
as to have in one piece bladder, rectum, and hind legs. (All 
this time the bladder must be kept moist with weak gold 
solution.) The bladder and the rest are now put into gold 
solution of 1 : 2000 for four hours ; the bladder is then ex- 
cised, slit open, and pinned (with hedgehog spines) on to a 
cork (outside downwards). Place it under running water 
until all the epithelium is washed away. Use a pencil if 
necessary. Put for twenty-four hours into gold solution of 
1 : 6000. Wash in pure water, and put away in the dark “ for 
some time” in acidulated water, and finally reduce in fresh 
water in common daylight. The muscles should be pale blue- 
red ; medullated nerves dark blue-red; sympathetic nerves 
and ganglia carmine-red. Ranvier (Traite, p. 854) recom- 
mends one or the other of his two gold processes. The 
bladder of frogs should be carefully distended by injection of 
the lemon juice or gold chloride and formic acid through the 
cloaca. 
See also the method followed by Berxheim, Arch. f. Anat. 
u. Physiol., Physiol. Abth., 1892, supp., p. 29; Zeit. f. wiss. 
Mik., x. 4, 1893, p. 484; likewise a gold method. 
The methylen-blue method would appear to be very much 
indicated for this object, but has not been used for it so far as 
I am aware. CHAPTER XXXI. 
NEUROLOGICAL METHODS INTRODUCTION AND SECTION 
ME'L’HODS. 
699. Introduction.—The technique of the microscopic ana- 
tomy of the nervous system is highly special. The ordinary 
methods of microscopic anatomy are not sufficient to elucidate 
either the delicate and complicated structure of nervous 
elements, or their anatomical relations. These problems 
can only be successfully attacked by means of special methods 
of hardening, and the employment of highly specific methods 
of coloration. 
Histological research into the structure of the nervous 
system pursues two ends. Either it is desired to elucidate 
the minute structure of the nervous elements or neurons 
(neurites—Fish), that is to say, the internal organisation of 
nerve-cells and nerve-fibres : the processes employed to this 
end forming a group of cytological methods. Or it is desired 
to study the form of nerve-cells, the exact distribution of the 
divers groups of nerve-cells in the grey matter, the connec- 
tions that are formed by means of nerve-fibres between these 
groups of nerve-cells or “ nuclei,” and to follow out the intri- 
cate course of the tracts of fibres that enter into the constitu- 
tion of the white matter of the cerebro-spinal axis. The pro- 
cesses employed in all these researches form a group of the 
anatomical methods of neurology. It is more especially in 
this group that we find highly special methods of selective 
coloration. This group may be divided as follows. 
A. Nerve-fibres. 
(a) Myelin stains; comprising the methods of Weigert, 
and similar methods. 
(b) Axis-cylinder stains, and axis-cylinder and myelin 
stains. 386 
CHAPTER XXXI. 
B. Nerve-cells. 
(c) Axis-cylinder and protoplasm stains, comprising the 
methylen-blue method and some rather old-fashioned general 
stains. 
(d) Axis-cylinder and protoplasm impregnations, con- 
sisting chiefly of the methods of Golgi (the sublimate method 
and the three bichromate of silver methods), and the gold 
methods that have been given in previous chaptei’S. 
Nature acknowledges no absolute distinction between a 
central and a peripheral nervous system ; and as, moreover, 
the chief neurological methods are applicable both to the 
study of central and of peripheral nerve-tissue, it does not 
seem advisable to make a formal distinction of that sort here. 
The three following chapters wear the aspect of being devoted 
chiefly to the “ Central Nervous System ” simply because a 
large proportion of the methods used in the study of nerve- 
tissue in peripheral organs have already been extensively 
treated of in the chapters on “Methylen Blue,” on “ Im- 
pregnation Methods,” on “ Tegumentary Organs,” and on 
“ Muscle and Tendon.” The reader will kindly bear in mind 
that a considerable part of the subject properly compre- 
hended under the term “ Neurological Methods ” is contained 
in those chapters, which should be referred to in order to 
complete the account given in the following pages. 
The remainder of this chapter will be devoted to the special 
section methods employed for the central nervous a 
knowledge of which is a necessary preliminary to further 
study, and to the Cytological Methods of Neurology. Group A 
of the Anatomical Methods will be given in Chap. XXXII, 
and Group B in Chap. XXXIII. 
For more minute details concerning the dissection and hardening of the 
voluminous encephala of Man and the larger Vertebrates than can be given 
here see Mercier, Les Coupes du Systeme Nerveux Central (1894, Paris, 
Rueff); Dejerine, Anatomie des Centres Nerveux, 1895 ; Be van Lewis, 
The Human Brain; Histological and Coarse Methods of Research, 
London, Chui’chill; and Obersteiner, Anleitung heim Studium des 
Banes d. nervosen Centralorgane im gesunden u. hranken Zustande, 
Leipzig, Toeplitz. These very welcome additions to the literature of the 
subject relieve me from the obligation of treating the matter with all the 
minuteness that might be desired by specialists ; the more so as they show 
that so to treat it would require a volume, not a chapter. NEUROLOGICAL METHODS. 
387 
Section Methods. 
700. Fixation by Injection.—Fixation, in the proper sense 
of the word, is of course out of the question in the case of 
the human subject. But in the case of the lower animals 
it is possible to introduce fixing liquids into the living nerve- 
centres by means of injection, thus ensuring a much more 
rapid penetration of the reagents than can be obtained by 
simple immersion. This method was, I believe, first suggested 
by Golgi (Arch. Ital. de Biologie, t. vii, p. 30). He injected 
2’5 per cent, solution of bichromate of potash, through the 
carotid if he wished to limit the hardening to the encephalon, 
or through the aorta if he desired to fix the spinal cord. 
De Queevain (Virchow’s Archiv, cxxxiii, 1893, p. 489 ; Zeit.f. 
iviss. Mik., x, 4,1893, p. 507) proceeds as follows with dogs or 
cats. The subject, lightly chloroformed, is fixed securely on the 
operating table. A carotid is laid bare and cut through, and a 
canula fitted with a stopcock is tied both into the central and the 
distal portion. The cock of the central portion is opened first, 
and blood is allowed to flow out till no more comes away. Then 
liquid of Muller, at the temperature of the body, is injected 
under a moderate pi*essure through the peripheral canula 
(therefore towards the brain). This kills the subject at once 
and begins the fixation. Then more liquid of Muller is in- 
jected alternately through the central canula and the peri- 
pheral one, until a quantity has been injected equal to the 
quantity of blood that has been abstracted. For dogs 
300 c.c. to 600 c.c. will be required, for cats from one third 
to one half of that quantity. As soon as the injection has 
been made, ligatures are placed on the cut ends of the vessel; 
the encephalon is removed, and is put for some weeks into 
liquid of Muller kept at 37° 0. 
Mann (Zeit.f. iviss. Mile., xi, 4, 1894, p. 482) proceeds in a 
similar manner. He injects through the aorta. Before throw- 
ing in the fixing liquid, he injects for about twenty seconds 
physiological salt solution warmed to 39° 0. This washes out 
the capillaries, and prevents the blood from coagulating there. 
The fixing solution employed by him consists of saturated 
solution of corrosive sublimate, warmed to 39° C. After five 
minutes of injection the brain ought to be fixed throughout. 
It is removed and put for twelve hours into the same sub- 388 
CHAPTER XXXI. 
limate solution, after which it is either put for permanent 
preservation into O’l per cent, solution of sublimate, or is at 
once passed through alcohol for imbedding in paraffin. 
Strong (New York Acad, of Sci., January 13th, 1896; 
Anat. Anz., xi, 21, 1896, p. 655) advises injecting formalin 
diluted with an equal volume of water into the cephalic 
vessels until it runs from the cut jugulars. After a few 
minutes the same quantity is again injected, and once or twice 
again after a lapse of fifteen to twenty minutes. When only 
the Golgi method is to be used, an equal volume of 10 per 
cent, solution of potassium bichromate is to be taken instead 
of the water. I must say this treatment seems to me very 
heroic (see § 86). 
Hardening. 
701. Hardening by the Freezing Method.—This is in many cases 
a very good method, and in particular may be of service for the histological 
study of the cortex. 
If it be desired to freeze an organ that has been already hardened by 
reagents, the freezing may be done by means of a freezing mixture of ice 
and salt; but in this case the preparation should first be penetrated by a 
mucilaginous or gelatinous freezing mass (§ 177, et seq.), in order to avoid 
the formation of ice crystals in the tissues. But in the case of fresh tissue 
the ether freezing method is to he preferred. This method allows of rapidly 
producing any desired degrees of hardness, and maintaining them or allow- 
ing them to diminish as occasion may require, and is perhaps the only 
method by which satisfactory sections of unhardened nerve-tissue can be 
obtained. 
The sections should be floated on to water, treated for a minute on the 
slide with 025 per cent, osmic acid solution, and stained or otherwise treated 
as desired. 
For a detailed description of these manipulations see Bevan Lewis’s The 
Unman Brain. 
702. Generalities on Hardening by Reagents.—If large pieces 
of nerve-tissue are to be hardened, it is necessary to take 
special precautions in order to prevent them from becoming 
deformed by their own weight during the process. Spinal 
cord or small specimens of any region of the encephalon may 
be cut into slices of a few millimetres thickness, laid out on 
cotton wool, and brought on the wool into a vessel in which 
they may have the hardening liquid poured over them. The 
wool performs two functions : it forms an elastic cushion on NEUROLOGICAL METHODS. 
389 
which the preparations may lie without being' distorted by 
their own weight; and it allows the reagent to penetrate by 
the lower surfaces of the preparations as well as by their ex- 
posed surfaces. A further precaution, which is useful, is to 
hang up the preparations, lying on or in the cotton wool, in a 
glass cylinder or other tall vessel; by hanging them near the 
top of the liquid the processes of diffusion and the penetra- 
tion of the reagent are greatly facilitated. 
If the preparations are placed on the bottom of the vessel, 
they should never be placed one on another. 
If it be desired to harden voluminous organs without divid- 
ing them into portions, they should at least be incised as deeply 
as possible in the less important regions. It is perhaps better 
in general not to remove the membranes at first (except the 
dura mater), as they serve to give support to the tissues. The 
pia mater and arachnoid may be removed partially or entirely 
later on, when the hardening has already made some pro- 
gress. 
The spinal cord, the medulla oblongata, and the pons Varolii 
may be hardened in toto. The dura mater should be removed 
at once, and the preparation hung up in a cylinder-glass, 
with a weight attached to its lower end. The weight has the 
double function of preventing any part of the preparation 
from floating above the level of the hardening liquid (a thing 
that easily happens where somewhat dense liquids, such as 
Muller’s solution, are used), and of preventing the torsions of 
the tissues that may otherwise be brought about by the elastic 
fibres of the pia mater and arachnoid. 
The cerebrum should be very delicately laid out on a layer 
of cotton wool, or, if possible, hung up in it. Plugs of the 
wool should be put into the fissure of Sylvius, and as far as 
possible between the convolutions. Unless there are special 
reasons to the contrary, the brain should be divided into two 
symmetrical halves by a sagittal cut passing through the 
median plane of the corpus callosum. Betz recommends that 
after a few hours in the hardening liquid the pia mater should 
be removed wherever it is accessible, and the choroid plexuses 
also. I have found this by no means easy, and think it is 
an operation that can only be recommended for experienced 
hands. 
The cerebellum should be treated after the same manner. 390 
CHAPTER XXXI. 
The temperature at which the preparations are kept in the 
hardening solution is an important point. The hardening 
action of most solutions is greatly enhanced by heat. Thus 
Weigert (Gentrail, f. d. med. Wiss., 1882, p. 819 ; Zeit. f. wiss. 
Mik., 1884, p. 388) finds that at a temperature of from 30° to 
40° C. preparatious may be sufficiently hardened in solution 
of Muller in eight or ten days, and in solution of Erlicki in 
four days; whilst at the normal temperature two or three 
times as long would be required. 
But it is not certain that this rapid hardening always gives 
the best definite results. Sahli, who has made a detailed 
study of the hardening action of chrome salts, is of opinion 
that it does not, and thinks it ought for this reason to be 
abandoned (see Zeit. f. wiss. Mik., 1885, p. 3). 
On the other hand, the slowness of the action of chromic 
salts at the normal temperature is such that decomposition 
may easily be set up in the tissues before the hardening and 
preserving fluid has had time to do its work. For this reason 
voluminous preparations that are to be hardened in the slow 
way should be put away in a very cool place—best of all in 
an ice-safe. 
703. The Reagents to be employed.—The hardening agents 
most used are the chromic salts. Chromic acid was much 
used at one time, but most workers now agree that its action, 
though much more rapid than that of the salts, is much more 
uneven, and frequently causes a disastrous friability of the 
tissues. Osmic acid is excellent—according to Bevan Lewis 
it is the best of hardening agents; but its employment is un- 
fortunately very restricted, as it can hardly be used for objects 
of more than a cubic centimetre in size. 
It has already been noted that the liquid of Erlicki has a 
more rapid action than the other solutions of chromic salts; 
for this reason it is one of the most commonly employed 
solutions. Sahli, however (1. c.), after having studied the 
action of the usual solutions, concludes that the best harden- 
ing agent for fresh tissues is ■pure bichromate of potash, in 
3 or 4 per cent, solution, the hardening being done in a cold 
place. And he does not approve of the addition of sodium 
sulphate (Muller), and rejects the liquid of Erlicki on account 
of the precipitates it so frequently gives rise to (see § 96). NEUROLOGICAL METHODS. 
391 
Obersteiner is of the same opinion, recommending pure 
bichromate for general hardening purposes; whilst for the 
study of the most delicate structural relations he recommends 
fixing in Fobs modification of Flemming’s liquid (p. 32) for 
twenty-four hours, followed by washing with water and 
hardening in 80 per cent, alcohol. 
In view of the slowness of penetration of chromic salts it is 
often advisable to treat preparations for twenty-four hours or 
more with alcohol of 80 to 90 per cent, before putting them 
into the hardening liquid, in order to avoid maceration of the 
deeper layers of tissue. 
Two recent observers, Fish (The Wilder Quarter-Century Booh, 1893, 
p. 335) and Donaldson (Journ. of Morphol., ix, 1894, p. 123; Journ. Boy. 
Mic. Soc., 1894, p. 642), have made numerous determinations of weight and 
volume with the object of ascertaining what changes are produced by 
reagents in encephala of sheep. They have found that bichromate of 
potash produces a slight increase both of weight and volume, whereas all 
the other reagents tried produce a diminution of both these factors. 
Formaldehyde is a reagent that has been too recently introduced for it to 
be desirable to enunciate a definitive opinion concerning its fixative and 
hardening properties. I have quoted in §§ 86 and 109 some authors who 
have found it advantageous, especially in the form of mixtures with bichro- 
mate or other reagents, in the hardening of nervous tissue, and some other 
formulae of authors will be given further on. I have already stated (§ 86) 
that I find that used pure it does not faithfully preserve the minute structure 
of cells. There is no doubt that it is excellent for the preservation of 
specimens intended only for macroscopical study, but that is another thing. 
See further §§ 711 and 765 below. 
For the question as to how far certain so-called pathological alterations 
of ganglion-cells should be attributed to putrefractive changes or to the 
influence of reagents see Neurolocjisches Centralb. for the years 1884 and 
1885. 
As to the so-called “ pigment spots ” produced by the liquid of Erlicki 
see supra, § 96. 
704. Strengths of the Reagents.—All hardening reagents 
(except osmic acid) should at first be taken as weak as is con- 
sistent with the preservation of the tissue, and be changed by 
degrees for stronger. 
Osmic acid may be taken of 1 per cent, strength, and will 
harden small pieces of tissue sufficiently in five to ten days 
(Exner) . 
Bichromate of 'potash should be taken at first of not more 
than 2 per cent, strength; this is then gradually raised to 3 392 
CHAPTER XXXI. 
or 4 per cent, for the cord and cerebrum, and as much as 5 
per cent, for the cerebellum. Obersteiner begins with 1 per 
cent., and proceeds gradually during six to eight weeks to 2 
or 3 per cent. (This is at the normal temperature : at a tem- 
perature of 35° to 45° 0. the hardening can be got through 
in one or two weeks.) 
Bichromate of ammonia should be taken of half the strength 
recommended for bichromate of potash, or even weaker at 
first; it may be raised to as much as 5 per cent, for cerebellum 
towards the end of the hardening. 
Chromic acid is not much used alone (see § 88). It forms part of some 
of the mixtures mentioned below. A very little chromic acid (say one to 
two drops of 1 per cent, solution for each ounce) added to bichromate solu- 
tion will do no harm, and will quicken the hardening. 
Nitric acid has been and still is employed in strengths of 10 to 12 per 
cent., and gives particularly tough preparations. 
Neutral acetate of lead in 10 per cent, solution affords an excellent pre- 
servation of ganglion-cells, according to Anna Kotlabewski (see Zeit. f. 
wiss. Mile., iv, 3,1887, p. 387). 
Tezebinski (Virchow’s Arch., 1887, p. 1; Zeit.f. wiss. Mile., iv, 4, 1887, 
p. 497) finds that, as regards the faithful preservation of ganglion-cells (of 
the spinal cord of the rabbit and dog), the best results are obtained by 
hardening for eight days in 7 per cent, solution of corrosive sublimate, 
followed by hardening in alcohol containing 0'5 per cent, of iodine. 
Diomidoff (ibid., p. 499) also obtained very excellent results by harden- 
ing small pieces of brain (as suggested by G-aule, Ogata, and Bechteeeff) 
for from five to nine days (not more in any case) in 7 per cent, sublimate 
solution, and then putting the tissues for twenty-four hours into 50 per 
cent, alcohol, and for the same time into 70 per cent, and 96 per cent, 
alcohol successively. (This process produces artificial “ pigment spots,” 
similar to those produced by solution of Erlicki; they may be dissolved out 
by prolonged treatment with warm water, or in five minutes by strong 
solution of Lugol.) The tissues are of a good consistence for cutting. 
Chloride of zinc has been recommended for some purposes (see below, 
§§ 710,711). _ . . . * 
The next following paragraphs give in detail some methods of hardening 
recommended by some of the most competent workers. 
705. Betz’s Methods (Arch. f. mik. Anat., 1873, p. 101).—The 
spinal cord, medulla oblongata, and pons Varolii are treated as 
follows :—The dura mater is removed, and they are hung up 
in a cylinder containing 75 to 80 per cent, alcohol, to which is 
added enough iodine to produce a light brown coloration. 
After from one to three days the preparation will be found to 
be somewhat surface-hardened; it is taken down, and the pia NEUROLOGICAL METHODS. 
393 
mater and arachuoid are removed. If the pia mater does not 
come away completely enough the preparation is put back for 
some days into the alcoholic iodine. The membranes having- 
been removed, the preparation is put back into the original 
fluid, which is found to have become colourless owing to ab- 
sorption of the iodine by the tissues. Fresh quantities of a 
strong solution of iodine in alcohol are from time to time 
added to the liquid in order to keep it at its original strength 
of iodine (as shown by the colour). If the membranes have 
been carefully removed, it will be found that after about six 
days the preparation ceases to take up further quantities of 
iodine. The preliminary hardening may now be considered 
complete. 
The preparation is now bi-ought into a 3 per cent, solution 
of bichromate of potash. (A small weight is attached to it 
to prevent any portion of it from floating above the surface 
of the liquid. After a day or two it will have lost much of 
its alcohol, and will sink to the bottom of the vessel, which 
is equally undesirable; this must be watched for, and the 
preparation hung up or otherwise supported.) The vessel is 
put away in a cool 'place. As soon as a brown turbidity is 
seen in the liquid, together with a brown deposit on the pre- 
paration, the hardening may be considered to be complete. 
The preparation must be at once washed with water, and 
put away until wanted in a | to 1 per cent, solution of 
bichromate. 
Cerebellum.—Must be quite fresh, and before placing in 
the iodine the membranes and vessels must as far as possible 
be very carefully removed. (If the pia mater does not come 
away freely the organ must be macerated for a few hours in 
iodine solution in which other preparations have been kept, 
and which is diluted before usiug for this purpose.) The 
membranes having been removed, the cerebellum is placed 
(supported on cotton wool, with which the different organs 
are so propped up as to preserve their natural position) in 
solution of iodine for two or three days, and fresh iodine 
solution frequently added. 
The pia mater is now removed from the rest of the pre- 
paration, which is put back for seven to fourteen days into 
the iodine solution. If at the expiration of this time it be 
found that the cerebellum can be supported on the finger 394 
CHAPTER XXXI. 
by the vermiculus alone without bending, the preliminary 
hardening is complete, and it is brought into a 5 per cent, 
solution of bichromate, where it remains until fit for cutting. 
Cerebrum.—The cerebrum is divided into two halves along' 
the median line of the corpus callosum, and put into the 
iodine solution. After a few hours the pia mater is removed 
from the fissure of Sylvius and from the corpus callosum, and 
if possible the choroid plexus is removed likewise. 
The preparation is now put away in the iodine solution in 
a cool place (in summer in a cool cellar), and fresh iodine 
added as soon as the liquid is seen to lose colour (which must 
be watched for). After twenty-four to forty-eight hours the 
remaining pia mater is carefully removed by means of scissors 
and forceps from the fissures and convolutions, and one half- 
volume of fresh iodine solution is added to the liquid. (To 
facilitate the penetration of the liquid, wads of cotton wool are 
stuffed into the fissure of Sylvius and between the “operculum” 
and the central lobe \_“ Centrallappen”~], in the direction of 
the descending cornu, and between the convolutions.) After 
twenty-four to seventy-two hours the brain is brought into 
fresh solution of iodine in 70 per cent, alcohol, where it re- 
mains until the hemispheres are hard enough to be supported 
on two fingers without bending. (This will not be before ten 
to fourteen days.) It is then put into 4 per cent, solution of 
bichromate and left to acquire its definitive hardness. If an 
excessive brown deposit make its appearance, and the brain 
be found notwithstanding to be not hard enough for 
cutting, it must be rinsed with water and the bichromate 
solution changed. When ripe for cutting the brain ought to 
show an almost equal intensity of yellow-brown stain over 
the whole surface of a cut made through the total thickness 
of a hemisphere. 
Brains that are not fresh require for hardening longer time 
and stronger alcohol. 
The methods of Betz are particularly adapted to the harden- 
ing of voluminous specimens, and of tissues that are in a state 
of post-mortem softening. 
706. Cerebrum (Bevan Lewis, The Human Brain, p. 102).— 
Methylated spirit, twenty-four hours in a cool place. Muller’s 
solution, three days in a cool place. Then change the liquid; NEUROLOGICAL METHODS. 
395 
and after three days more change it again, or, preferably, 
substitute a 2 per cent, solution of potassium bichromate. 
At the end of the second week a solution of double the 
strength may be added; and if at the termination of the 
third week the mass is still pliable, and of the consistence of 
ordinary rubber, it as yet unfit for section cutting, and the 
reagent should be replaced by a solution of chromic acid. 
707. Brain (Hamilton, Journ. of Anat. and Physiol., 1878, 
p. 254).—Take a fresh brain and make a series of incisions 
into different parts, still keeping everything in situ ; or slice 
it into any number of segments about one inch thick, but of 
the whole length or breadth of the organ, as may be desired. 
Do not remove the membranes; they form a protection for 
the superficial layers, and do not interfere with the hardening 
process. The large segments are placed flat in a large vessel 
padded with cotton; do not put them one above the other. 
Cover them with the following fluid : 
Muller’s fluid ... 8 parts. 
Methylated spirit . . 1 part. 
(Heat is evolved on mixing these liquids, and the mixture 
must be allowed to cool before pouring it over the brain 
tissue.) Put the preparations away in an ice-safe. Turn the 
segments over next day. Change the solution in a fortnight 
or three weeks; or if on examining a section of one of the 
pieces it is found that the hardening reagent has penetrated 
to the interior, they may be at once removed to the following 
mixture: 
Bichromate of ammonia . . 1 grm., 
Water .... 400 c.c., 
in which they remain for one week. Then change the solu- 
tion to one of 1 per cent, for one week; and let this be 
followed by a solution of 2 per cent, for another week, or 
longer if required. The pieces will now be sufficiently hard 
for cutting; they may be kept permanently in solution of 
chloral hydrate, twelve grains to the ounce. 
Probably the chloral hydrate serves to attenuate the yellow 
coloration produced by the chromic liquids. 
This is a process particularly adapted to the preparation of 
large segments of brain. The consistence is very tough and 
firm. 396 
CHAPTER XXXI. 
708. Entire Encephalon (Deecke, Journ. Roy. J\fic. Soc., 1883, 
p. 449).—To harden the entire brain so that the inside and 
the outside shall be hardened equally and properly, Dr. Deecke 
finally adopted bichromate of ammonia in to 1 per cent, 
solution, according to the consistence of the brain. If it 
happens to be soft he adds say jr to T\j- per cent, of chromic 
acid to the solution, and always to j of the whole volume 
of alcohol. It is then placed in a refrigerator and the fluid 
changed frequently. After a month add a little more alcohol 
from week to week until the alcohol is 90 per cent. This is 
changed as often as it is discoloured. The treatment requires 
from twelve to eighteen months. 
709. Encephalon (M. Duval, Robin’s Journal de VAnatomie, 
1876, p. 497).—First Method.—Place the fresh tissues in 
solution of bichromate of potash 25, water 1000; change the 
liquid after the first twenty-four hours, and again after three 
or four days. After two or three weeks place the prepara- 
tions in chromic acid of 3 per 1000, change the liquid every 
day for the first week, and after that every eight days until 
the middle of the second month, after which time it is no 
longer needful to change the liquid. The preparations must 
remain at least two months in the chromic acid; the longer 
they remain in it the better. A few fragments of camphor 
should be added to the liquid in order to prevent the growth 
of mould. 
710. Encephalon (Fish, from an interesting paper on “ Brain 
Preservation ” in The Wilder Quarter-Century Book, 1893, p. 
393). The following is said to be not ideal in its effects, bnt to 
answer the requirements of economy, fixation of the struc- 
tural elements, differentiation of tissue, firmness of texture, 
and rapidity of action : 
Water ..... 400 c.c. 
95 per cent, alcohol . . 400 ,, 
Glycerin .... 250 ,, 
Zinc chloride .... 20 grms. 
Sodium chloride . . . 20 ,, 
Immerse in this, filling the cavities of the brain with it, and 
if practicable also injecting the blood-vessels with it, for NEUROLOGICAL METHODS. 
397 
about three days, then transfer for a week or more to a mix- 
ture of equal parts of the fluid and 70 per cent, alcohol, 
and finally store in 90 per cent, alcohol. 
711. Formaldehyde.—The liquids employed by Strong and 
Dorig are given below in § 765. See also § 109. 
Weigert (Beitr. zur Kenntn. d. normalen menschlichen 
Neuroglia, 1895, quoted from Neurol. Centralb., 1895, p. 1146) 
puts portions of material of not more than half a centimetre 
in thickness for four days into a 4 per cent, solution of formol 
(by which is presumably meant commercial formol diluted 
with 9 volumes of water). 
Marcus (quoted from Fish, see below) recommends harden- 
ing the spinal cord for two or four weeks in a | per cent, 
solution of formalin, then small pieces one half-centimetre 
thick are cut out and placed in Muller’s fluid for a week in 
an oven at 37° C. 
Van Gieson {Anat. Anz., x, 1895, p. 494) states that he 
obtained good results by using “ solutions of formalin of 4, 
6, and 10 per cent.,” followed by 95 per cent, alcohol. Myelin 
was found to be well preserved and to give the characteristic 
blue reaction with Weigert’s haematoxylin (the 1885 method), 
just as if a chrome salt were present. 
Lachi (cf. Zeit. f. wiss. Mih., xii, 1895, p. 32) states that he 
has had good results with “ 20 per cent, solutions of formol.” 
Fish (Proc. Amer. Mic. Soc., xvii, 1895, p. 319) recommends 
the following formula : 
Water .... 2000 c.c. 
Commercial formalin . . 50 ,, 
Sodium chloride . . .100 grms. 
Zinc chloride . . 15 ,, 
Brains should be left in this mixture for a week or ten days 
or more, then transferred to a 2’5 per cent, solution of 
formalin (water 2000 c.c., formalin 50 c.c.), in which they may 
remain indefinitely if the jar be kept tightly covered. After 
an immersion of two weeks in these solutions a human brain 
lost only 6'8 per cent, of its weight; but after an immersion 
for a similar period of eight days in 50 per cent, alcohol and 
eight days in 70 per cent, alcohol it was found to have lost 
22 per cent, of its first weight. 
Parker and Floyd {Anat. Anzeiger, Bd. xi, 1895, Vo. 5, 398 
CHAPTER XXXI. 
p. 156) find that a “ 2 per cent, solution of formol,” by which 
is meant a mixture of two volumes of formol with 98 of 
water, will harden a sheep’s brain in a week or ten days in a 
satisfactory manner as regards consistency, but with a marked 
increase of volume, which may amount to as much as 40 per 
cent. To obviate this they advise a mixture of— 
Alcohol 95 per cent. . . . 6 volumes, 
Formol 2 per cent, (the above mixture) 4 „ 
which has the same excellent and rapid hardening qualities 
and gives only a hardly perceptible increase of volume. 
Brains may be kept for months in the mixture (ibid., 1896, 
p. 568). 
712. Berkley, cortex of cerebellum of the dog (cf. Zeit.f. 
wiss. Mik., x, 3, 1893, p. 088). Liquid of Flemming, twenty- 
four to thirty-six hours ; absolute alcohol; celloidin. 
713. Nervous Centres of Reptiles and Amphibia (Mason, Central 
Nervous System of Certain Reptiles, &c. ; Whitman’s Methods, p. 196). 
Iodised alcohol, six to twelve hours; 3 per cent, bichromate, with a piece of 
camphor in the bottle, and to be changed once a fortnight until the harden- 
ing is sufficient (six to ten weeks). 
Burckhakdt (Das Centralnervensystem von Protopterus, Berlin, 1892 ; 
Zeit.f. unss. Mik., ix, 3, 1893, p. 347) recommends a liquid composed of 
300 parts of 1 per cent, chromic acid, 10 parts of 2 per cent, osmic acid, and 
10 parts of concentrated nitric acid, in which brains of Protopterus are 
hardened in twenty-four to forty-eight hours. 
Pish (Journ. of Morphol., x, 1, 1895, p. 234) employed for the encephalon 
of Desmognatlms fusca a mixture of 100 c.c. of 50 per cent, alcohol, 5 c.c. 
of glacial acetic acid, 5 grins, of corrosive sublimate, and 1 grm. of picric 
acid, fixing for twelve to twenty-four hours, and passing through the usual 
alcohols. 
Imbedding and Cutting. 
714. The Methods of Imbedding.—The paraffin infiltration 
method can only be used for the smaller objects of this class. 
Human spinal cord (which is quite at the upper limit as re- 
gards size) can be properly penetrated with paraffin by taking 
the precaution of first cutting it up into slices of not more 
than a few millimetres—preferably not more than one—in 
thickness. Ihe laigest objects of this class, such as entire 
hemispheres of man, cannot be properly penetrated by any 
known imbedding mass ; and the anatomist must be content 
with simple superficial imbedding—a proceeding which is NEUROLOGICAL METHODS. 
399 
here of the greatest service. For intermediate objects—those 
whose size varies between that of a small nut and a walnut— 
it appears to me that no hesitation as to the proper course is 
possible; such objects should be treated by the collodion 
method, which is at once the safest, the most convenient, and 
the most advantageous as regards the ulterior treatment of 
sections. 
Imbedding is not a necessary process. Sections can be 
obtained from any part of the central nervous system without 
imbedding. The material should be very well hardened, and 
a suitable piece should be glued on to a piece of wood or cork 
by means of a rather thick solution of gum arabic. As soon 
as it begins to stick to the support the whole is thrown into 
80 per cent, alcohol to harden the joint, after which it may 
be fixed in the object-holder of the microtome and cut. 
If the collodion method has been taken a difficulty may 
arise. It may be found that, notwithstanding every pre- 
caution, the collodion has not thoroughly penetrated the 
tissues. Good sections may, however, still be obtained by 
Duval’s method of collodionisiug the sections. The cut sur- 
face of the tissue is dried by blowing on it, and is covered 
with a thin layer of collodion laid on it with a brush. As 
soon as this layer has somewhat dried, which happens very 
rapidly, a section is cut, and the cut surface collodionised as 
before, and so on for each section. This process gives very 
good results, and may be advantageously employed even with 
material that has been successfully imbedded, as it gives a 
better consistency to the tissue, and enables thinner sections 
to be obtained (Van Gehuchten, in litt.). 
Beyax Lewis recommends the ether freezing method for fresh brain. 
For hardened brain he recommends some of the old-fashioned wax-and-oil 
and other fatty mixtures, a doctrine at which I am surprised. 
Hamilton recommends freezing in the gum and syrup mass given above, 
§ 178. He also recommends a method of penetrating with collodion, which 
is hardened in the usual way, the hardened mass being cut with an ice-and- 
salt freezing microtome (see Journ. of Anat. and Physiol., 1887, p. 444; or 
Journ. Boy. Mic. Soc., 1888, p. 1051). 
Goodall’s Rapid Method for preparing Spinal Cord (Brit. Med. Journ., 
May, 1898, p. 947; Journ. Boy. Mic. Soc., 1893, p. 405).—Cut sections of 
fresh tissue with a freezing microtome ; float them on to water, and as soon 
as possible drain them and float them on to pyridin. After a quarter of an 
hour wash in water; stain with 025 per cent, aqueous solution of anilin blue- 400 
CHAPTER XXXI. 
black, followed by picro-carmine; dehydrate and clear in pyridin; mount 
in balsam thinned with pyridin. See also ante, § 107. 
Benda (Centralb. f. Allgem. Path. u. path. Anat., vi, 1895, p. 803) finds 
there is great advantage in using formaldehyde for removing the alcohol 
from hardened tissues before freezing. Small pieces of material should he 
left for from a quarter of an hour to several hours in 1 per cent, formal- 
dehyde solution (2J per cent, of commercial formalin), and washed in dis- 
tilled water and frozen therein. The tissues after thawing are not found 
to be brittle, as is the case with those that have not been treated with 
formaldehyde, but have a peculiar tough soapy consistency. 
For sections of entire human brain, Deecke (1. c., § 708) proceeds as fol- 
lows :—The brain to be cut is placed upon the piston of the microtome (a 
Banvier model) and held in situ by several pieces of soft cork. It is then 
imbedded in a cast of paraffin, olive oil, and tallow, which, after it has 
become hard, is held in position by a number of small curved rods attached 
to, and projecting upwai'ds from, the piston to the height of about an inch. 
Before cutting, and as it proceeds, the cast is carefully removed from around 
the specimen to the depth of about half an inch (which is easily done by the 
use of a good-sized carpenter’s chisel), so that the knife never comes in con- 
tact with the cast. 
Cutting is done under alcohol, the entire microtome being immersed in a 
copper basin. The sections are floated, with the aid of a fine camel-hair 
brush, on to sheets of glazed writing-paper. They are removed thereon 
successively into staining, washing, and clearing fluids. After clearing, 
they are brought on the paper on to a slide, and the paper is gently pulled 
away from them ; they are then mounted in chloroform- or benzol-balsam. 
It should be noted that the membranes should not be removed from the 
brain; they present no obstacle to cutting if this is done with a slight 
sawing movement, or with a series of short cuts, instead of one sweep of 
the knife. By this plan the sections are much more perfect and uniform in 
thickness, and the loss in a series of from four to five hundred to the inch 
through the entire cerebrum of man may not amount to more than 2 or 3 
per cent. 
Osborn (Proc. Acad. Nat. Sci. Philadelphia, 1883, p. 178, and 1884, 
p. 262: Whitman’s Methods, p. 195) found advantage in employing Ruge’s 
egg-mass (for the brain of Urodela). He recommends that the mass be 
injected into the ventricles. 
The Dejerines (Anat. des Centres Nerveux, t. i, 1895, p. 29) have 
found advantage in the employment of both collodion and paraffin at the 
same time, for cutting entire hemispheres. The preparation is thoroughly 
impregnated with the collodion, which after hardening should form a wall 
of a couple of centimetres in thickness all around it. On the lower surface 
of this wall of collodion, incisions of about one centimetre in depth are made, 
running in various directions. The whole is then plunged into the cylinder 
of a Gludden microtome containing melted paraffin, and oriented according 
to the direction in which it is desired to make the sections. It is not neces- 
sary that the preparation should be entirely covered by the paraffin; it is 
sufficient that it should plunge therein for about one third of its depth. NEUROLOGICAL METHODS. 
401 
The paraffin penetrates into the cuts that have been made in the collodion, 
and after it has solidified is found to hold the preparation firmly. A very 
considerable quantity of paraffin is required for this operation, and as in 
drying it undergoes great shrinkage, it is well to wait for twenty-four hours 
before making the sections. 
Feist (Zeit. f. wiss. Mih., viii, 4, 1892, p. 492) gives a 
useful hint for marking the right and left sides of spinal cord. 
He imbeds with each segment of the cord a small cylinder 
(of about 1 square millimetre in section) of hardened liver, 
stuck vertically in the imbedding mass (either celloidin or 
paraffin) against the side of the cord that it is desired to 
mark. 
Cytological Methods. 
715. General Cytological Methods.—For the general prin- 
ciples of cytological research as applicable to nervous ele- 
ments the reader is referred to the chapter on “ Cytological 
Methods.” The following paragraphs are of a more special 
nature. 
(a) Nerve-cells. 
716. Nissl’s Fuchsin Method {Neurol. Gentralb., quoted 
from Mercier’s Coupes du Systeme Nerveux Central, 1894, 
p. 188).—Perfectly fresh material is to be cut into pieces of 
not more than one cubic centimetre in size. These are put 
for two days into a “ chromic solution in 70 per cent, alcohol ” 
(strength not stated). They are then transferred for five 
days to absolute alcohol, and may then be sectioned. 
The sections are brought into a saturated solution of fuchsin 
contained in a deep watch-glass, only one section being 
treated at a time. The watch-glass is then warmed over a 
flame, care being taken not to overheat the preparation, until 
vapours begin to be given off from the liquid. Then the 
section is removed and plunged for one to two minutes into 
absolute alcohol. It is then got on to a slide and covered 
abundantly with clove oil, in which it remains until no more 
colour is given off. It is then drained, washed once with 
fresh clove oil, drained and mopped up, and mounted in 
balsam. 
Nuclei and the chief protoplasmic processes are demon- 
strated, as also neuroglia cells, but not myelin. 402 
CHAPTER XXXI. 
717. Nissl’s Methylen-blue Method {Neurol. Gentralb., 1894, 
p. 508).—Fresh material is hardened in 96 per cent, alcohol, 
and sectioned. The sections are brought into a watch-glass 
with the following stain : 
Methylen blue (Methylenblau pat.) . 3'75 parts. 
Venice soap . . . 1‘75 ,, 
Distilled water . . . lOOO'O „ 
The watch-glass is warmed over a flame to about 65° to 70° C., 
till bubbles are given off which burst at the surface of the 
liquid. The sections are then brought into a mixture of ten 
parts of anilin oil with 90 parts of 96 per cent, alcohol, and 
are differentiated therein until colour is no longer given off 
from them. They are got on to a slide, dried with filter- 
paper, cleared with oil of cajeput, dried again with filter- 
paper, treated with a few drops of benzin, and mounted in 
benzin- colophonium. 
Professor van G-ehuchtbn, to whom I am indebted for this 
process, obtains the same results in a more commodious 
manner. Sections are cut by the paraffin method, and fixed 
on a slide by the distilled-water method (see § 186). The 
paraffin is removed by means of xylol, the slide is passed 
rapidly first through absolute alcohol, then through 80 
per cent, alcohol, and is put into a dish with NissFs staining 
mixture. The whole is put for five or six hours into a stove 
kept at about 35° to 40° C. Differentiation is performed as 
in Nissl’s process, and the preparation is mounted in xylol- 
damar. In this way a considerable series of sections can be 
treated at one time. 
718. Rosin’s Method (Neurol. Centralbl., xii, 1893, p. 1; Zeit.f. wiss. 
Mik., xii, 1, 1895, p. 77) consists in staining with the Ehrlich-Biondi 
mixture. 
719. The methods of Rehm (Miinchener med. Wochenschr., 1892, No. 13 ; 
Zeit.f. wiss. Mik., ix, 3, 1893, p. 390) are as follows:—Sections are stained 
for a few minutes in concentrated aqueous solution of Congo, washed in 
alcohol, and treated for ten minutes, until they become blue, with alcohol 
acidulated with hydrochloric or nitric acid, then cleared with origanum oil 
and mounted. A sharp axis-cylinder stain with considerable richness of 
other detail. 
But Rehm prefers the following simple process:—Sections of alcohol- 
hardened material are placed for one to two days in an aqueous solution of 
htematoxylin of 05 per cent, strength, then washed out in aqueous solution 
of carbonate of lithia (strength not given) until no more colour comes away NEUROLOGICAL METHODS. 
403 
from them, dehydrated and mounted. Axis-cylinders, cells, and processes, 
grey-black. The sections may be after-stained for a few minutes with OT 
per cent, aqueous solution of Bismarck brown. 
(h) Nerve Fibres. 
720. Structure of Medullated Nerve.—In order to demonstrate 
the axis-cylinder and the sheath of Schwann, the myelin may 
be removed. This may be done by boiling in caustic spda, 
and then neutralising; by boiling in a mixture of absolute 
alcohol and ether, and adding caustic soda; by boiling in 
glacial acetic acid; by boiling in fuming nitric acid, and 
adding caustic potash; or by treating with eau de Javeile; 
or (van Gehuchten, in litt.) the myelin may be extracted in 
the cold by leaving the nerves for some time in a mixture of 
alcohol and ether. 
721. Kuppfer’s Method (Sitzb. math. phys. Kl. h. Bayr. Ahad. TFiss., 
1884, p. 446; Zeit.f. iviss. Mih., 1885, p. 106).—A nerve is stretched on a 
cork and treated for twenty-four hours with 0‘5 per cent, osmic acid. It is 
then washed in water for two hours and stained for twenty-four to twenty- 
eight hours in saturated aqueous solution of Saurefuchsin ; after which it is 
washed out for from six to twelve hours (not more in any case) in absolute 
alcohol, cleared in clove oil, imbedded in paraffin, and cut. Sections are said 
to show the axis-cylinder as a bundle of fibrils (stained red) floating in an 
albuminous liquid. 
722. Neuroceratin Structures (Galli, Zeit.f. wiss, Mih., iii, 1,1886, 
p. 467).—An ischiatic nerve is excised, and fixed and hardened for eighteen 
to twenty minutes in solution of Muller. Small portions of the nerve are 
then further treated for one or two days with solution of Muller diluted 
with 2 parts of water, then for a quarter of an hour with glycerin contain- 
ing 1 or 2 drops of glacial acetic acid for each cubic centimetre, and finally 
(without previous washing with water) are stained for fifteen to twenty 
minutes in aqueous solution of China blue (the best China blue for this pur- 
pose is that supplied by the Badische Anilin- und Soda-Fabrik, at Stuttgart). 
They are washed out in alcohol, cleared in essence of turpentine, and 
mounted in dammar. 
For the results see the paper and the plate, 1. c. 
Platnek (Zeit.f. wiss. Mik., vi, 2, 1889, p. 186) obtains a specific stain 
of the neuroceratin framework of medullated nerve in the following way:— 
Small nerves are fixed and hardened for several days in a mixture of 1 part 
of Liq. Ferri Perchlor. (Ph. G., ed. 2) and 3 to 4 parts of water or alcohol. 
They are then to be washed out in water or alcohol till no traces of iron 
remain in them (the reaction of the washings with rhodanide of potassium 
being a good test of this), and are stained for several days or weeks in a con- 
centrated solution of “Echtgrtin” (dinitrosoresorcin, not “ dinitroresorcin,” 404 
CHAPTER XXXI. 
as erroneously in Platner’s paper) in 75 per cent, alcohol; after which they 
are dehydrated, imbedded, and sectioned. 
See also the papers of Gedoelst in La Cellule, iii, 1887, p. 117, and v, 
1889, p. 126 (good details of digestion methods); also the report in Zeit. f. 
wiss. Mik., vii, 1, 1890, p. 57. 
723. Other Methods.—Ranvier, Traite, p. 718, et. seq.; Rezzonico, 
Arch, per le Sci. Med., 1879, p. 237 ; Tizzoni, ibid., 1878, p. 4 (a process 
of boiling in chloroform for an hour or two, then staining and mounting in 
glycerin); Boveri, Zeit. f. wiss. Mik., iv, 1, 1887, p. 91; Jakimovitch, 
Journ. de VAnat., xxiii, 1888, p. 142, or Zeit. f. wiss. Mik., v, 4, 1888, 
p. 526 (instructions for impregnating the axis-cylinder with silver, followed 
by reduction in formic acid and amyl alcohol) ; Schiefferdecker, in 
Behrens, Kossel, u. Schiefferdecker, Das Mikroskop, Bd. ii, p. 227; 
Huber, Zeit. f. wiss. Mik., x, 3, 1893, p. 394 (stains with Benda’s safranin 
and Lichtgriin) ; Rabl, ibid., xi, 1, 1894, p. 42 (the lines of Frommann 
are artefacts due to the silver nitrate); Fischee, ibid., p. 48 (similar con- 
clusion) ; Tirelli, ibid., xi, 3, 1894, p. 391; Segall, Journ. de VAnat., 
xxix, 1893, p. 586. CHAPTER XXXII. 
NEUROLOGICAL METHODS—NERVE-FIERE STAINS (WEIGERT 
AND OTHERS). 
a. Myelin Stains. 
724. Introduction.—The most important of the methods for 
the study of tracts of medullated nerve-fibres are the haema- 
toxylin methods of Weigert. 
There have been in all three methods of Weigert; the 1884 
method, the 1885 method, and the 1891 method. The ordi- 
nary methods of staining with haematoxylin depend on the 
employment of an aluminium lake of haematoxylin. Weigert’s 
method depends on the formation of another lake, a chromium 
or copper lake. In consequence of the formation of these 
lakes haematoxylin acquires the property of staining the 
myelin of nerves in a quite specific way. 
In Weigert’s process the formation of these lakes takes 
place in the tissue itself. The details of the process have 
been considerably modified, both by other workers and by 
Weigert himself. The 1884 method (Fortschr. d. Med., 1884, 
pp. 113, 190; Zeit. f. wiss. Mik., 1884, pp. 290, 564), which 
depends on the formation of a chrome lake, may be considered 
to be superseded. Not so the two others, which depend on 
the formation of a copper lake. 
725. Weigert’s 1885 Method (Fortschr. cl. Med., 1885, p. 
136; Zeit. f. wise. Mik., 1885, pp. 399, 484).—The tissues are 
to be hardened in bichromate of potash (the solutions of 
Muller or Erlicki will do as well, so far as I know). The 
hardening need only be carried to the point at which the 
tissues have acquired a brown, not a green coloration (but 
green tissues may be used, provided they have once passed 
through the brown stage). The preparation is then (but this 
is not necessary) imbedded by infiltration with celloidin, 
and the celloidin block fastened on cork and hardened in CHAPTER XXXII. 
the usual way. The hardened block is put for one or two 
days into saturated solution of neutral acetate of copper 
diluted with one volume of water, the whole being kept at the 
temperature of an incubating stove. By this treatment the 
tissues become green, and the celloidin bluish green. The 
mordantage of the tissues is now terminated, and the pre- 
paration may be kept, till wanted for sectioning, in 80 per 
cent, alcohol. 
Sections are made with a knife wetted with alcohol, and 
are brought into a stain composed of— 
Hasmatoxylin . . . . 0*75 to 1 part. 
Alcohol . . . . . .10 parts. 
Water 90 „ 
Saturated solution of lithium car- 
bonate 1 part. 
(A trace of any other alkali may be added in the place of lithium carbon- 
ate. The object of adding a little of some base is to “ ripen ” the hasma- 
toxylin solution.) 
The sections remain in the stain for a length of time that 
varies according to the nature of the tissues :—Spinal cord, 
two hours; medullary layers of brain, two hours; cortical 
layers, twenty-four hours. 
They are then rinsed with water, and brought into a de- 
colourising solution composed of— 
Borax ...... 2-0 parts. 
Ferricyanide of potassium . . 2*5 ,, 
Water 200-0 „ 
They remain in the solution until they are decoloured to 
the right degree—that is, until complete differentiation of the 
nerves (half an hour to several hours)—and are then rinsed 
with water, dehydrated with alcohol, and mounted in balsam. 
They may be previously stained, if desired, with alum-carmine 
for the demonstration of nuclei. 
For very difficult objects, sucli as pathological nerves, the decolouring 
solution should he diluted with water, and the immersion in it prolonged. 
G-elpke (Zeit.f. wiss. Mik., 1885, p. 489) states that for transverse sections 
of atrophied nerves the solution should be diluted with fifty volumes of 
water, and the immersion be prolonged to twelve hours at the least; for 
longitudinal sections it should be diluted with ten volumes of water. 
The process is applicable to tissues that have been hardened in alcohol or 
in any other way, provided that they be put into a solution of a chromic NEIJKOLOGICAL METHODS. 
407 
salt until they become brown, before mordanting them in the copper solu- 
tion. 
As above stated, it is not necessary that the mordantage be done in bulk 
with tissues imbedded in celloidin. Max Flesch (Zeit.f. wiss. Mik., iii, 1, 
1886, p. 50) finds that this practice is unfavorable to subsequent staining 
with other reagents than hsematoxylin, and prefers (following Lichtheim) 
to make the sections first, bring them on closet-paper into the mordant, and 
after mordanting bring them on a spatula into 70 per cent, alcohol, and 
thence into the stain. 
In the process given above, a copper lake is formed in the tissues. In the 
earlier form of the pi’ocess the mordantage with the copper salt was omitted, 
and the stain depended on the formation in the tissues of a chromic lake. 
The results were not quite so good, and the process may he taken to be 
superseded by the copper process. 
If very many large sections have to he prepared, and if the staining solu- 
tion be thrown away after using, the process may be found somewhat 
expensive. The following method for regenerating the staining solution is 
given by Fanny Berlinerblau (Zeit.f. wiss. Mik., 1886, p. 50) :—About 
2'5 to 5 per cent, of baryta water is added to the used solution ; it is well 
shaken and allowed to stand for twenty-four hours ; carbonic acid (obtained 
from the action of crude hydrochloric acid on marble) is led through it, it is 
allowed to stand for twenty-four hours more, and then filtered. 
Paneth {ibid., 1887, p. 213) makes the stain with extract of logwood 
instead of pure hsematoxylin. One part of commercial extract of logwood is 
dissolved in 90 parts of water and 10 of alcohol. To the filtered solution is 
added 8 drops of concentrated solution of lithium carbonate for each 100 c.c. 
Sections require from eighteen to twenty-four hours in the stain at the 
normal temperature. 
Breglia (ibid., vii, 2, 1890, p. 236 ; see also Journ. Boy. Mic. Soc., 1890, 
p. 817) stains with liquid extract of logwood or Pernambuco wood, prepared 
by extracting 7 to 10 grms. of the wood for five or six days with 100 c.c. 
of alcohol of 90 to 95 per cent. 
The results obtained by Weigert’s method are most splendid. 
The blue-black nerves stand out with admirable boldness on 
a golden ground. The method is applicable to the study of 
peripheral nerves as well as to nerve-centres, and is likely to 
be of great utility in Vertebrate embryology. 
Nerve-tissue is not the only tissue stained by the process, 
which can be usefully applied to lymphatic glands and to 
skin (see Schiefferdecker, in Anat. Anz., ii, 1887, p. 680). 
726. Weigert’s 1891 Method (Deutsche mecl. Wochenschr., 
42, 1891, p. 1184; Zeit. f. wiss. Mik., viii, 3, 1891, p. 392).— 
The material is to be hardened in bichromate and imbedded 
in celloidin in the usual way. The hardened blocks of cel- 
loidin are brought into a mixture of equal parts of a cold 408 
CHAPTER XXXII. 
saturated solution of neutral acetate of copper and 10 per cent, 
aqueous solution of potassio-tartrate of sodium (C4H406KNa 
-f4H30, salt of Seignette). They are left in the mixture for 
twenty-four hours in an incubator. (Large specimens [Pons] 
will require forty-eight hours, the mixture being changed for 
fresh at the end of twenty-four hours.) They are then 
brought for twenty-four hours into aqueous solution of neutral 
acetate of copper, either saturated or diluted with 1 volume of 
water, being kept as before in the incubator. They are then 
rinsed with water and brought into 80 per cent, alcohol, in 
which they may either remain till wanted or be cut after half 
an hour. 
The sections are stained for from four to twenty-four hours 
at the temperature of the room in a freshly prepared mixture 
of 9 vols. of (a) a mixture of 7 c.c. of saturated aqueous solu- 
tion of carbonate of lithium with 93 c.c. of water, and 1 vol. 
of (b) a solution of 1 grm. of hmmatoxylin in 10 c.c. of alcohol 
(a and b maybe kept in stock, but a must not be too old). 
The sections must be loose ones, not such as have been seriated 
in celloidin, and must not be thicker than 0-025 mm. The 
stain is poured off and the sections are washed in several 
changes of water poured on to them. They are then treated 
with 90 per cent, alcohol followed by carbolic-acid-and-xylol 
mixture (for a short time only), or by a mixture of 2 parts of 
anilin oil with 1 of xylol, then pure xylol and xylol balsam 
(not chloroform balsam, which injures the stain). 
Medullated fibres dark blue on a light, sometimes rosy 
ground. If it be wished to have the ground particularly 
colourless, take instead of the second wash-water a mixture 
of I to | volume of common (not glacial) acetic acid with 
100 volumes of water. Thick sections or series in celloidin 
require a special differentiation. They may be differentiated 
either with the above-mentioned acetic acid mixture, or in 
the usual borax-ferricyanide mixture diluted with water. In 
the latter case the ground will be yellow. 
If the impregnation with the copper be imperfect (as, for 
instance, may happen if the treatment with the copper salt 
be performed at the normal temperature instead of in an in- 
cubator) some instructive differentiations of ganglion-cells 
may be obtained, the processes of the cells of Purkinje in the 
cerebellum, for instance, being very sharply brought out; NEUROLOGICAL METHODS. 
409 
but such preparations have a tendency to after-blackening, 
which does not happen with those that have been thoroughly 
impregnated with the copper. 
The advantages of the improved method are that differen- 
tiation after staining is not necessary; that the annoying 
precipitates formed on the surface of the preparations by the 
copper in the old method do not appear ; that the divers 
manipulations are simpler and easier; the preparations are 
equal in beauty to those of Pal, and can be obtained with 
greater certainty. 
Since the first publication of this method, it has been dis- 
covered (Weigekt, Ergebnisse der Anat., iii, 1894, p. 21) that 
preparations made as above, without differentiation in the 
ferricyanide liquid, do not leep well. Weigert therefore now 
advises that they be mordanted as above with salt of Seignette, 
which has the advantage of preventing the formation of pre- 
cipitates on the surface of the preparations, but that they be 
also differentiated in the ferricyanide, as in the 1885 method. 
Modifications of Weigert’s Method. 
727. Pal’s Method (Wien. med. Jahrb., 1886; Zeit. f. wi-ss. 
Mil., iv, 1, 1887, p. 92; Med. Jahrb., 1887, p. 589 ; Zeit. f. 
wiss. Mil., 1888, p. 88).—This is a chrome-lale process. You 
proceed at first as in Weigert’s process, but omitting the 
copper bath, and you stain as in Weigert’s process. After 
staining in the hsematoxylin solution the sections are washed 
in water (if they are not stained of a deep blue a trace of 
lithium carbonate must be added to the water). They are 
then brought for twenty to thirty seconds into 0 25 per cent, 
solution of permanganate of potash, rinsed in water, and 
brought into a decolouring solution composed of— 
Acid. Oxalic, pur. . . . 1*0 
Potassium Sulphite* (Kalium Sul- 
furosum [S03K2]) . . 1‘0 
Aq. Desfc. . . . . . 200-0 
In a few seconds the grey substance of the sections is de- 
colourised, the white matter remaining blue. The sections 
should now be well washed out, and may be double-stained 
* Not “ sulphide,” as erroneously given in Meeciee’s Les Coupes du 
Systeme Nerveux Centred, p. 190. 410 
CHAPTER XXXII. 
with Magdala red or eosin, or (better) with picro-carmine or 
acetic-acid-carmine. 
For further details as to the somewhat elaborate minutiae of 
the process see the papers quoted, or Behrens, Kossel, and 
Schiefferdeceer’s Das Mihroshop, i, p. 199. 
Pal’s process gives more brilliant results than that of 
Weigert, the ground of the preparations being totally colour- 
less. But it has a defect; it is less certain, or, to put it in 
another Avay, less easy to control. The differentiation is 
more energetic and rapid than is desirable. The whole pro- 
cess of differentiation only lasts some seconds; evidently, then, 
an error of judgment of only a feAV seconds may entirely 
vitiate the result. 
728. Kaiser’s Modification of Weigert (Neurol. Centralb., xii, 
1893, No. 11, pp. 364, 368; Zeit. f. wiss. Mih, xi, 2, 1894, 
p. 249; anterior methods suppressed).—Put the material into 
liquid of Muller. After two or three days divide it into slices 
of two to four millimetres in thickness, and put them back into 
the liquid of Muller for five or six days more. Then put them 
for eight days into liquid of Marchi (§ 733). Wash, pass 
through alcohol, and imbed in celloidin. Make sections, and 
mordant them for five minutes in the following mixture : 
Liquor Ferri Sesquichlorati . 1 part. 
Aq. Dest. . . . . . 1 ,, 
Spirit. Rect. . . . .3 parts. 
Wash them in Weigert’s hsematoxylin, then warm them in 
a fresh quantity of the same (not to boiling-point) for a few 
minutes. Wash with water and differentiate in Pal’s liquid. 
Neutralise the oxalic acid by washing in Avater containing a 
little ammonia. 
729. Kultschitzky (Anat. Anz., 1889, p. 223, and 1890, 
p. 519 ; Zeit. f. wiss. Mih., vi, 2, 1889, p. 196, and vii, 3, 1890, 
p. 367) has given two forms of his well-known process, of 
which the following is the later :—Specimens are hardened 
for one or two months in solution of Erlicki, imbedded in 
celloidin or photoxylin, and cut. Sections are stained for 
from one to three hours, or as much as twenty-four, in a stain 
made by adding 1 grm. of haematoxylin dissolved in a little 
alcohol to 100 c.c. of 2 per cent, acetic acid. They are washed 
out in saturated solution of carbonate of lithium or sodium. NEUROLOGICAL METHODS. 
411 
By adding to the carbonate of lithium solution 10 per cent, 
of a 1 per cent, solution of red prussiate of potash, and de- 
colourising therein for two or three hours or more, a finer 
differentiation is obtained. After this the sections are well 
washed in water and mounted in balsam. 
730. AVolters {Zeit. f. wiss. Mile., vii, 4, 1891, p. 466) proceeds as Ivult- 
schitzky, except that he stains in a solution kept warm by placing it on the 
top of a stove kept at 45° C. for twenty-four hours, after which time the 
sections are dipped in solution of Muller, and differentiated by the method 
of Pal. 
K aes {ibid., viii, 3, 1891, p. 388; Neurol. Centrcdb., 1891, No. 15) modi- 
fies this by staining for as much as two or three days, and performing the 
differentiation several times over. It appears doubtful whether either of 
these modifications is an improvement. 
731. Berkley’s Rapid Method (Neurol. Centralb.,x\,9, 1892, 
p. 270 ; Zeit. f. wiss. Mih., x, 3, 1893, p. 370).—Slices of tissue 
of not more than two and a half millimetres in thickness are 
hardened for twenty-four to thirty hours in mixture of 
Flemming, at a temperature of 25° C. Without washing out, 
they are brought into absolute alcohol, which is changed 
twice during the first twenty-four hours. After sufficient 
hardening they are imbedded in celloidin and cut. After 
washing in water the sections are put overnight into a satu- 
rated solution of acetate of copper (or they may be simply 
warmed therein to 35° to 40° 0. for half an hour). They are 
then washed, and stained for fifteen to twenty minutes in the 
fluid given below, warmed to 40° C., allowed to cool, and 
differentiated for one to three minutes in Weigert’s ferri- 
cyanide liquid, which may be diluted if desired with one third 
of water. Water, alcohol, bergamot oil, xylol-balsam. 
The stain is made as follows :—Two cubic centimetres of 
saturated solution of carbonate of lithia are added to 50 c.c. 
of boiling water, and the solution boiled for two minutes more, 
when to 2 c.c. of 10 per cent, solution of haematoxylin in 
absolute alcohol are added. 
This method is most suited to fresh material, and does not 
give good results with tissues that have suffered post-mortem 
changes. 
732. Other Modifications or Similar Methods.—Flechsig {Arch, 
f. Anat. u. Phys., Phys. Abth., 1889, p. 537; Zeit.f. wiss. Mih.. vii, 1890, 
p. 71; Journ. Boy. Mic. Soc., 1890, p. 538; Breglia, Zeit., vii, 2, 1890, 412 
CHAPTER XXXII. 
p. 36; Rossi, ibid., vi, 2, 1889, p. 182 ; Mercier, ibid., vii, 4, 1891, p. 480 ; 
Haug, ibid., vii, 2, 1890, p. 153 ; Walsem, ibid., xi, 2, 1894, p. 236. 
Other Myelin Stains. 
733. Marchi’s Method for Degenerate Nerves (Rivista sperim. 
di Fren. e di Med. legale, 1887, p. 208 ; taken from the report 
by Schiefferdecker of a paper by Eijkmax, in Zeit.f. wiss. Mik., 
ix, 3, 1893, p. 350).—Nerves are first hardened for a week in 
solution of Muller, and then put for a few days into a mixture 
of 2 parts solution of Muller and 1 part 1 per cent, osmic 
acid solution. The treatment with the chrome salt deprives 
the medullary sheath of normal fibres of the faculty of im- 
pregnating with osmium, whilst the degeneration products 
in diseased sheaths retain that faculty. In consequence the 
sheaths in normal nerves acquire a yellow coloration, those of 
degenerated tracts a black one. 
For the study of degenerate nerve-tracts the method of 
Marchi has an advantage over that of Weigert, in that it 
gives positive images of the degenerated elements, Weigert’s 
process only giving negative ones. 
734. Azoulay’s Osmic Acid Method (Anat. Anz., x, 1, 1894, 
p. 25).—An application of the osmic acid stain given § 377. 
(a) Material that has been for several months in liquid of 
Muller is washed for a couple of days in water, imbedded in 
celloidin, and sectioned. The sections washed in water are 
put for five to fifteen minutes into solution of osmic acid of 
1 : 500 or 1 : 1000 strength. Rinse with water and put them 
for two to five minutes into a 5 or 10 per cent, solution of 
tannin, warming them therein over a flame till vapours are 
given off, or in a stove at 50° to 55° C. Wash for five 
minutes in ivater, double-stain if desired with carmine or 
eosin, and mount in balsam. Thin sections are necessary to 
ensure good results. If they should be too thick it will be 
necessary after staining to differentiate by Pal’s process, or 
by eau de Javelle diluted with 50 vols. of water. (b) 
Material that has been in an osmic mixture (liquid of Flem- 
ming, of Marchi, or of Golgi). Sections as before, then the 
tannin bath, warming for three to ten minutes, and the rest 
as before. 
735. Osmic Acid (Exner, Sitzb. Tc. AJcad. TEiss. Wien, 1881, lxxxiii, 
3 Abth. ; Bevast Lewis, The Human Brain, p. 105).—A small portion of NEUROLOGICAL METHODS. 
413 
brain, not exceeding a cubic centimetre in size, is placed in ten times its 
volume of 1 per cent, osmic acid. The solution should be replaced by fresh 
after two days, a proceeding which may advantageously be repeated at the 
end of the fourth day. In from five to ten days the piece is usually 
hardened throughout, and may be washed with water, treated with alcohol, 
and imbedded. The sections may be treated by a drop of caustic ammonia, 
which clears up the general mass of the brain substance, leaving uiedullated 
fibres black. B. Lewis says that this method exhibits a wealth of structure 
which no other method displays. The sections may be mounted in soluble 
glass. The chief value of this method is for tracing the course of medul- 
lated fibres. 
Bellonci (Arch. Ital. de Biol., vi, p. 405) employed this method in his 
researches on the optic nerve of mammalia. He used an osmic acid solution 
of 0‘5 to 1 per cent., hardened for only fourteen to twenty hours, made 
sections, and treated them for three or four hours with 80 per cent, alcohol, 
and then with ammonia. 
736. The methods of Paladin o (next section) and Ziehen (§ 780) are 
also more or less specific myelin stains. 
b. Myelin-and-axis-cylinder Stains. 
737. Paladino’s Iodide of Palladium Method (Rendic. R. 
Accad. Scienze Fis. e Mat., Napoli, iv, 1890, p. 14, and 1891 
[1892], p. 227; Zeit. f. wiss. Mih., vii, 2, 1890, p. 237, and ix, 
2, 1892, p. 238 ; Journ Roy. Mic. Soc., 1890, p. 817, and 1892, 
p. 439).—Pieces of material hardened in bichromate, chromic 
acid, or corrosive sublimate, and not more than 5 to 8 mm. in 
thickness, are put for two days into a large quantity (at least 
150 to 200 c.c. for each piece) of 0T per cent, solution of 
chloride of palladium (which may be made as directed in 
§ 66). They are next put for twenty-four hours into a 1 per 
cent, solution of iodide of potassium (the later paper quoted 
says the iodide solution should be of 4 : 100 strength, and 
that a relatively small volume of it should be taken; other- 
wise the iodide of palladium, which is rapidly formed in the 
tissues, may be again extracted by the liquid : small pieces of 
tissue should not remain in it for more than one or two hours). 
Dehydrate; imbed, if necessary, in paraffin by the chloroform 
method; mount in balsam. 
A brown stain, being both a myelin stain and an axis- 
cylinder and cell-process stain, and applicable both to central 
and to peripheral nervous structures. It is very well spoken of. 
738. Sahli (Zeit.f. wiss. Mih., 1885, p. 1) gives the following method: 
—Sections of tissue hardened in bichromate to the degree required for 414 
CHAPTER XXXII. 
Weigert’s hsematoxylin process are washed for not more than five or ten 
minutes in water, and stained for several hours, until they are of a dark blue 
colour, in concentrated aqueous solution of methylen blue. They are then 
rinsed with water, and stained for five minutes in saturated aqueous solution 
of Saurefuchsin. If now they be rinsed with alcohol and brought into a 
liberal quantity of water, the stain becomes differentiated, axis-cylinders 
being shown coloured red and the myelin sheaths blue. If, instead of rins- 
ing with pure alcohol, alcohol containing from O'l to 1 per cent, of caustic 
potash be taken, the stain differentiated in water, and the sections cleared 
with cedar oil and mounted in balsam dissolved in cedar oil, still finer images 
are obtained. Axis-cylinders are red as before, but the myelin sheaths are 
blue in some places, red in others. Sahli thinks that this reaction points to 
some difference of kind in the nerve-tubes that exhibit it. 
The same author (1. c., p. 50) also gives a method for obtaining a specific 
stain of nerve-tubes by means of methylen blue alone. Sections of material 
hardened as before are stained for a few minutes or hours in the following 
liquid: 
AVater ........ 40 parts. 
Saturated aqueous solution of methylen blue . 24 „ 
5 per cent, solution of borax . . . 16 „ 
(Mix, let stand a day, and filter.) 
The sections are then washed either in water or alcohol until the grey 
matter can be clearly distinguished from the white, are cleared with cedar 
oil, and mounted in balsam. Nerve-tubes are stained blue, ganglion-cells 
greenish, nuclei of neuroglia blue. Micrococci are stained, if any be present 
in the tissues. The preparations are not perfectly permanent. 
739. Safranin followed by methylen blue gives a very special stain of 
spinal cord. The method is due to Adamkiewics (Sitzb. Ic. Akad. Wiss. 
Wien. Math. Naturw. Kl., 1884, p. 245; Zeit.f. wiss. Mik., 1884, p. 587). 
Sections (of material hardened in liquid of Muller for not less than one 
month and not more than three) are washed first with water, then in water 
acidified with a little nitric acid, and stained in concentrated solution of 
safranin. They are then treated with alcohol and clove oil till no more 
colour comes away, and are brought back again into water, washed in water 
acidified with acetic acid, stained in methylen blue, and cleared as before. 
Myelin (“ erythrophilous substance” of Adamkiewics) is red, nuclei of 
nerves, of neuroglia, and of vessels violet. The erythrophilous substance 
of pathological nerve-tubes does not take the stain, so that the method is 
valuable for the study of degenerative changes. 
740. Nikiforow (Zeit.f. iviss. Mik., v, 3, 1888, p. 338) has a modifica- 
tion of the foregoing method, which consists in impregnating with gold 
chloride or other metallic salt after the safranin stain. 
741. Similar to the methods of Adamkiewics are those of Ciaglinski 
(.Zeit.f. iviss. Mik., viii, 1, 1891, p. 19) and of Stkoebe (ibid., x, 3, 1893, 
p. 336), both of them employing safranin followed by anilin blue. For 
Nissl’s Congo-red method, see Munchener med. Wochensclir., 1886, p. 528, 
or Zeit.f. iviss. Mik., iii, 3, 1886, p. 398. CHAPTER XXXIII 
NEUROLOGICAL METHODS, AXIS-CYLINDER AND PROTOPLASM 
STAINS (GOLGI AND OTHERsh 
742. Introduction.—There are three chief methods for the 
study of axis-cylinders and protoplasmic nerve-cell processes, 
viz. the methylen blue method, the sublimate method of 
Golgi, and the bichromate-of-silver method of Golgi. The 
methylen blue method may be considered to be a staining 
method in the proper sense of the word, whereas the methods 
of Golgi are true impregnation methods. The methylen blue 
method having been given in Chap. XVII, it remains to 
group together here some other subordinate but useful 
methods that are also stains proper; after which will be 
given the methods of Golgi and some other impregnation 
methods. 
c. Stains Proper. 
743. Ammonia-carmine is old-fashioned, but may be used for general 
views. Beale’s formula is a good one, especially where prolonged staining 
is required. The secret of success lies in staining very slowly in extremely 
dilute solutions. Bichromate material ought to be brought direct into the 
stain without passing through alcohol (see § 94). 
Picro-carmine has much the same action as ammonia-carmine, but gives 
a better demonstration of non-nervous elements. 
Chromic objects stain very slowly in both these media. Sections may, 
however, be stained with them in a few minutes if they be put into a watch- 
glass with the stain, and the whole be kept on a wire net over a water-bath 
heated to boiling-point (Obersteiner). 
Henle (Handb. cl. Nervenlehre, 1871) gives the following, after Merkel. 
In order to do away with the slowness of staining of tissues hardened in 
chromates, sections should be placed in solution of chloride of palladium 
(1 in 300 to 1 in 600) till they are of a straw-colour (one or two minutes), 
rinsed in water, and stained in strong ammonia-carmine. Myelin, yellow; 
axis-cylinders, nerve-cells, and neuroglia, deep red. 
Borax-carmine is chiefly useful when employed for double-staining with 
indigo-carmine or an anilin blue to follow. I have obtained some superb 
stains with Seiler’s borax-carmine and indigo-carmine process (§ 339). 
Merkel’s mixture of borax-carmine and indigo-carmine (§ 340) has been 416 
CHAPTEE XXXIII. 
strongly recommended by Max Flesch {Zeit. f. wiss. Mik., 1884, p. 566, 
and 1885, p. 349), who says that it gives extremely rich and instructive 
images. 
Alum-carmine (Grenadier’s or Csokor’s) may be used as a nuclear stain 
(Obersteiner). The stain principally takes effect on non-nervous nuclei. 
See also Schmaus {Munch, med. Wochensclir., 1891, No. 8; see Zeit.f. 
wiss. Mik., viii, 2, 1891, p. 230, and Journ. Boy. Mic. Soc., 1892, p. 439); 
Upson (Neurolog. Central!)., 1888, p. 319; Zeit.f. iviss. Mik., v, 4, 1888, 
p. 525); Freeborn {Amer. Mon. Mic. Journ., 1888, p. 231; Journ. Boy. 
Mic. Soc., 1889, p. 305). 
744. Anilin blue-black was first recommended by Sankey 
(Quart. Journ. Mic. Sci., 1876, p. 69). He stained in a 05 
per cent, solution, and, in order to obtain a differential stain, 
washed out for twenty to thirty minutes in solution of chloral 
hydrate. Be van Lewis (Human Brain, p. 125) considers this 
to be one of the most valuable stains for nervous centres. He 
stains sections for an hour in 025 per cent, aqueous solution, 
and clears and mounts (in the case of brain or cord sections); 
for the cortex of the cerebellum he washes out for twenty to 
thirty minutes in 2 per cent, chloral solution. Sankey and 
Stirling have also used anilin blue-black in a much weaker 
solution, which Bevan Lewis does not recommend. Vejas, 
however (Arch. f. Psychiatrie, xvi, p. 200), obtained good 
results by staining from eighteen to twenty-four hours in a 
solution of 1 in 3000. 
Gierke (Zeit. f. wiss. Mik., 1884, p. 379) was not able to 
obtain good results with anilin black procured in Germany, 
and finds that the treatment with chloral is injurious to the 
preservation of the tissues. Martinotti (ibid., p. 478) comes 
to the same conclusion. 
Luys (Gaz. med. de Paris, 1876, p. 346) greatly recommends 
the anilin colour known as Noir Colin. He stains for three 
to four minutes in a 0T per cent, solution. 
Jelgersma (Zeit.f. wiss. Mik., 1886, p. 39) finds that anilin 
bine-black gives excellent results ‘provided that the English 
preparation of the colour be alone employed. He makes solu- 
tions of 1 : 100, 1 : 800, and 1 : 2000, of which the first stains 
sections in a quarter of an hour, the second in five hours, 
the third in twelve hours. The stain takes effect on ganglion- 
cells and their processes, and on axis-cylinders, but does not 
demonstrate neuroglia or connective tissue. 
Schmaus (Munch, med. Wochensclir., No. 8, 1891, p. 147; NEUROLOGICAL METHODS. 
417 
Zeit.f. iviss. Mih., viii, 2, 1891, p. 280) recommends English 
blue-black in 0'25 per cent, solution in 50 per cent, alcohol, 
with the addition of a little picric acid; sections to be stained 
for an hour. The addition of picric acid has the advantage 
of leaving celloidin almost colourless, whilst pure aqueous 
solutions of blue-black stain it strongly. 
745. Marttstotti (1. c., 1884, p. 478) finds that picro-nigrosin 
gives very good results, especially for pathological objects. 
He stains for two or three hours or days in a saturated 
solution of nigrosin in saturated solution of picric acid in 
alcohol, and washes out in a mixture of 1 part of formic 
acid with 2 parts of alcohol until the grey matter appears 
clearly differentiated from the white to the naked eye. 
746. Rosin (Neurol. Centralb., xii, 1893, p. 1; Zeit.f. wiss. Mih., xii, 
1, 1895, p. 77) recommends Ehrlich-Biondi mixture. Lindsay-Johnson 
(in litt.) adds to it about one third of a 20 per cent, (saturated) solution of 
nigrosin. 
747. Kaiser (Zeit.f. wiss. Mih., vi, 4,1889, p. 471) advises, for celloidin 
sections of spinal cord, naphthylamin brown (obtainable from Griibler)* 
Sections are stained for a few hours in a solution containing 1 part of 
naphthylamin brown, 200 parts of water, and 100 parts of alcohol, washed 
with alcohol, cleared with origanum oil, and mounted. Chromophilous 
ganglion-cells, dark brown; chromophobous cells, light on a dark ground. 
748. Rehm (Munch, med. Wochenschr., 1892, No. 13; Zeit.f. wiss. Mile., 
ix, 3, 1893, p. 389) gives the following:—Sections (of alcohol-hardened 
material) stained for five minutes in 1 per cent, ammonia-carmine, and 
washed out in 70 per cent, alcohol acidified with 1 per cent, of nitric acid; 
the acid removed by pure alcohol; the sections stained for half a minute 
in O'l per cent, solution of methylen blue, differentiated in alcohol, cleared 
in origanum oil, and mounted in colophonium. Nuclei and axis-cylinders, 
red. This stain is said to prove that the nuclei of the ganglion-cells of the 
rabbit contain only one true nucleolus, instead of two or more, as has been 
hitherto believed. 
749. For a plasma-stain, Rehm (1. c.) gives a method modified from 
Nissl. Sections of alcohol-hardened material are stained for half a minute 
to a minute in a hot O'l per cent, solution of methylen blue, washed in 96 
per cent, alcohol till no more colour comes away, cleared with origanum oil, 
and mounted in balsam or benzin-colophonium. Nerve-cells, dark blue; 
connective-tissue cells lighter, and greenish. 
750. To obtain a sharper distinction between nerve-cells and connective- 
tissue cells, Rehm (1. c.) stains as before in the hot methylen-blue solution 418 
CHAPTER XXXIII. 
for not more than half a minute, and washes out as before with 96 per cent, 
alcohol. The sections are then stained for fifteen to thirty minutes in a OT 
per cent, solution of fuchsin in 96 per cent, alcohol, washed out fora minute, 
until no more red colour comes away, in alcohol, cleared in clove oil, and 
mounted. Nerve-cells blue-red, their nuclei being unstained; nuclei of 
connective tissue and of vessels, brilliant red. 
Nuclei of connective tissue and vessels may also be brought out by stain- 
ing for a few minutes in 1 per cent, aqueous solution of eosin, followed by 
a few minutes in warm 0T per cent, aqueous solution of dahlia, dehydration, 
and mounting (the nuclei blue, all else red). Or 1 per cent, aqueous solu- 
tion of nigrosin may he taken instead of the eosin, and OT per cent, alcoholic 
solution of fuchsin (half an hour) instead of the dahlia (nuclei red, all else 
grey-blue). 
This distinction is not obtained in embryonic tissues. 
751. For Mallory’s Phosplio-molybdic Acid Hsematoxylin Stain, see 
Anat. Anz., 1891, p. 375 ; Zeit.f. wiss. Mile., viii, 3, 1891, p. 341; Mercier, 
Les Coupes du Systeme nerveux Central, p. 228. Schiefferdeckee and 
Vobis find that celloidin sections of material hardened in Muller stain well 
in it. 
752. Wolters’s Chloride of Vanadium process for axis- 
cylinder and cell-staining is as follows (Zeit. f. wiss. Mih., 
vii, 4, 1891, p. 471): 
The material (either central or peripheral nervous tissue) 
is hardened in liquid o/Kultschitzky, § 53, followed by alcohol, 
as there described. It is imbedded either in celloidin or 
paraffin, and cut. The sections are mordanted for twenty- 
four hours in a mixture of 2 parts of 10 per cent, solution of 
chloride of vanadium and 3 parts of 3 per cent, solution of 
acetate of aluminium, washed for ten minutes in water, and 
stained for twenty-four hours in a solution of 2 grammes of 
ho3matoxylin (dissolved in a little alcohol) in 100 c.c. of 2 per 
cent, acetic acid. They are washed out until they are of a 
light blue-red colour (it is not possible to specify exactly the 
time required) in 80 per cent, alcohol acidulated with 05 per 
cent, of hydrochloric acid. Kemove the acid thoroughly by 
washing with pure alcohol, dehydrate, clear with origanum 
oil, and mount. 
A sharp axis-cylinder stain, myelin being coloured only if 
the differentiation in the acid alcohol is insufficient. 
753. Alt (Munch, tried. Wochenschr., 1892, No. 4; Zeit.f. vriss. Mile.,. 
ix, 1, 1892, p. 81) stains for a couple of hours in solution of Congo in abso- 
lute alcohol, and washes out with pure alcohol. The results are said to be 
specially adapted to the study of peripheral axis-cylinders. Schieffer- NEUROLOGICAL METHODS. 
419 
decker, reporting on the method, does not recommend it. Squire (using 
a 2 per cent, aqueous solution) says it is one of the best stains he has tried. 
d. Impregnations 
754. The Methods of Golgi. There are two methods of 
Golgi, viz. the Corrosive Sublimate Method, and the Bichromate 
and Nitrate of Silver Method. The corrosive sublimate method 
will be given later on. 
The bichromate and nitrate of silver method has been 
worked out by Golgi in three forms. These are known as 
the slow process, the rapid process, and the mixed process.* 
The rapid process is the one that is the most in use at the 
present time for researches into the distribution and relations 
of axis-cylinders and protoplasmic processes; it may be taken 
to be the classical method of inquiry into the finer relations 
of the neurons in hardened tissue. It may be said that it is 
to the study of hardened tissue that which the methylen blue 
method is to the study of fresh tissue. 
Before proceeding to describe tliese processes it is desir- 
able to indicate briefly the general characters of the impreg- 
nations obtained by them. The preparations have not in the 
least the appearance of stains, and are even very different in 
aspect from the impregnations obtained on fresh tissue by 
the ordinary methods of impregnating with nitrate of silver 
or chloride of gold. The impregnation is a partial one, by 
which is meant that of all the elements, whether nervous or 
not, that are present in a preparation, only a limited number 
are coloured. That is the peculiar quality—not by any 
means the defect, but rather the advantage—of the method. 
For if all the elements present were coloured equally with 
the great intensity with which they take the colour in this 
method, you would not be able to see the wood for the trees, 
in fact you would hardly be able to distinguish any detail at 
all in the preparations. But Golgi's method selects from 
* In a recent text-book, the Leitfaden of Rawitz, the sublimate method 
is called “the slow method of Golgi,” and the bichromate and silver nitrate 
method is given under the form of the slow process, and called “ the rapid 
method of Golgi.” That is a very “ nice derangement of epitaphs” indeed. 
Rawitz further attributes the rapid method to Ramon y Cajal, which is 
equally erroneous. Similar confusions are made by Meecier in his Coupes 
du Sy8t'eme Nerveux Central. 420 
CHAPTER XXXITI. 
among the elements present a small number which it stains 
with a great intensity and very completely, that is to say, 
throughout a great length, so that they are at the same time 
very clearly separated from those elements that have remained 
uncoloured, such as supporting cells and the like, and can be 
followed out for a great distance. 
There is no other method which will allow cell-processes to 
be followed out for such great distances. But the method 
does not demonstrate at the same time the histological detail 
of other tissues that may be present in the preparations, 
and all cytological detail is lost. It is par excellence a special 
method. 
Nervous tissue is not the only thing that is impregnated 
in these preparations; indeed, the method has been applied 
with success to the study of such things as bile-capillaries, 
gland-ducts, and the like. Both on account of this character, 
and on account of the capriciousness with which the impreg- 
nation takes hold of only certain elements of the preparations, 
care must be exercised in the interpretation of the images 
obtained. It has been constantly taught in the chapter de- 
voted to gold impregnations in this book (see § 366) that “ the 
very best gold preparations give images that are only worthy 
of credence as to what they show, and furnish absolutely no 
evidence whatever as to the non-existence of anything that 
they do not show; for you can never be sure that the imbibi- 
tion of the salt has not capriciously failed, or its reduction 
capriciously stopped, at any point.” This warning applies 
with at least equal force to Golgi’s impregnations. And in 
their case a further source of error is found in the fact that 
the method frequently gives precipitation-forms of chromate 
of silver that simulate dendrites and other structures (see 
the paper of Frtedlaender in Zeit. f. wins. Mik., xii, 2, 1895, 
p. 168, and the plate in the following number). A corre- 
spondent writes me that he has “ Golgified a potato, and 
obtained beautiful nerve-fibres,” and Friedlaender’s paper 
describes similar results obtained with white of egg, &c. 
Cleaidy, then, caution is necessary in the interpretation of the 
images. 
It has been said that the method does not give good results 
with the tissues of invertebrates. It has, however, been 
applied with success by Ramon y Cajal to the study of the NEUROLOGICAL METHODS. 
421 
muscles of insects; by von Lenhossek to the nerves of 
Lumbricus; by Retzius to the retina of Cephalopods; and 
other anatomists have been successful with various objects. 
The details of the method have been considerably modified 
at the hands of various woi’kers, the most important modifica- 
tion being that of the “ double ” or “ intensified ” impreg- 
nation of Ramon y Cajal. 
The method has been described at length by Golgi in the 
Archives Italiennes de Biologie, t. iv, p. 32, et seq. The follow- 
ing account is from the paper by Golgi himself in the Archives 
de Biologie, t. vii, p. 15, et seq. The account given in former 
editions of this work represents an earlier form of the method, 
and should not be followed. 
755. Golgi’s Bichromate and Nitrate of Silver Method, 
SLOW Process (1. c., p. 17).—(a) The hardening.—This must 
be done in a bichromate solution. Either pure bichromate 
of potash may be employed or liquid of Muller (the reaction 
can be obtained with liquid of Erlicki, but it is not to be 
recommended). The normal practice is to take bichromate 
of potash, beginning with a strength of 2 per cent., and 
changing this frequently for fresh solution of gradually 
increased strength, 3, 4, and 5 per cent. The tissue to be 
operated on should be as fresh as possible; though satisfactory 
results may be obtained from material taken twenty-four to 
forty-eight hours after death. It should be in pieces of not 
more than 1 c.cm. or H c.cm. in size. 
The most difficult point of the method consists in hitting 
off the exact degree of hardening in the bichromate that should 
be allowed before passing to the next stage of the process, 
the silver bath. The degree of hardening arrived at in any 
given time depends on so many factors (state of the tissues, 
temperature, &c.) that it is impossible to formulate rules in 
this respect. In summer good results may be obtained after 
fifteen to twenty days, and the material may continue in a 
favorable state for impregnation up to thirty, forty, or fifty 
days. In cold weather good results can seldom be obtained 
under a month ; when obtained, the material may continue 
to give good results up to two, three, and even four months 
of hardening. The only way to make sure is to pass trial 
portions of the tissue at intervals into the silver bath, in 422 
CHAPTER XXXIII. 
summer frequently, in winter every eight or ten days, and 
observe whether the reaction is obtained. 
Good results are obtained by injecting the organs with 
the hardening fluid (2'5 per cent, bichromate). 
Stoving at a temperature of 20° to 25° C. is useful for 
abridging the hardening, but there is risk of over-hardening; 
and Golgi thinks the results are never quite so delicate as 
after hardening in the cold. 
(b) Impregnation.—As soon as the pieces of tissue have 
attained the proper degree of hardening in the bichromate, 
they are brought into a bath of nitrate of silver. The usual 
strength of this bath is 0'75 per cent., but it is not necessary 
to hold rigorously to this strength : 050 per cent, may be 
taken, and even seems to be better for material that has not 
been quite enough hardened, and solutions of 1 per cent, 
may also be used and even seem to be better adapted for 
material that has been slightly over-hai'dened. 
A relatively large quantity of solution should be taken 
for the bath. For two or three pieces of tissue of 1 c.cm. 
each, Golgi says that about “ half a glassful of solu- 
tion ” should be taken (that distinguished anatomist having 
evidently forgotten for the moment that glasses vary in capa- 
city from a liqueur-glass to a soda-water tumbler). 
The moment the pieces of tissue are put into the silver 
bath, an abundant yellow precipitate of chromate of silver is 
formed. This of course weakens the bath pro tanto. It is 
therefore well, before putting the pieces into the final silver 
bath, to first wash them well in a weaker silver solution, until 
on being put into a fresh quantity of it no further precipitate 
is formed. Used solutions will do for this purpose. The 
final silver bath in general needs no further attention, unless 
it. be that sometimes, in the case of tissues that have taken 
up a great deal of bichromate of potash, the solution may 
after six to ten hours become somewhat yellow, in which case 
it should be changed for fresh. 
it is not necessary to keep the preparations in the dark 
during the impregnation-bath; in winter it is well to keep 
them in a warm place. 
The time necessary for impregnation by the silver salt is 
from twenty-four to forty-eight hours. The normal time is 
from twenty-four to thirty hours, forty-eight being quite ex- NEUROLOGICAL METHODS. 
423 
ceptional. By this is meant that the reaction is not obtained 
in less time, but tissues may remain in the bath without hurt 
for days, weeks, or months. 
(c) Preservation.—As soon as a trial has shown that a suffi- 
ciently satisfactory impregnation has been obtained, the 
pieces are brought into alcohol. The alcohol is changed two 
or three times, or even more, until it remains transparent even 
after the preparations have been two or three days in it. For 
in view of good preservation it is necessary that the ex- 
cess of nitrate of silver should be washed out from them 
thoroughly. 
Sections are now made. They are to be washed very 
thoroughly in three or four changes of absolute alcohol. 
They are then cleared, first in creosote, in which they should 
remain only a few minutes, then in oil of turpentine, in wrhich 
they should remain for ten to fifteen minutes (they may re- 
main there for days without hurt). They are then mounted 
in Damar (rather than in balsam), and without a cover. Pre- 
parations mounted under covers in the usual way always go 
bad sooner or later, whilst those that are mouuted without a 
cover keep very well, especially if they be kept in the dark. 
Golgi states that he has a large number that have kept with- 
out change for nine years. 
The order in which the elements of tissues impi-egnate is 
—first, axis cylinders, then ganglion cells, and, lastly, neu- 
roglia cells. 
756. Golgi’s Bichromate and Nitrate of Silver Method, RAPID 
Process (op. cit., p. 33).—Small pieces of very fresh tissue are 
thrown into the following mixture : 
Bichromate solution of 2 to 2*5 per cent, strength, 8 parts. 
Osmic acid of 1 per cent, strength . . . 2 ,, 
The hardening being much more rapid than with the slow 
process, the tissues will begin to be in a fit state for taking 
the silver impregnation from the second or third day; in 
the next following days they will be in a still more favor- 
able state, but the favorable moment does not last long, the 
faculty of impregnation soon declines, and is generally quite 
lost by the tenth or twelfth day. 
The silver impregnation is conducted exactly as in the slow 424 
CHAPTER XXXIII. 
process, and sections are prepared and mounted in the same 
manner. 
There is this difference, that the impregnated material can- 
not be preserved for any length of time in alcohol, but must 
not remain for more than two days in it. But it may be 
kept until wanted for sectioning in the silver solution. 
This process has the advantage of great rapidity, and of 
sureness and delicacy of result, and is the one that has found 
the most favour with other workers. But for methodical 
study of any given part of the nervous system, Golgi himself 
prefers the following. 
757. Golgi’s Bichromate and Nitrate of Silver Method, MIXED 
Process (op. cit., p. 34).—Fresh pieces of tissue are put for 
periods varying from two to twenty-five or thirty days into 
the usual bichromate solution. Every two or three or four 
days some of them are passed on into the osmio-bichromate 
mixture of the rapid process, hardened therein for from three 
or four to eight or ten days, and finally impregnated with 
silver, and subsequently treated exactly as in the rapid 
process. 
The reasons for which Golgi prefers this process are—the 
certainty of obtaining samples of the reaction in many stages 
of intensity, if a sufficient number of pieces of tissue have 
been operated on; the advantage of having at one’s disposition 
a notable time—some twenty-five days—during which the 
tissues are in a fit state for taking the silver, and the possi- 
bility of greatly hastening the process whenever desired by 
simply bringing the pieces over at once into the osmic mix- 
ture ; lastly, a still greater delicacy of result, especially 
remarkable in the demonstration of the “ functional ” or 
nervous process of nerve-cells. 
758. Critique of Golgi’s Method.—The above-described 
methods have been found extremely valuable in the most 
Various departments of nervous anatomy. They have given 
brilliant results in the study of peripheral nerves and their 
origins or terminations, and in the study of the relations of 
fibres and cells in the central nervous system. It has been 
found, at the same time, that they have the defect of con- 
sidei’able uncertainty in the production of the desired reac- NEUROLOGICAL METHODS. 
425 
tion, and in the preservation of the stain. These defects have 
given rise to a most elaborate discussion, which unhappily 
has not as yet led to very satisfactory results. 
Golgi’s method is apparently (but this is by no means 
certain) a double decomposition method, based on the com- 
bination of the bichromate of potassium in the tissues with 
the silver nitrate, and formation of a brown precipitate of bi- 
chromate of silver (K2Cr207 + 2AgN03 = Ag2Cr207 + 2KNO ; 
the precipitate is brown by reflected light, but appears black 
by transmitted light). The problem is to preserve this pre- 
cipitate in the tissues free from chemical or molecular change. 
And the problem is not an easy one; without special precau- 
tions the preparations will not resist the processes necessary 
for imbedding, will not always resist those necessary for 
merely mounting in balsam, and even then may easily “ go 
bad ” after they have been mounted for a short time. 
Modifications concerning the Impregnation of the Tissues. 
759. Ramon y Cajal, who has done a great deal of im- 
portant work by Golgi’s method, has always used the rapid 
process. For the times and strengths used by him in his 
researches on the cerebral cortex of mammals, see his paper in 
La Cellule, vii, 1891, p. 125, or Zeit. f. wiss. Mik., ix, 2, 
1892, p. 239; also Journ. Roy. Mic. Soc., 1892, p. 154. He 
found it useful to adopt Sehrwald’s gelatin process [infra, 
section 766) for avoidance of peripheral precipitates. He 
prefers not to adopt Greppin’s treatment with hydrobromic 
acid, nor Obregia’s treatment with gold chloride, finding 
that although they serve to render the preparations per- 
manent, they obscure the finer relations of fibres. 
For embryos of the fowl he employs the same process; 
see his paper in Anat. Anz., v, 1890, p. 85, or Zeit. f. wiss. 
Mik., vii, 2, 1890, p. 235. 
760. Ramon y Cajal’s Double-Impregnation Process.—In a 
paper on the structure and relations of the sympathetic gan- 
glia (which I have not seen, and quote from Zeit. f. wiss. 
Mik., 1. c.) Ramon y Cajal describes a process of “ intensified ” 
or “ double ” impregnation. After hardening for three days 
(embryos of fowl) in the osmium-bichromate mixture, the 
preparations are put for thirty-six hours into nitrate of silver 426 
CHAPTER XXXIII. 
solution (0-5 to 0'75 per cent.) They are then brought back 
into the same osmium-bichromate mixture, or into a weaker 
one containing only 2 parts of osmic acid solution to 20 of 
the bichromate. After treatment with this they are washed 
quickly with distilled water, and put for a second time into 
the silver solution for thirty-six to forty-eight hours. It is 
important to hit off the proper duration of the first impreg- 
nation in the bichromate. If it has been too long (four days) 
or too short (one day), the second impregnation will not 
succeed. In this case a third impregnation must be resorted 
to, the objects being again treated with the weak osmium- 
bichromate mixture, and afterwards again with the silver 
solution. 
This modification of tbe original process is, perhaps, the 
most important that has hitherto been made. 
761. Kallius (Anat. Hefte, x, 1894, p. 527 ; Zeit.f. wiss. Mik., xi, 2, 
1894, p. 154) states that he has often found it advantageous to employ 
bichromate of ammonia or of soda instead of the bichromate of potash, and to 
perform all the reactions in the dark. Preparations made by the ammonia 
or soda salt rarely require a double impregnation. 
762. Smirkow (Intern. Monattssclir., x, 2, 1893, p. 241; Zeit. f. wiss. 
Mik., x, 2, 1893, p. 254), for the study of nervee ndings in the human skin, 
performs the hardening in Altmann’s mixture (equal parts of 5 per cent, 
bichromate of potash and 2 per cent, osmic acid), leaving the pieces therein 
for 5 to 10 days, and impregnating with 1 per cent, solution of nitrate of 
silver. 
Tor nerve endings in the skin of the earthworm, he hardens (Anat. Anz., 
ix, 9, 1894, p. 571) in a mixture of equal parts of 5 per cent, bichromate and 
1 per cent, osmic acid, for from 5 to 28 days. 
763. Reviving Over-Hardened Tissues.—Tissues that have 
been too long (three to four weeks) in the osmium-bichromate 
mixture will no longer take on the silver impregnation, as 
has been explained above. They can, however, be re- 
vivified, and made to impregnate, in the following manner, 
due to Golgi, and published by Sacebdotti (Intern. 
Monatsschr., xi, 1894, 6, p. 326; Zeit. f. wiss. Mile., xi, 3, 
1894, p. 389). They are washed in a half-saturated solution 
of acetate of copper until they no longer give a precipitate, 
and are then put back again for five or six days into the 
osmium-bichromate mixture. Sections of the impregnated 
material give remarkably fine images, and will bear mounting 
in thickened oil of cedar under a cover. NEUROLOGICAL METHODS. 
427 
764. Boehm, and afterwards Oppel (Anat. Anz., v, 1890, p. 148, and 
vi, 1891, p. 165; Zeit. f. wiss. Mik., vii, 2, 1890, p. 222, and viii, 2, 1891, 
p. 224) have modified the hardening part of the process by taking instead of 
bichromate of potash (slow process) the one, an 0'5 per cent, solution of 
chromic acid (forty-eight hours), the other, an 0 5 per cent, solution of 
neutral chromate of potash (twenty-four hours, the pieces having been pre- 
viously hardened in alcohol). This is for liver. 
765. Formaldehyde Mixtures.—Strong (Anat. Anz., x, 15, 
1895, p. 494) states that formaldehyde can with advantage be 
substituted for the osmic acid in the osmio-bichromic mixture 
of Golgi’s impregnation process. He adds from 2’5 to 5 per 
cent, of “formaline” to the 3'5 to 5 per cent, bichromate 
solution. 
The advantage of using formaldehyde instead of osmic acid 
is stated by Strong to be that the stage of hardening favor- 
able for impregnation lasts longer; in other words, the 
formaldehyde-bichromate does not over-harden. 
Durig {ibid., p. 659) makes the same recommendation. He 
obtained the best results by means of 3 per cent, bichromate 
solutions containing 4 to 6 per cent, of formaldehyde (if I 
understand rightly). Durig hardens therein for three days, 
then performs double impregnation by Ramon y Cajal’s 
process. 
Fish (Proc. Amer. Mic. Soc., xvii, 1895, p. 319) has also 
obtained good results with the following mixtures for the 
Golgi process: 
Formalin ...... 2 c.c. 
3 per cent, bichromate . . . 100 ,, 
leaving the tissues three days in this liquid and three days in 
the silver nitrate (§ per cent.). 
Or, with advantage: 
Liquid of Muller .... 100 c.c. 
10 per cent, formalin . . . 2 ,, 
1 per cent, osmic acid . . . 1 ,, 
The formalin and bichromate mixtures should be kept in 
the dark. It is well only to make them up at the instant of 
using them. 
Kopscb (Anat. Anz., xi, 1896, p. 727) states that he has 
obtained good impregnations with a mixture of 4 parts of 
3'5 per cent, bichromate solution, and one of commercial 428 
CHAPTER XXXIII. 
formaldehyde solution. He considers the results more certain 
than with the osmic acid mixture. 
Van Gehuchten {in litt.) has tried the substitution of 
formaldehyde for the osmic acid in the Golgi process, and 
has not obtained good results. 
766. Sehrwald’s Gelatin Process {Zeit. f. wiss. Mik., vi, 
4, 1889, p. 456). 
One of the annoyances of Golgi’s process is that it fre- 
quently gives rise to the formation at the surface of the 
preparations of voluminous precipitates that are destructive 
of the clearness of the images. Sehrwald finds that this 
evil can be avoided by putting the tissues into gelatin solution 
before bringing them into the silver-bath. A 10 per cent, 
solution of gelatin in water may be made. The tissues are 
imbedded in this in a paper imbedding box with the aid of a 
little heat (the gelatin melting at a sufficiently low tem- 
perature), and are brought therein into the silver-bath. After 
the silvering the gelatin is removed by warm water saturated 
with chromate of silver. Martinotti wraps the tissues simply 
in blotting-paper. 
Modif cations concerning the Preservation of the Preparations. 
767. Sehrwald, in an elaborate paper (Zeit. f. iviss. Mik., 
vi, 4, 1889, p. 443), develops the theory that the deterioration 
of the preparations is due to solution of the precipitate in the 
reagents employed in the ulterior processes of preparation— 
in water, in chlorides (which may be present in alcohol), in 
the xylol, in the pai’affin, in the very balsam itself. He made 
elaborate attempts (fruitless, but instructive) to turn the diffi- 
culty by transforming the soluble chromic salt of silver into 
some insoluble compound. He tried to replace the acid of 
the salt; he tried to replace the metal of the salt; he tried to 
make a sulphide of it; he tried to reduce it to the metallic 
state with photographic developing solutions (and had some 
success with hydroquinone, but does not recommend the pro- 
cess on account of its giving rise to deceptive precipitates 
that simulate organised elements). Hone of these experi- 
ments gave the desired result; and the upshot of the paper is 
that it is better not to attempt to modify the state of the pre- NEUROLOGICAL METHODS. 
429 
cipitate, but rather so to modify the preparation liquids as to 
reduce their solvent action on the precipitate. This may be 
done by supersaturating them with bichromate of silver before 
using. The powdered salt should be added to each of them 
in excess, including the clearing media, the medium used for 
fixing the sections to the slide, the paraffin itself, the solvent 
used for removing it, and the balsam, and made to dissolve 
by the aid of heat. By this means thin sections may be pre- 
pared and mounted in balsam without injury to the stain. 
Sehrwald does not say how long they will keep. 
768. Samassa (op. cit., vii, 1, 1890, p. 26) points out that 
bichromate of silver is not soluble either in absolute alcohol, 
toluol, xylol, paraffin, or Canada balsam, nor otherwise chemi- 
cally affected by them ; and that therefore Sehrwald’s ex- 
planation is untenable. He thinks that the true explanation 
of the deterioration of the preparations is that the precipitate 
forming the impregnation is little by little floated away from 
the tissues by the mechanical force of the diffusion currents 
set up on the passage of the preparations through the dif- 
ferent reagents, and particularly those long-continued ones 
caused by the slow drying of balsam under a cover-glass. 
He recommends, therefore, simply that the preparations be 
preserved without a cover, as directed by Golgi. 
769. Tick (Zeit. /. wiss. Milt., viii, 2, 1891, p. 168) does not 
admit Samassa’s theory of the deterioration of sections through 
diffusion currents (supra). He points out that bichromate of 
silver is soluble in water, especially with the aid of heat; and 
after an elaborate series of experiments, concludes that the 
water of the reagents or damp confined by the cover-glass is 
the cause of the ruin of the preparations. Watery fluids 
should be avoided, and sections should be mounted without a 
cover, or on a cover raised free of contact with the slide by 
means of wax feet, or the like. Or sections mounted without 
a cover may be later on provided with one if the balsam be 
first rendered perfectly anhydrous by careful heating. 
This last method is also recommended by Hubek (Anat. 
Anz., vii, 1892, p. 587; Journ. Roy. Mic. Soc., 1892, p. 707 ; 
Zeit. f. wiss. Milt., ix, 4, 1893, p. 479). The reader will of 
course understand that the balsam is to be heated on the 430 
CHAPTER XXXIII. 
slide, with the section in it, until it immediately sets hard on 
cooling. 
770. Gbeppin (Arch.f. Anat. u. Entw., Anat. Abth., 1889, Supp., p. 55 ; 
Zeit.f. wiss. Mik., vii, 1, 1890, p. 66) finds that by means of hydrobromic 
acid, suggested by Neumann, preparations made by the slow method may 
be rendered sufficiently resistent to bear mounting under a cover. After 
silvering, sections are made with a freezing microtome and treated for thirty 
to forty seconds with 10 per cent, solution of hydrobromic acid, and may 
then be well washed in several changes of water and mounted in the usual 
way. If they be cleared in clove oil and exposed therein to sunlight for 
ten or fifteen minutes, they will take on a violet tone, and details will be 
more strongly brought out. It is sometimes well to treat them, after the 
10 per cent, acid, with 40 per cent, acid for twenty to thirty seconds. They 
may also be treated by Pal’s modification of Weigert’s hsematoxylin 
process. 
771. Obregia [Virchow’s Archiv, cxxii, 1890, p. 387; Zeit. 
f. wiss. Mik., viii, 1, 1891, p. 97 ; Journ. Roy. Mic. Soc., 1891, 
pp. 536, 830; Amer. Mon. Micr. Journ., 1891, p. 210) gives 
the following. Sections of silvered material are made, either 
without imbedding or after imbedding either in paraffin or 
celloidin, care being taken in either case not to use alcohol of 
a lower grade than 94 or 95 per cent. They are brought 
from absolute alcohol into a mixture of eight to ten drops of 
1 per cent, solution of gold chloride with 10 'c.c. of absolute 
alcohol, which should be prepared half an hour beforehand 
and exposed to diffused light until the sections are placed in 
it, when it should be put into the dark. After fifteen to 
thirty minutes therein, according to their thickness, the 
sections are quickly washed in 50 per cent, alcohol, then in 
water, then treated for five or ten minutes with 10 per cent, 
solution of hyposulphite of soda. They are lastly washed 
well with water, and may then be mounted at once in balsam 
under a cover, or if desired may be previously stained with 
carmine or hasmatoxylin, or Pal’s modification of Weigert’s 
process, or the like. 
Obregia thinks that the reason why Sehrwald did not 
succeed in substituting gold for the silver in his preparations 
(vide supra) is that he took the gold salt in aqueous solution. 
This method is also applicable to material treated by G-olgi’s 
sublimate process, § 774. 
772. Sala (Zeit. f. wiss. Zool., lii, 1, 1891, p. 18; Zeit. f. NE GEOLOGICAL METHODS. 
431 
wiss. Mih., viii, 3, p. 389), in a paper written in Golgi’s labora- 
tory, finds Greppin’s hydrobromic acid variation not merely 
useless, but hurtful. And he thinks that Sehrwald’s process 
for imbedding the material in paraffin with the object of get- 
ting very thin sections is a mistake. The chief quality of 
Golgi’s process is that it admits of the following of nerve-cell 
processes for a very great distance. Evidently this cannot be 
done with very thin sections. It is better simply to wash 
the preparations taken from the silver-bath with water, fix 
them to a cork with gum, put the whole into alcohol for a few 
hours to harden the gum, and cut with a microtome without 
imbedding. 
773. Kallius (Anat. Hefte, ii, 1892, p. 269; Zeit.f. wiss. Mile., ix, 4,1893, 
p. 477) has worked out the following process. Take 20 c.c. commercial 
hydroquinone developing solution and 230 c.c. distilled water (the hydro- 
quinone solution may be made up with 5 grms. hydroquinone, 40 grms. 
sodium sulphite, 75 grms. carbonate of potassium, and 250 grms. distilled 
water). At the instant of using, further dilute the solution with one third 
to one half its volume of absolute alcohol, and put the sections into it for 
several minutes; they become dark grey to black. In order to ascertain 
whether reduction is complete, throw a section into a solution of hypo- 
sulphite of soda (about 10 parts to 50 of water): chromate of silver will 
quickly dissolve, whilst metallic silver will not be attacked. As soon as re- 
duction is complete the sections are put for ten to fifteen minutes into 70 
per cent, alcohol, then brought for five minutes into the above-given solu- 
tion of hyposulphite of soda, and thence into a large quantity of distilled 
water, where they should remain for twenty-four hours or more. Lastly, 
dehydrate in the usual way, and mount under a cover. All the details which 
in the undeveloped preparations were brown are now black on a light ground. 
After-staining with carmine, &c., may be employed. Other developers were 
tried, and gave satisfactory reductions, but they caused a red or brownish 
discoloration of the preparations. 
774. Golgi’s Bichromate and Sublimate Method (Archivio per 
le Scienze Mecliche, 1878, p. 3; Archives italiennes de Biologie, 
t. iv, p. 32; Arch, de Biol., t. vii, p. 35).—This method, 
which may be said to be in principle identical with the bi- 
chromate of potash and silver nitrate method of the author, 
consists, like the latter, of two processes : 1, hardening in 
bichromate; 2, treatment with bichloride of mercury. 
For hardening, use either a solution progressively raised 
in concentration from 1 per cent, to 2| per cent., or Muller’s 
solution. It is best to take small pieces of tissue (not more 
than 1 to 2 c.c.), large quantities of liquid, and change the 432 
CHAPTER XXXIII. 
latter frequently, so as to have it always clear. But the re- 
action can be obtained with much larger pieces, even entire 
hemispheres. In this case the brain should at first be treated 
by repeated injections of the liquid, so as to ensure as rapid 
a permeation of the interior as possible. Fifteen to twenty 
days’ immersion will suffice, or even six to eight, but twenty 
to thirty should be pi’eferred, and an immei’sion of several 
months is not injui’ious. 
The tissues when hardened are passed direct from the bi- 
chromate into the bichloride of mercury. The solutions of 
the latter, first employed by Golgi, varied from 0‘25 per cent, 
to 050 or 1 per cent.; he now always takes 0'50 per cent. 
The immersion in the bichloride must be much longer than 
the immersion in the nitrate of silver bath of that process : 
for the latter, twenty-four to forty-eight hours suffice; but 
in the bichloride an immersion of eight to ten days is necessary 
in order to obtain a complete reaction through the whole 
thickness of the tissues, or for entire hemispheres two months 
or more. During the bath the bichromate will diffuse out 
from the tissues into the bichloride, which must at first be 
changed every day, and later on as often as it becomes 
yellow. At the end of the reaction the preparations will be 
found decolourised, and offering the aspect of fresh tissue. 
They may be left in the bichloride for any time. 
In Rendiconti R. 1st. Lombardo di Sci. Milano, 2, xxiv, 1891, pp. 594, 
656 (see Zeit. f. wiss. Mile., viii, 3, 1891, p. 388) Golgi says that for the 
study of the diffuse nervous reticulum of the central nervous system the 
best results are obtained by keeping the preparations in 1 per cent, sub- 
limate for a very long time, two years being not too much in some cases. 
The reaction may be said to have begun by the time the 
tissues are nearly decolourised. From that time onwards 
sections may be made day by day and examined, and those 
which it is desired to preserve may be mounted. 
Before mounting, the sections that have been cut must be 
repeatedly washed with water (if it be wished to mount them 
permanently), otherwise they will be spoilt by the formation 
of a black precipitate. In the last place quoted Golgi says 
that after washing they may be toned by putting them for a 
few minutes into a photographic fixing-and-toning bath, after 
which it is well to wash them again, and stain them with NEUROLOGICAL METHODS. 
433 
some acid carmine solution. Mount in balsam or glycerin; 
the latter seems the better preservative medium. 
The result of this process is not a true stain, but an “ appa- 
rently black reaction,” the tissues appearing black by trans- 
mitted light, white by reflected lig-ht. Golgi thinks that 
there is formed in the tissue elements a precipitate of some 
substance that renders them opaque. The elements acted on 
are (1) the ganglion-cells, with all their processes and rami- 
fications of the processes. These are made more evident than 
by any other process except the bichromate and silver-nitrate 
process. An advantage of the mercury process is that it 
demonstrates nuclei, which is not the case with the silver 
process. (2) Connective-tissue corpuscles in their charac- 
teristic radiate form. But the reaction in this case is far less 
precise and complete than that obtained by the silver process. 
(3) The blood-vessels, and particularly their muscular fibre 
cells. 
The method gives good results only with the cortex of the 
cerebral convolutions, hardly any results at all with the 
spinal cord, and very scanty results with the cerebellum. 
And, on the whole, the method shows nothing more than 
can be demonstrated by the silver-nitrate method, but it is 
superior to it as regards two points : the reaction can always 
be obtained with perfect certainty in a certain time, and 
the preparations can be perfectly preserved by the usual 
methods. 
But Golgi holds {Arch, de Biol., t. vii, p. 41), that the 
method deserves an honorable place amongst neurological 
methods, by the side of the silver-nitrate methods. 
See also Flatatj, in Arcli.f. mik. Anat., xlv, 1895, p. 158; Zeit. f. wiss. 
Mik., xii, 2, 1895, p. 257. 
The method is recommended by Blochmann (Biol. Centralbl., xv, 1895, 
p. 14; Zeit.f. wiss. Mik., xii, 2, 1895, p. 226) for the nervous system of 
Cestodes. 
Modifications of Golgi’s Bichromate and Sublimate Method. 
774a. Tal (Gazz. degli Ospitali, 1886, Ifo. 68) finds that if sections made 
by this process be treated with solution of sodium sulphide, a much darker 
stain is obtained. Sections may then advantageously be double-stained with 
Magdala red. 
Grolgi’s method may be combined with Weigert’s nerve stain (see Pal, 
Wien. med. Jcihrb., 1886 ; Zeit.f. wiss. Mik., v, 1, 1887, p. 93). 434 
CHAPTER XXX11I. 
775. Cox (Arch. f. mih. Anat., xxxvii, 1891, p. 16 ; Journ. Roy. Mic. 
Soc., 1891, p. 420) finds the sublimate and bichromate may be used together, 
and give a uniform impregnation. He used a fluid consisting of 20 parts 
5 per cent, bichromate, 20 parts 5 per cent, sublimate, 16 parts 5 per cent, 
simple chromate of potash, and 30 to 40 parts of water. The mixture 
should be as little acid as possible. The duration of the impregnation is 
from two to three months. There is considerable difficulty in preserving 
sections, which must be made with a freezing microtome, alcohol being 
avoided, treated with solution of sodium carbonate, and mounted without a 
cover. 
776. Magini (Boll. Accad. Med. di Roma, 1886 ; Zeit.f. wiss. Mih., 1888, 
p. 87) recommends a development of Golgi’s process in which zinc chloride 
is used in place of sublimate. Portions of tissue of 2 to 3 cm. cube ai-e 
hardened for at least two or three months in Muller’s solution. They are 
well washed with distilled water, and brought into a 01 to 1 per cent, solu- 
tion of chloride of zinc. This is changed for fresh every day for seven to 
ten days (until it does not become yellower than bichromate solution). Sec- 
tions are then made, washed quickly with alcohol, imperfectly cleared with 
kreasote, and mounted in dammar. This process is said to demonstrate better 
than Golgi’s the finer structure of ganglion-cells and their processes. 
777. Flechsig’s modifications, see Arch. f. Anat. u. Phys., Physiol. 
Abth., 1889, p. 537; Zeit. f. wiss. Mih., vii, 1, 1890, p. 71. 
778. Monti (Atti d. JR. Accad. dei Lincei Roma, Rendic., v, 1889, 1 sem., 
p. 705; Zeit. f. wiss. Mih., vii, 1, 1890, p. 72) describes a brown impregna- 
tion brought about bj the combined action of bichromate of potash and sul- 
phate of copper. The method does not appear to have been yet brought to 
a practical form. 
779. Ziehen’s gold and sublimate method (Neurol. Centralb., 
x, 1891, No. 3, p. 65 ; Zeit.f. wiss. Mih., viii, 3, 1891, p. 385). 
Zielieu gives the following derivative of Glolgi’s sublimate 
method as an improvement on it:—Small pieces of fresh 
material are thrown into a large quantity of a mixture of 1 per 
cent, sublimate solution and 1 per cent, chloride of gold solu- 
tion in equal parts. They remain therein for at least three 
weeks, preferably for several months (up to five), by which 
time they will have become of a metallic red-brown colour. 
They are gummed on cork and sectioned without imbedding. 
The sections are treated either with Lugol’s solution (§ 73) 
diluted with four volumes of water, or with dilute tincture of 
iodine, until duly differentiated, which will require more or 
less time according to the thickness of the sections. They 
are then washed and mounted in balsam. The result is a 
bluish-grey impregnation; both medullated and non-medul- NEUROLOGICAL METHODS. 
435 
lated nerve-fibres are stained, also nerve- and glia-cells and 
their processes. 
Other Methods. 
780. Weigert’s Specific Neuroglia Stain (Weigert, Beitr. zur 
Kenntniss der normalen menschlichen Neuroglia, Frankfurt- 
a-M., 1895; quoted from Neurol. Centralbl., 1895, xxiii, p. 
1146).—Pieces of tissue of not more tlian lialf a centimetre 
in thickness are put for at least four days into “ 10 per cent, 
solution of formol.” They are then mordanted for four or five 
days in an incubating stove (or for at least eight days at the 
temperature of the laboratory) in a solution containing five 
per cent, of neutral acetate of copper, five per cent, of acetic 
acid, and two and a half per cent, of chrome alum, in water. 
(Add the alum to the water, raise to boiling point, and add 
the acetic acid and the acetate, powdered.) 
After the mordanting, the tissues are washed with water, 
dehydrated, imbedded in celloidin and sectioned. The sec- 
tions are treated for ten minutes with a one third per cent, 
solution of permanganate of potash, and well washed in water. 
They are then treated for two to four hours with a solution 
of “ Chromogen.” “ Chromogen ” is a naphthalin compound 
prepared by the Hoechst dye manufactory. The solution of 
“ Chromogen ” to be used is prepared as follows : five per 
cent, of “ Chromogen” and five per cent, of formic acid are 
dissolved in water and the solution carefully filtered. To 
90 c.c. of the filtrate are added 10 c.c. of ten per cent, solu- 
tion of sodium sulphite. 
After this bath, the sections are put till next day into a 
saturated (5 per cent.) solution of Chromogen. 
They are next carefully washed and submitted to the stain. 
The stain is a modification of Weigert’s fibrin stain. Instead 
of saturated aqueous solution of methyl violet, you take a 
warm-saturated solution in 70 per cent, or 80 per cent, alcohol, 
decant it after cooling, and add to it 5 per cent, of 
aqueous solution of oxalic acid. And instead of treating 
with pure anilin, you take a mixture of equal parts of anilin 
and xylol. This is afterwards removed from the sections by 
means of pure xylol, and they are mounted in balsam. 436 
CHAPTER XXXIII. 
Weigert lias concluded from the reactions obtained by this 
process that neuroglia is not a network of nerve-cell pro- 
cesses, but an intercellular substance of the nature of con- 
nective tissue substance. 
781. See also Aronson, a gallein stain, in Mercier’s Coupes du systeme 
nerveux Central, p. 255 ; Kultschizky, Saurerubin for neuroglia, Anat. 
Anzeig., viii, 1893, p. 357, or Zeit. f. wiss. Mik., x, 2, 1893, p. 256. 
782. Upson’s Methods (Mercier, in Zeit. f. wiss. Mik., vii, 4, 
1891, p. 474; or, in his “ Coupes du systeme nerveux Central, 
p. 234; I pass over the older methods given in Neurol. Cen- 
tralb., 1888, p. 319; and in Zeit. f. wiss. Mik., 1888, p. 525). 
a. The G-old and Iron Method.—Material must be very carefully 
hardened for from four to six months in bichromate of potash (not solution 
of Muller, which does not allow of the same precision of stain). A 1 per 
cent, solution should be taken at first, and should at first he frequently 
changed. Only after some weeks should the strength be gradually increased 
up to 2 or 2'5 per cent. All the hardening should be done in the dark. 
Over-hardened material is not available. The surface of section of a pro- 
peidy hardened piece of material should show macroscopically no marked 
difference of colour between the white and the grey matter. If the white 
substance appear very dark, almost black, and the grey substance whitish 
grey, good results will probably not be obtained; a medullary stain will 
probably result, whereas the method ought to give an axis-cylinder and cell- 
process stain. 
After hardening, the specimens are washed with water, and put for two or 
three days into 50 per cent, alcohol, changed as often as necesssary, and then 
into 95 per cent, alcohol, in which they should remain until they show a de- 
cidedly green coloration (two to four weeks), the alcohol being changed as 
often as precipitates occur. Sections are made either under water or by the 
celloidin process (in the former case they should not be allowed to remain in 
contact with the water longer than is absolutely necessary, and should he put 
back into alcohol for two or three days before passing to the next stage). 
The next stage consists in a bath of from one to two hours’ dui'ation in 1 
per cent, solution of chloride of gold acidified with 2 per cent, of hydrochloric 
acid. After this the sections are rinsed with water, and treated for half a 
minute with a freshly prepared solution of ferricyanide of potassium in 10 
per cent, potash solution (a lump of ferricyanide not half so big as a pea to 
5 c.c. of the potash solution). They are then washed for half a minute 
more in pure 10 per cent, potash solution, and after that for some time in 
distilled water. 
The following reducing mixture (which must be freshly made up at the 
very instant of using it for each section) is now to be prepared : 
Acidum Sulfurosum ...... 5 c.c. 
Tinctura Iodi (3 per cent, strength) . . . 10—15 drops. 
Mix, and add— 
Liquor Ferri Chloridi ...... 1 drop. NEUROLOGICAL METHODS. 
437 
You must mix quickly in a graduated glass. The section should now be 
brought on a piece of filter-paper into a watch-glass, and the reducing mix- 
ture should be poured over it evenly at one pour, just as developing solution 
is poured on to a photographic negative. The section should remain in this 
bath until it is of a fine rosy red tint, not a second longer, or it will become 
reddish black and useless. As soon as the rose-red colour appears the section 
is removed into distilled water. The water is changed once, the section is 
placed on a slide and brought into absolute alcohol, and after five to fifteen 
minutes therein into clove oil and mounted in balsam. The sections must 
be kept in the dark. 
(The treatment with ferricyanide of potassium sometimes gives rise to the 
formation later on of precipitates of Berlin blue in the tissues; and as this 
treatment is not essential it may be omitted, the sections being simply 
treated with pure potash solution as directed.) 
783. Upson’s Methods.—b. The Gold and Vanadium Method— 
Sections (made as before) are placed, in the following solution : 
1 per cent, chloride of gold solution . . .5 c.c. 
Saturated solution of vanadate of ammonium . . 10 drops. 
Hydrochloric acid . . . . . . . 3 „ 
After two hours therein they are washed with distilled water, and put for 
half to one minute into a mixture of— 
10 per cent, solution of caustic potash . . .5 c.c.' 
10 per cent, solution of permanganate of potash . 10 drops. 
Vanadate of ammonium . . . . .a trace; 
They are rinsed in distilled water, and treated until they become red, as 
in method a, with the following freshly prepared reducing fluid : 
a. 3 per cent, tincture of iodine, to which has been 
added chloride of tin until of a white or yellowish 
tint (may be kept in stock) . . . .15 drops. 
b. Distilled water ....... 3 c.c. 
c. Saturated solution of phosphate of iron in distilled 
water (the usual pharmaceutical solution) . . 3—5 drops. 
d. Sulphurous acid ...... 3 c.c. 
Mix a, b, and c, and add d, which will cause a voluminous precipitate, at 
which instant the mixture is to be poured over the section, or the section 
brought into it, as in method a, the remaining treatment being exactly as 
there described. 
The principle of the two methods is the decoloration of the impregnated 
elements (especially glia-cells) up to a certain point by means of the potash, 
or potash and ferricyanide, or the vanadate and permanganate. It is im- 
portant to take care that this decoloration be not overdone, as if carried too 
far the axis-cylinders and ganglion-cells will be decolourised. 
784. Giacomini’s “Dry” Process for Preserving Brains (Arch.per 
le Scienze Mecliche, 1878, p. 11).—Although this is in intention a macro- 
scopic method, it appears worth while, both on account of its thorough success 
and on account of its suggestiveness, to give a description of it here. 
The object is to make “dry” preparations of the encephalon, by which 438 
CHAPTER XXXIII. 
is meant preparations that can be permanently preserved in the air. The 
methods hitherto employed were not successful because they consisted in 
making preparations that were “ dry” in the literal sense of the word—that 
is, deprived of their natural water; and since brain-substance contains 88 per 
cent, of water, such preparations could not of course be obtained without so 
great an amount of shrinkage as to most seriously diminish the scientific 
value of the result. The principle of Giacomini’s method is, on the contrary, 
to retain the natural water of the tissues, or an equivalent for it, by means 
of impregnation with a hygroscopic substance—glycerin. 
The process consists of two divisions:—1, hardening; 2, impregnation 
with glycerin. 
1. For hardening may be used zinc chloride, bichromate of potash, chromic 
acid, nitric acid, or alcohol. 
Chloride of zinc gives the best results. Perfectly fresh brain is put into 
a saturated aqueous solution of the salt (if there be reason to fear that the 
tissues are somewhat softened through having been left too long after the 
death of the subject, it is well first to inject 600 grms. of the solution 
through the internal carotid arteries). After forty-eight hours’ immersion 
(during which time the floating brain must be turned over three or four 
times, so that all parts of it may duly come into contact with the liquid) the 
surface of the brain will have attained a consistency that will allow of the 
removal of the arachnoid and pia mater. The meninges having been removed 
the encephalon is put back into the solution for two or three days more, 
during which time it will be seen that, increasing in specific gravity, it tends 
towards the bottom of the vessel containing it. When this is seen to happen 
it must be removed into commercial alcohol, as if allowed to remain longer 
in the chloride of zinc solution it would take up too much water. 
In the alcohol it may remain for an indefinite time, or it may be removed 
if desired after ten or twelve days. (During the alcohol-bath it must be 
frequently turned over in order that no malformation may arise from con 
tinuance of pressure on the same part.) 
It is then removed into glycerin (either pure or with 1 per cent, of carbolic 
acid). It floats at first, but gradually sinks as the alcohol evaporates. As 
soon as it has sunk just below the surface it may be removed and exposed 
to the air. 
It is set aside to “ evaporate” in a convenient place for a few days. As 
soon as the surface has become dry, it is varnished with india rubber or 
(better) with marine glue varnish diluted with a little alcohol. This com- 
pletes the process. 
If it be desired to make dissected preparations, the necessary dissection 
should be made on removing the encephalon from the alcohol before putting 
into glycerin. 
Bichromate of potash may be used for hardening in solutions gradually 
increasing in concentration from 2 to 4 per cent. The liquid must be fre- 
quently changed ; the immersion must be of not less than a month’s dura- 
tion. Six to eight days will suffice for the alcohol-bath, or this may be 
altogether omitted. 
Nitric acid is used in solutions of from 10 to 12 per cent, for twelve to 
fifteen days. (Encephala float in this liquid, and must therefore be fre- NEUROLOGICAL METHODS. 
439 
quentlv turned over. It is this reagent that gives the toughest prepara- 
tions.) 
Concerning the value of the process, Golgi (from whose abstract I take 
the foregoing account) states that after a series of experiments he is able to 
affirm that for preservation of the volume, the colour, the finer relations of 
the parts, and the physiognomy proper to the organ, the process is far 
superior to any hitherto known. I am able to add that I saw specimens of 
Giacomini’s preparations at the Milan International Exhibition of 1881, and 
think it would be hard to over-praise their beauty of aspect. 
It should be added that histological detail is preserved to a remarkable 
extent by this process, and that excellent sections may be cut at any time 
from the hardened brains. And as the preparations take up as little room 
as possible, there seems no reason why the process should not be generally 
adopted in medical schools, lunatic asylums, and similar institutions. 
The method may also be applied, with most perfect success, to the pre- 
servation of small animals entire, such as Batrachia, Beptilia. It is well to 
inject them with the zinc solution. 
Another “ dry ” method has been given by Max Elesch (Mt. Naturf. 
Ges. Bern., 1887, p. xiii). He hardens in alcohol, and then brings the 
brains through Calberla’s mixture (§ 425) into glycerin containing (1 part 
to 3000 of) sublimate. He does not appear to varnish his preparations. 
See Journ. Boy. Mic. Soc., 1888, p. 507. 
Retina. 
785. Fixation and Hardening.—For section cutting, the 
retina of small eyes is best prepared by fixing* the entire un- 
opened bulb with osmium vapour. According* to Ranvier 
(Traite, p. 954) you may fix the eye of a triton (without 
having previously opened the bulb) by exposing it for ten 
minutes to vapour of osmium. The sclerotic being very thin 
in this animal, such a duration of exposure is generally suffi- 
cient. Then divide it by an equatorial incision, and put the 
posterior pole for a few hours into one-third alcohol. 
Somewhat larger eyes, such as those of the sheep and calf, 
may be fixed in solutions without being opened. But it is 
generally the better practice to make an equatorial incision, 
and free the posterior hemisphere before putting it into the 
liquid. 
The older practice was to use strong solutions of pure osmic 
acid; but most of the best recent work has been done with 
chromic mixtures. 
Lindsay Johnson (in litt.) gets the best results by fixing 
the globe over the steam of a 2 per cent, osmic acid solution 440 
CHAPTER XXXIII. 
raised nearly, but not quite, to boiling point, for five minutes; 
or, if the eye be that of an adult, by removing the cornea 
and lens and injecting three to six drops of a mixture of equal 
parts of acetic acid and 2 per cent, platinic chloride into the 
vitreous cavity of the eye. After that the eye is put for 
twelve hours into the mixture, § 97; it is then washed in 
running water, and suspended in a large volume of 2'5 per 
cent, bichromate of potash for two days, then passed gradu- 
ally through successive alcohols, beginning with 20 per cent., 
and ending with absolute, taking five days from first to last. 
This is the treatment recommended for adult human eyes. 
The eyes of children, small animals, e.g. rabbits, monkeys, 
and cats, and especially foetal eyes, will require a much gentler 
treatment or the retina will shrink into folds and the bulb 
collapse owing to the small amount of supporting and con- 
nective tissue present, and the thinness of the sclerotic. 
Ihese eyes should not he opened at all during the fixing stage. 
After steaming for two or three minutes over osmic vapour, the 
bulb should be put entire into mixtui*e of Flemming or Her- 
mann, or the mixture No. 97, for three to six hours, and then 
into bichromate solution, and only after two or three days 
should the bulb be opened. 
He also finds that eyes are preserved perfectly in a mixture 
of formaldehyde and platinic chloride, the best proportions 
being:—Commercial formol or formalin, 4 parts, and 1 per 
cent, solution of platinic chloride, 30 parts. This mixture 
hardens slowly, but the retina remains nicely adherent, and 
the fovea well preserved, provided that the eye has previously 
been fixed by steaming over a 2 per cent, osmic acid solution 
raised to nearly boiling point, or solid osmic acid fumes pro- 
duced by heating the crystals in a test tube. Johnson finds 
as the result of repeated trials that the preliminary rapid 
fixing of the bulb over osmic vapour is absolutely essential 
to prevent the swelling and folding over of the retina at the 
macula, and to a less extent of the retina generally, nor has 
he been able to find any reliable substitute for this prelimi- 
nary treatment. As regards the after-hardening, a number 
of processes may be employed with good results, Flemming’s 
mixture, Muller’s fluid, bichromate solution, formol and 
platinic chloride, and his mixture (§ 97) being especially 
recommended. NEUROLOGICAL METHODS. 
441 
Leber (Munch, med. Wochenschr., xli, 30,1894; cf. Zeit. f.tviss. Mik., xii, 
2.1895, p. 256) confirms Hermann’s observation concerning eyes (§ 109). He 
advises a 4 per cent, solution (formol 1, water 10). After a few days’ hard- 
ening in this, the eyes may be cut through, it is said, without derangement 
of the parts. The retina lies flat, and is at least as well preserved as with 
solution of Muller. The eyes may be passed without hurt direct into suc- 
cessive alcohols; the vitreous will shrink a little, but less rather than more 
than after solution of Muller. I must he allowed to doubt the correct cyto- 
logical preservation of the elements by this process. 
786. Staining.—Ramon y Cajal employs the rapid chromate 
of silver method of Golgi, double impregnation process, as 
given above, § 760. 
Kuhnt {Jen. Zeit. f. Naturw., Bd. xxiv, H. 1, 1889, p. 177; 
Zeit. f. wiss. Mik., vii, 1, 1890, p. 65) employs Pal’s modifica- 
tion of Weigert’s hsematoxylin process. 
Dogiel employs the methylen-blue method, as given in 
§§ 291, 293. 
Schaffer (Sitzb. k. Akad. wiss. Wien, xcix, 1890, 3, p. 110; 
Zeit. f. wiss. Mik., viii, 2, 1891, p. 227) recommends mordant- 
ing sections in 1 per cent, chromic acid for some hours, 
washing for a short time only with water, staining for twenty 
hours in Kultschitzky’s acetic acid hsematoxylin (§ 729), and 
differentiating for twelve hours in Weigert’s ferricyanide 
solution. 
Krause (1. c., § 788) obtains instructive preparations by treating fresh 
retina with perchloride of iron or of vanadium in 1 per cent, solution, and 
then with a 2 per cent, solution of tannic or pyrogallic acid. These reagents 
only stain the granule layers and the nuclei of the ganglion-cells. The ele- 
ments of the other layers may then be stained with Saurefuchsin, or some 
other anilin. 
Lennox {Arch. f. Ophtlialm., xxxii, 1; Zeit.f. iviss. Mik., iii, 3, 1886, 
p. 408; and Journ. Boy. Mic. Soc., 1887, p. 339) has been applying 
Weigert’s hsematoxylin method to the retina, with some remarkable results. 
Cuccati stains with concentrated aqueous solution of Saurefuchsin, and 
mounts in balsam. 
See also Bernheimer, Sb. k. Akad. wiss. Wien, 1884; or Journ. Boy. 
Mic. Soc., 1886, p. 167; and Ramon y Cajal, Bev. trim, de Hist. Norm, y 
Path., i, 1888, p. 1; Anat. Anz., 1889, p. Ill ; Zeit. f. wiss. Mik., v, 3,1888, 
p. 373, and vi, 2, 1889, p. 204. 
Colucci (Zeit.f. wiss. Mik., xii, 1, 1895, p. 87) recommends above every- 
thing Paladino’s iodide of palladium impregnation, § 737. 
787. Sections.—Some workers recommend celloidin ; but I 
see no reason whatever for not employing paraffin. Sections 
may be mounted in dammar or (Flemming) in glycerin. 442 
CHAPTER XXXIII. 
<88. Dissociation Methods.—For maceration preparations 
you may use weak solutions (0-2 to 05 per cent.) of osmic 
acid for fixation, and then macerate in 0*02 per cent, chromic 
acid (M. Schultze), or in iodised serum (M. Schultze), or in 
dilute alcohol (Landolt), or in Muller’s solution, or (Ranvier, 
Praite, p. 957) in pure water, for two or three days. Thin 
{Journ. of Anat., 1879, p. 139) obtained very good results by 
fixing for thirty-six to forty-eight hours in one-third alcohol, 
or in 25 per cent, alcohol, and then staining and teasing. 
8>chiefferdecker macerates fresh retina for several days in 
the methyl mixture, § 559. 
Krause {Intern. Monatsch. f. Anat. u. Hist., 1884, p. 225; 
Zeit. J. wiss. Mih., 1885, pp. 140,396) recommends treatment for 
several days with 10 per cent, chloral hydrate solution. Barrett 
finds that this process preserves the rods and cones admirably. 
789. Schwalbe (Beitr. z. Phys., 1887; Zeit. f. iviss. Mih., 
iv, 1, 1887, p. 90; Journ. Roy. Mic. Soc., 1887, p. 840).—Fix 
(cochlea of guinea-pig) for eight to ten hours in Flemming,” 
wash in water, decalcify (twenty-four hours is enough) in 1 
per cent, hydrochloric acid, wash the acid out, dehydrate, 
and imbed in paraffin. 
Prenant {Intern. Monatsschr. f. Anat. u. Physiol., ix, 1, 
p. 6 ; Zeit. f. wiss. Mih., ix, 3, 1893, p. 379).—For sections, 
open the cochlea in solution of Flemming or of Hermann, 
and fix therein for four to five hours. Avoid decalcification 
as far as possible, as it is inimical to good preservation of 
elements;, but if necessary, take 1 per cent, palladium 
chloride. Make paraffin sections and stain with safranin, or 
with methyl violet B, or with anilin green and orange, or 
with Renaut’s eosin-haematoxylin. 
Isolation preparations of the stria vascularis may be made 
by putting a cochlea for a day into 1 per cent, solution of 
osmic acid, then for four to five days into Ol per cent, solu- 
tion ; the stria may then be got away whole. 
Inner Ear. 
790. Other Methods.—Waldeyee, Strieker’s Hcindb., p. 958 (decalci- 
fication either in O'OOl per cent, palladium chloride containing 10 per cent, 
of HC1, or in chromic acid of 025 to 1 per cent.). 
Ueban Peitchaed (,Journ. Roy. Mic. Soc., 1876, p. 211).—Decalcification 
in 1 per cent, nitric acid. 
Lavdowsky (Arch.f. mile. Anat., 1876, p.497).—Fresh tissues (from the NEUROLOGICAL METHODS. 
443 
cochlea) are treated with 1 per cent, solution of silver nitrate, then washed 
for ten minutes in water containing a few drops of 0-5 or 1 per cent, osmic 
acid solution, and mounted in glycerin. 
Max Flesch (Arch. f. mik. Ancit., 1878, p. 300). 
Tafani (Arch. Ital. de Biol., vi, p. 207). 
Politzer, “ Die anatomische u. histologische Zergliederung 
d. menschlichen Gehororganes,” Stuttgart (Enke), 1889 (see 
Zeit. f. wiss. Mile., vii, 3, 1890, p. 364).—The reviewer (Haug) 
here calls attention to the admirable qualities of phloroglucin 
as a decalcification agent for this object (see § 587). 
Eichler (Abh. d. math-phys. Cl. d. h. Sdchsischen Ges. d. 
Wiss., Bd. xviii, 1892, p. 311; Zeit.f. wiss. Mik., ix, 3, 1893, 
p. 380).—Detailed account of manipulations for injection of 
blood-vessels of the labyrinth. 
See also Siebenmanx, Die Blutgefdsse im Labyrinthe des 
menschlichen Ohres, Wiesbaden, Bergmann, 1894; cf. Zeit.f. 
wiss. Mik., xi, 3, 1894, p. 386. 
790a. Methylen Blue for Central Nervous System (Semi Meyer, 
Arch. f. mik. Ancit., xlvi, 1895, p. 282).—Methylen blue has 
not hitherto given good results with the central nervous 
system of the higher animals, either by the method of intra- 
venous injection or by that of staining by immersion. Meyer 
has obtained better results (for the central nervous system, 
not for the peripheral) by means of subcutaneous injection. 
The following points should be observed:—Large quantities 
of solution must be injected, but they may be divided into 
fractional doses. A young rat will require at least 5 c.c. of 
1 per cent, solution; a rabbit of a few weeks about 40 c.c.; 
a cat of the same size about three times as much. Mice are 
not good subjects. The total dose should be given in several 
portions, at intervals of one to several hours. If 20 c.c. of 
1 per cent, solution be injected into a young rabbit, and again 
the same quantity after the lapse of two hours, the subject 
will generally be found to be dead or dying at the end of two 
hours more. It is not necessary to wait till death by intoxica- 
tion has taken place, and after a suitable interval the subject 
may be killed. It is not necessary to expose the organs to 
the air for the sake of “ oxydising ” the stain. They should 
be thrown direct into the liquid of Bethe, § 294. The liquid 
ought to be well cooled before use. The preparations should 
remain in it till the next day. CHAPTER XXXIV. 
SOME OTHER HISTOLOGICAL METHODS. 
Connective Tissues. 
791. Connective Tissue.—S. Mayer (Sitzh. k. Akad. Wiss., 
Ixxxv, 1882, p. 69) recommends, for staining fresh tissue, a 
solution of 1 gramme of “ Violet B ” (Bindschedler and 
Busch, Bale) in 300 c.c. of 0-5 per cent, salt solution. In 
this liquid connective-tissue cells stain rapidly and energeti- 
cally. Elastic fibres and smooth muscle also stain, but of 
different tints. 
For Banvier’s method of artificial oedemata for the study 
of areolar tissue, see his Traite, p. 329. 
For Flemming’s observations on the development of connective-tissue 
fibrils, see Festschr. B. Virchow gewidmet, &c., 1, 1891, p. 215; Zeit.f. 
wiss. Mile., ix, 2, 1892, p. 225. 
For Pianese’s very complicated double stain with carmine and picro- 
nigrosin, see Journ. Boy. Mic. Soc., 1892, p. 292. 
Freeboen (Amer. Mon. Mic. Journ., 1888, p. 231; Journ. Boy. Mic. 
Soc., 1889, p. 305) recommends (for sections) picro-nigrosin, made by mix- 
ing 5 c.c. of 1 per cent, aqueous solution of nigrosin with 45 c.c. of aqueous 
solution of picric acid. Stain for three to five minutes, wash with water, 
and mount in balsam. Connective-tissue fibres bright blue, nuclei blackish, 
all the rest greenish yellow. 
792. Benecke’s Stain for Fibrils.—Benecke (Verb. d. anat. 
Ges.,vii Vers., 1893, p. 165; Zeit. f. wiss. Mik., xi, 1, 1894, 
p. 7 9) finds that by a slight modification of Weigert’s Fibrin 
Stain (post, § 813) good stains of connective-tissue fibrils and 
some other elements may be obtained. The modification 
consists in taking for the decoloration a mixture of anilin 
with xylol instead of pure anilin, as in Weigert’s form of the 
process. Benecke takes a mixture of two parts of anilin with 
three of xylol. The process is therefore essentially similar to 
that of Ivromayer given above, § 665. Connective-tissue 
fibrils are stained of a deep blue, elastic tissue red or violet, 
glia-fibres of the same tone as the connective tissue. 
793. Unna’s Specific Stains for Collagen (Monatsh. /. prakt. 
Dermatol., xviii, 1894, p. 509; Zeit. f wiss. Mik., xi, 4, 1894, SOME OTHER HISTOLOGICAL METHODS. 
445 
p. 518). Unna finds that the method of Benecke (last §) 
is unsurpassed for the demonstration of collagen fibrils alone, 
but prefers the following- whenever it is desirable to obtain 
at the same time good images of the ground substance and of 
other elements of preparations. 
1. The Orcein Method.—Sections of alcohol material are 
stained for five minutes in Grubler’s strong solution of poly- 
chromatic methylen blue. They are then brought for fifteen 
minutes into a neutral 1 per cent, solution of orcein in abso- 
lute alcohol, rinsed in pure alcohol, cleared in bergamot oil, 
and mounted. Nuclei blue, collagenous ground-substance 
dark red, granules of Mastzellen carmine red, protoplasm of 
Plasmazellen blue. 
2. The Method of Sulphosalts. —(a) Stain for five or ten 
minutes in an aqueous 2 per cent, solution of Saurefuclisin, 
rinse, treat for one or two minutes with saturated aqueous 
solution of picric acid, dehydrate (two minutes) in absolute 
alcohol saturated with picric acid, rinse with pure alcohol, 
clear and mount. (6) Stain for twenty seconds in aqueous 
1 percent, solution of Wasserblau, rinse, treat for five minutes 
with neutral aqueous 1 per cent, solution of safranin. Rinse 
and put into absolute alcohol until the blue colour reappears, 
clear with bergamot oil and mount. Collagen light blue, 
nuclei red. 
794. Fat.—Dekhuysen (see Flemming, in Zeit. f. wiss. Mik., 1889, 
pp. 39, 178) has discovered that fat that has been stained black by treat- 
ment with chromo-aceto-osmic acid (not with pure osmic acid) is dissolved 
in the course of a few hours in turpentine. It is dissolved also in xylol, 
ether, and kreasote. Flemming finds that very good demonstration pre- 
parations may be made by treating fatty tissue with chromo-aceto-osmic 
acid, staining with safranin or gentian, and then treating for a few hours 
with turpentine until all the fat is dissolved. The optical hindrance caused 
by the high refraction of the fat being thus eliminated, nuclei and cytoplasm 
may be studied to far greater advantage than in the usual preparations. 
795. Granule-cells (“ Plasmazellen ” and “ Mastzellen ”). — In 
1874 there were described by Waldeyer (Arch. f. mik. Anat., Bd. xi) cer- 
tain special cells existing between the bundles of connective tissue, besides 
the flat cells, and lymphatic and fat cells. They are large round cells con- 
taining large granules ; Waldeyer called them plasma cells (“ Plasma- 
zellen”). Later on, Ehrlich (ibid., xiii, 1877) distinguished in the same 
tissue and in other places certain cells containing large granules, which 
have a superficial resemblance to Waldeyer’s plasma cells, but which differ 446 
CHAPTER XXXIV. 
from them in staining reaction (Verhandl. Berl. Physiol. Ges., January 17th, 
1879 ; Reichert, n. Du Bois Reymond’s Arch., 1879, p. 166). Ehrlich 
named these food cells (“ Mastzellen ”), intending to express thereby the 
opinion that these cells are derived from fixed connective-tissue cells by a 
transformation brought about by exalted nutrition. 
If (Korybutt-Daszkiewicz, Arch. f. mik. Anat., xv, 1878, 
p. 7) frogs be kept for two months (in summer) without food, 
then placed in a reservoir of running water and well fed for 
four weeks, “ plasma-cells ” will be found in abundance, 
especially in the neighbourhood of the nerves. 
It is a pretty general character of these elements that they 
take the stain of anilin colours and retain it on treatment with 
alcohol with greater energy than other tissue cells. And 
further (at least so far as regards the true Mastzellen of 
Ehrlich) that in successful preparations they show the nucleus 
unstained, the general mass of cytoplasm unstained or but 
slightly coloured, and, in the cytoplasm, the characteristic 
granules very intensely stained. 
The staining reactions of the granules are very similar to 
those of bacteria. In order to distinguish them from these 
it may be observed that the stain of anilins is removed from 
the granules, but not from bacteria, by treatment with a 
weak solution of carbonate of potassium (Squire, Methods and 
Formulae, p. 46). 
796. Ehrlich’s Classification of Granules.—Cell-granules in 
general have been classified by Ehrlich, according to their 
respective degrees of affinity for one or the other of the three 
groups of dyes defined in § 268, into (1) « granulations, or 
eosinophilous granulations, being such as in a mixture of dyes 
select the most acid stain there present; (2) the j3 granula- 
tions, or amphophilous granules,' being such as in a mixture 
of basic and acid dyes take up both; (3) the y or basophilous 
granules, being such as take up basic dyes only (such are 
the granules of Mastzellen; (4) the 3 granulations, also baso- 
philous ; and (5) the £ granulations, or neutrophilous granules, 
being such as take up neutral anilin dyes, such as methyl 
blue and “acid” fuchsin [acid fuchsin is, however, a weakly 
acid dye]. I do not propose to enter minutely into the subject 
of these reactions, as it is one that cannot profitably be treated 
apart from the histology of the elements in question, and will 
confine myself to mentioning a few well-known methods. SOME OTHER HISTOLOGICAL METHODS. 
447 
Some of these have already been given; viz., the Ehrlich- 
Biondi mixture, § 306; Ehrlich’s C mixture, or acidophi- 
lous mixture, § 323, for eosinophilous granulations; and his 
triacid mixture, § 307, which stains the a, j3, and e granula- 
tions. 
797. Ehrlich’s “ Mastzellen ” (Arch. f. mik. Anat., 1876, p. 
263).—The tissues must be first well hardened in strong 
alcohol (chromic acid and its salts must be avoided). They 
are then placed for at least twelve hours in a staining fluid 
composed of— 
Absolute alcohol .... 50 c.c. 
Aqua ...... 100 c.c. 
Acid. acet. glacial . . . . 12| c.c. 
—to which has been added enough dahlia to give an almost 
satui’ated solution. After staining, the preparations are 
transferred to alcohol, which washes out the stain from all 
but the plasma-cells, and may then be mounted in resin- 
turpentine solution. 
Mucus-cells and fat-cells are also sometimes stained by 
these solutions. 
Other Media.—In a similar way other soluble anilins may 
be employed (in the form of a fluid containing 7h per cent, 
of acetic acid), — primula, iodine violet, methyl violet, pur- 
purin, safranin, fuchsin ; of these, methyl violet gives the 
best results. 
798. “Mastzellen.”—Schiefferdecker (Schiefferdecker and 
Kossel’s Gewebelehre, p. 329) recommends the following, 
after Orth. A piece of mesentery of a rat is brought into a 
solution of gentian violet in anilin water (p. 190), which is 
carefully heated over a flame until vapour begins to be given 
off and then allowed to remain for a couple of hours (or the 
heating may be omitted, and the preparation allowed to stand 
for twenty-four hours). It is then rinsed in water, washed 
out until almost colourless in hydrochloric acid alcohol, rinsed 
in water, eounterstained (if desired) in carmine, and mounted 
in balsam. Nuclei red, granules blue. 
799. Plasma-cells (Nordmann, Beitr. z. Kenntniss d. 3Iast- 
zellen, Inauguraldiss., Helmstedt, 1884).—Nordmann finds it 
useful to employ a solution of vesuvin containing 4 to 5 per 448 
CHAPTER XXXIV. 
cent, of hydrochloric acid. Sections should remain for a few 
minutes in the solution, and then be dehydrated with absolute 
alcohol. The paper quoted contains a detailed discussion of 
the microchemical reactions of granule-cells. 
800. Plasma Cells and Mastzellen.—Unna, in his paper, Zeit. 
f. wiss. Mik., vii, 4, 1892, p. 475, gives the following: 
a. For Plasma Cells. 
Methylen blue . . . . DO 
Caustic potash . . . . 0’05 
Distilled water . . . 100‘0 
Add a few drops of this to ten, fifty, or one hundred vols. 
of anilin water (p. 190) in a watch-glass, and stain (alcohol 
material, or at most sublimate and alcohol material, not 
chromic material) for half an hour, several hours, or over 
night. Dehydrate rapidly in absolute alcohol, differentiate 
in creosol (details not given), rinse in xylol, and mount in 
balsam. 
b. General Stain, also bringing out Plasma Cells. 
Methylen blue . . . . 1*0 
Carbonate of potash . . . 1*0 
Distilled water. . . . 100*0 
Alcohol .... 20*0 
Heat on a water-bath until reduced to lOO’O. Use for 
staining undiluted, or diluted with one vol. of anilin water. 
Differentiate (details not given) with glycol, styron, or 
creosol. Mastzellen are not differentiated. 
c. Stain giving Red Mastzellen with Blue Plasma Cells. 
Methylen blue , . . . 1*0 
Kali Carbon, (natron carbon, ammon. 
carbon) . . . . 1*0 
Aq. dest. (Aq. carbolisata, chloroforma) 100*0 
Dilute about 100-fold, stain slowly, treat with. 70 to 80 per 
cent, alcohol, differentiate in styron, and bring through ber- 
gamot oil or xylol into balsam. In this process the granules SOME OTHER HISTOLOGICAL METHODS. 
449 
of the Mastzellen stain red in consequence of the formation 
of methylen red in the staining bath. 
In a paper “Ueber Plasmazellen, insbesondere bei Lupus” 
(for which see Monatsh. f. pralet. Dermatol., xii, 1891, p. 296, 
or Zeit. f. wiss. Mile., ix, 1, 1892, p. 92), Unna gives some 
directions concerning the process of differentiation with 
creosol. Creosol only differentiates the stain, it does not 
dehydrate the sections. Sections should, therefore, first be 
dried with blotting-paper, and should then be treated with 
absolute alcohol for a few seconds, or with anilm oil before 
applying the creosol. The time required for differentiation 
in the creosol is from a few minutes to a few hours. When 
the proper stage of differentiation is attained, it should be 
fixed with xylol and the sections mounted. The method 
shows Mastzellen of a cherry-red tone. 
In a paper, 1. c., xiii, 1891, p. 364 (see Zeit. f. wiss. Mik., xi, 1, 1892, 
p. 89), tan der Spek and Unna describe some further experiments. Main- 
taining the method of staining given above under a, they find that besides 
the differentiating agents given under b, a lengthy series of other reagents 
are available. In all of them Mastzellen are distinguishable from plasma 
cells by the reddish coloration of their granules. 
See also Unna, Monatsh. f. prakt. Dermatol., xix, 1894, p. 225 ; Zeit. f. 
wiss. Mik., xii, 2, 1895, p. 58. 
801. Plasma Cells and Mastzellen.—Bergonzini (Anat. Anz., 
vi, 1891, pp. 595—600; Zeit.f. wiss. Mile., ix, 1, 1892, p. 95) 
gives the following:—Mix 1 volume of 0‘2 per cent, solution 
of Saurefuchsin with 2 volumes of a like solution of methyl 
green, and 2 volumes of a like solution of gold-orauge, 
and filter through cotton wool. Stain alcohol or sublimate 
sections (after washing in water) for three to four minutes, 
wash for one or two minutes in water, bring into absolute 
alcohol for two minutes, clear in bergamot oil or pure creo- 
sote, wash in turpentine, and mount in balsam. 
One sort of gold-orange precipitates methyl green, and 
therefore cannot be used in this mixture. Orange G may be 
used instead (thus giving something very like Ehrlich-Biondi 
mixture), but in this case the acidophilous granules will stain 
greyish instead of red or orange. Basophilous granules ought 
to be green, weakly acidophilous granules red, strongly 
acidophilous ones orange or brown, nuclei always green. 
Other details 1. c. 450 
CHAPTER XXXIV. 
802. Plasma Cells.—Jadassohn {Arch. f. Dermatol, u. Syphilis, 
Erganzungsheft 1, 1892, p. 58; Zeit.f. wiss. Mile., ix, 2, 1892, p. 226) re- 
commends staining for not too long in a 1: 2000 strongly alkaline or borax 
solution of Thionin, and washing out with acidulated water. 
See also von Maeschalko, Arch. f. Dermatol. u. Syphil., xxx, 1895, 
p. 3 ; Zeit.f. wiss. Mik., xii, 1, 1895, p. 64. 
803. Clasmatocytes.—Ranvier (C. B. Acad, des Sci., 1890) 
has described under the name of “ clasmatocytes ” certain 
cells that occur in the thin connective membranes of verte- 
brates, and that show a close affinity to Mastzellen, possessing 
processes that break up and form islands of granules. He 
demonstrates them as follows :—A piece of suitable membrane 
(epiploon of mammalia, mesentery of batrachia) is stretched 
secundum artem on a slide, and a few drops of 1 per cent, 
solution of osmic acid are allowed to fall on to it. After one 
or two minutes it is washed with water and stained with 
concentrated aqueous solution of methyl violet 5b diluted with 
ten parts of distilled water. When sufficiently stained the 
preparation is covered with a thin glass cover and examined. 
Glycerin may be added to make the preparation permanent, 
but does not succeed very well, as it causes the stain to 
diffuse. Brun’s glucose medium (§ 420) would probably be 
preferable. 
804. Elastic Tissue.—Two of tlie most salient characters of 
elastic fibres are that they have a great affinity for osmium, 
staining with much more rapidity than most other tissue- 
elements, and that they are not changed by caustic soda or 
potash. A further character is that they have a great affinity 
for certain anilin dyes, especially Victoria blue. 
For a review of the older methods of Balzer, Unna, Lust- 
garten, and Herxheimer, see the paper by Martinotti in Zeit. 
f. wiss. Mik., iv, 1, 1887, p. 31. 
The method of Lustgarten has been given in § 281. The 
colour used by him was called “ Victoriablau 4B./’ and this 
is probably an important detail. 
The method of Martinotti (l.c.) is as follows :—Fix in a 
■chromic liquid, wash, stain for forty-eight hours in strong 
(5 per cent. Pfitzner’s) solution of safranin, wash, dehydrate, 
clear, and mount in balsam. Elastic fibres are stained an 
intense black, the rest of the preparation showing the usual 
characters of a safranin stain. SOilB OTHER HISTOLOGICAL METHODS. 
451 
The staining will be performed quicker if it be done at the temperature 
of an incubating stove (GrRiESBACH, ibid., iv, 1887, p. 442). And Ferria 
(ibid., v, 3, 1888, p. 342) says that clearer preparations will be obtained if 
the sections be left for a long time, say twenty-four hours, in the alcohol, or 
be treated for a short time with very dilute alcoholic solution of caustic 
potash. This decolourises more completely the ground of the preparations. 
Another safranin method, which seems to have the fault of requiring a 
too minute attention to details, is that of Mibelli, see Mon. zool. italiano, 
1, p. 17, or Zeit. f. wiss. Mik., vii, 2, 1890, p. 225 (the report in Journ. Boy. 
Mic. Soc., 1890, p. 803, is vitiated by a misprint). 
805. Unna’s Modified Orcein Method (Monatsh. /. 'pralct. 
Dermatol., xix, 1894, p. 397; Zeit.f. iviss. Mik., xii, 2, 1895, 
p. 240).—The following solution is made Griibler’s orcein, 
1 part; hydrochloric acid, 1 part; absolute alcohol, 100 parts. 
The sections are put into a porcelain capsule with just enough 
of the stain to cover them, and the whole is warmed in a 
stove or over a naked flame to about 30° C. After ten to 
fifteen minutes the stain becomes quite thick, owing to the 
evaporation of the alcohol. The sections are then well rinsed 
in alcohol, cleai’ed and mounted. Elastin dark brown, 
collagen light brown. 
For Unna’s earlier orcein method, see last edition, or Monatsh. f. prakt. 
Dermatol., xii, 1891, p. 394 (Zeit.f. wiss. Mik., ix, 1, 1892, p. 94). 
See also Zenthoefer, in Unna’s Dermatol. Studien, 1892, or Zeit. /• 
wiss. Mik., ix, 4, 1893, p. 509 ; Ivoppen, Zeit. f. iviss. Mik., vi, 4, 1889, p. 
473; and vi, 1, 1890, p. 22, or last edition; Btirci, Journ. Boy. Mic. Soc., 
1891, p. 831, and 1892, p. 292, or last edition ; Hansen, Virchow’s Archiv, 
cxxxvii, 1894, p. 25 ; Zeit.f. iviss. Mik., xi, 3, 1894, p. 383; Kttltschizky, 
ibid., xiii, 1, 1896, p. 74, or the original, Arcli.f. mik. Anat., xlvi, 1895, p. 
673. 
Bone.* 
806. Bone, Non-decalcified (Ranvier, Traite, p. 297).— 
Ranvier points out certain precautions that it is necessary to 
take in the preparation of sections of dry bone. In general, 
the bones furnished by “ naturalists,” or procured in ana- 
tomical theatres, contain spots of fatty substance that prevent 
good preparations from being made. Such spots are formed 
* For a minutely detailed review (40 pages, with references to 80 
memoirs) of the whole subject of the technique of hone, see the paper of 
Schaffer, Die Methodik der histologischen Untersuchung. des Knochen- 
gewebes, in Zeit.f. wiss. Mile., x, 2, 1893, p. 167. 452 
CHAPTER XXXIV. 
when bones are allowed to dry before being put into water 
for maceration; when a bone is left to dry the fat of the 
medullary canals infiltrates its substance as fast as its water 
evaporates. 
Bones should be plunged into water as soon as the sur- 
rounding soft parts have been removed, and should be divided 
into lengths with a saw whilst wet. The medulla should 
then be driven out from the central canal by means of a jet 
of water; spongy bones should be submitted to hydrotomy. 
This may be done as follows :—An epiphysis having been 
removed, together with a small portion of the diaphysis, a 
piece of caoutchouc tubing is fixed by ligature on to the 
cut end of the diaphysis, and the free end of the piece of 
tubing adapted to a tap through which water flows under 
pressure. 
As soon as the bones, whether compact or spongy, have 
been freed from their medullary substance they are put to 
macerate. The maceration should be continued for several 
months, the liquid being changed from time to time. As 
soon as all the soft parts are perfectly destroyed, the bones 
may be left to dry. When dry, they should be of an ivory 
whiteness, and their surfaces exposed by cutting of a uniform 
dulness. 
Thin sections may then be cut with a saw and prepared by 
rubbing down with pumice-stone. Compact pumice-stone 
should be taken and cut in the direction of its fibres. The 
surface should be moistened with water and the section of 
bone rubbed down on it with the fingers. When both sides 
of the sections have been rubbed smooth in this way, another 
pumice-stone may be taken, the section placed between the 
two, and the rubbing continued. As soon as the section is 
thin enough to be almost transparent it is polished by rubbing 
with water (with the fingers) on a Turkey hone or lithographic 
stone. Spongy bone should be soaked in gum and dried 
before rubbing down (but see von Koch’s copal process, ante, 
§ 174, and Ehrenbaum’s colophonium process, § 175). 
The process of Weil {Zeit. f. wiss. Mik., r, 2, 1888, p. 200), which is 
intended for bone and teeth, has been given § 176. 
Rose (Anat. Anz., vii, 1892, pp. 512—519; Zeit. f. wiss. Mik., ix, 4, 
1893, p. 506) points out some precautions that it is well to take. He 
penetrates first with a mixture of cedar oil and xylol, then with pure xylol, SOME OTHER HISTOLOGICAL METHODS. 
453 
and imbeds in solution of dammar in chloroform or xylol. The method can 
be combined with Golgi’s impregnation. 
Nealey (Amer. Mon. Mic. Journ., 1S84, p. 142 ; Journ. Roy. 
Mic. Soc., 1885, p. 348) says that perfectly fresh portions of 
bone or teeth may be ground with emery on a dentist’s lathe, 
and good sections, with the soft parts in situ, obtained in half 
an hour. 
AFhite (Journ. Roy. Mic. Soc., 1891, p. 307) recommends 
the following:—Sections of osseous or dental tissue should 
be cut or ground down moderately thin, and soaked in ether 
for twenty-four hours or more. They should then be put for 
two or three days into a thin solution of collodion stained 
with fuchsin (made by dissolving the dye in methylated spirit, 
adding the requisite quantity of ether, then the pyroxylin). 
The sections are then put into spirit to harden the collodion. 
After this they are ground down to the requisite thinness 
between two plates of old ground glass, with water and 
pumice powder, and mounted, surface dry, in stiff balsam or 
styrax, care being taken to use as little heat as possible. 
Lacunae, canaliculi, and dentinal tubuli are found infiltrated 
by the coloured collodion. 
For the method of Matschixsky (Arch. f. mile. Anat., xxxix, 
1892, p. 151; Zeit. f. wiss. Mile., ix, 3, 1893, p. 353), see last 
edition. 
For a similar infiltration and grinding method of Ruprecht, 
see Zeit. f. wiss. Mile., xiii, 1, 1896, p. 21, wherein see also 
quoted (p. 23) a method of Zimjiermann. 
Hopewell-Smith (Journ. Brit. Dent. Ass., xi, 1890, p. 310; 
Journ. Roy. Mic. Soc., 1890, p. 529) says that for preparing 
sections of teeth showing odontoblasts in situ the best plan is 
to take embryonic tissues. A lower jaw of an embryonic 
kitten or pup may be taken, and hardened in solution of 
Muller followed by alcohol, then cut with a freezing micro- 
tome. The knife cuts quite easily the thin cap of semi- 
calcified dentine and bone. 
807. Vitante (Intern. Monatssch.f. Anat. u. Phys., ix, 1892, p. 394; 
Zeit.f. wiss. Mile., ix, 3, 1893, p. 351) has made out that thin specimens of 
bone can be successfully treated by Golgi’s bichromate of silver process. He 
places portions of frontal bone of four to six months calves, which are not 
more than 3 to 4 mm. thick, for eight days in solution of Muller, then in the 
osmium bichromate mixture, and then in the silver solution. After impreg- 454 
CHAPTER XXXIV. 
nation the specimens should be decalcified, which may be done by putting 
them for twenty days into von Ebner’s mixture (§ 577), after which they 
should be well washed with water and brought into solution of carbonate of 
soda, and finally imbedded in paraffin. 
Another method recommended by Vivante is as follows:—Fix for five or 
six ’days in solution of Flemming, wash and decalcify with von Ebner’s 
mixture, wash, treat with carbonate of soda, imbed in paraffin, cut, and stain 
the sections for an hour in 02 per cent, solution of quinole'in blue (§ 321). 
Wash out in equal parts of alcohol and water followed by pure water, dry 
the sections in a stove at 40° C. (both alcohol and glycerin must be avoided), 
clear with bergamot oil, and mount in dammar. Nuclei violet, protoplasm 
and ground substance different shades of blue. 
For Underwood's gold process for teeth, see p. 257; and for that of 
Lepkowsky, see Anat. Anz., vii, 1892, p. 274; Zeit.f.wiss. Mile., ix, 3, 
1893, p. 355 ; or the last edition of this work. 
808. Bone, Decalcified (Flemming, Zeit. /. wiss. Mik., 1886, 
p. 47).—Sections of decalcified bone are made with the free 
hand. They are soaked in water, and brought in a drop of 
water on to a glass plate, where they are spread out flat. 
The excess of water is removed with blotting-paper, and the 
sections are covered with another glass plate, to prevent them 
from rolling. The whole is brought into a plate and covered 
with alcohol. After the lapse of half an hour the sections 
have become fixed in the flat position, and may be brought 
into absolute alcohol without risk of their rolling. To mount 
them, wash them with fresh alcohol (which may be followed 
by ether); lay them again flat on glass, and cover them with 
a double layer of blotting-paper and a somewhat heavy glass 
plate, and let them dry for a day in the air or in a stove. 
When they are dry, put a drop of melted balsam on a slide, 
and let it spread out flat and cool. Prepare a thin glass 
cover in the same way, put the section on the prepared slide, 
cover it with the prepared cover, put on a clip, and warm. 
By this process sections can be very expeditiously pre- 
pared, which show the lacunar system injected with air in 
quite as instructive a manner as non-decalcified sections. 
Kolliker {Zeit. f. wiss. Zool., xliv, 1886, p. 662) recom- 
mends the following process for the demonstration of the 
fibres of Sharpey in decalcified bone. Sections are treated 
with concentrated acetic acid until they become transparent, 
and are then put for one quarter to one minute into a con- 
centrated solution of indigo-carmine, then washed in water 
and mounted in glycerin or balsam. In successful prepara- SOME OTHER- HISTOLOGICAL METHODS. 
455 
tions the fibres of Sharpey appear stained of a pale or dark 
red, the remaining bone-substance blue. 
Zachariades (Zeit.f. wiss. Mile., x, 4,1893, p. 447) has the following :— 
Bone is decalcified by means of picric acid, is repeatedly washed until all 
the acid is removed, then put into alcohol and sectioned. The sections are 
placed on a slide and treated for a few seconds with 1 per cent, solution of 
osmic acid. They are then stained, either for twenty-four hours in a weak 
aqueous solution of quinolein blue, or for a few minutes in a saturated 
aqueous solution of safranin. They are then treated on a slide with a drop 
of 40 per cent, solution of caustic potash, the slide being warmed over 
a flame until the sections spread out flat. The excess of potash is then 
removed and the sections are carefully washed with water, covered and 
examined. The safranin preparations may be permanently preserved in 
glycerin containing a small proportion of safranin. It is said that prepara- 
tions thus made demonstrate that there exist in fresh bone, at all ages of 
the subject, ramified cells possessing a membrane, and easily isolated by the 
action of potash. Their ramifications anastomose with one another and 
with the ramifications of other cells, the whole forming a protoplasmic 
network enveloped in a membrane. 
809. Stains for Cartilage (and Decalcified Bone).—For an 
excellent discussion (especially as regards staining) of the 
methods that have been recently recommended for these 
objects, see the exhaustive paper of Schaffer in Zeit. f. wiss. 
Mile., v, 1, 1888, which gives in sufficient detail all the methods 
in question. The following appear to be the best :— 
Schaffer’s safrcinin method. This method is due in its 
principle to Bouma (Centralb. f. d. med. Wiss,, 1883, p. 866). 
I give it in the form to which it has been brought by the 
careful study of Schaffer (Zeit. f. wiss. Mile., v, 1, 1888, p. 17). 
Sections of bone decalcified with nitric acid (chromic acid 
may be used, but the stain will be less brilliantly contrasted) 
are stained for half an hour to one hour in 0'05 per cent, 
aqueous solution of safranin, washed with water, put for two 
or three hours inOT per cent, solution of corrosive sublimate, 
and examined in glycerin. In order to make permanent 
preparations, the sections on removal from the sublimate 
are rinsed with alcohol, pressed on to a slide with filter- 
paper, cleared for a long time in bergamot oil or clove oil, 
and mounted in xylol balsam. 
This is a double stain ; cartilage, orange; bone, uncoloured 
(or green in chromic objects) ; marrow, red. 
Bayerl’s method for ossifying cartilage {Arch. f. mik. Anat., 456 
CHAPTER XXXIV. 
1885, p. 35), is as follows:—Portions of ossified cartilage are 
decalcified as directed § 578, cut in paraffin, stained in 
Merkel s borax-carmine and indigo-carmine mixture, § 340, 
and mounted in balsam. 
Max Flesch (Zeit. f. wiss. Mik., 1885, p. 351) particularly 
recommends this process for the study of the development of 
dental tissue. 
Aqueous solution of benzoazurin has been commended as a 
stain for ossifying cartilage by Zschokke, see Zeit. f. wiss. 
Mik., x, 3, 1893, p. 381. 
A process recommended by Baumgakten has been given, 
§ 342. 
Moerner (Slcandinavisches Arch. f. Physiol., i, 1889, p. 216; Zeit. f. 
wiss. Mik., vi, 4, 1889, p. 508) gives several stains for tracheal cartilage, 
chiefly as microchemical tests, for which see the last edition. 
See also a critique of these methods by Woltees in Arch.f. mik. Anat., 
xxxvii, 1891, p. 492; Zeit.f. wiss. Mik., viii, 3, 1891, p. 383; and on the 
whole subject of cartilage, see Schiefferdeckee’s Gewebelehre, p. 331. 
Blood. 
810. It might be supposed that for the study of blood it 
would suffice to prick a finger, place a drop of blood on a 
slide, cover, and examine it. But this is by no means the 
case. “ Blut ist ein ganz besonderer Saftand will not 
yield up its secrets to such simple wooing. The technique of 
blood is most elaborate; see, for instance the voluminous 
work of Hayem, Du sang et de ses alterations anatomigues, pp. 
1035, with 126 figures, Paris, Masson, 1889 (a report of over 
twenty pages on this important woi'k is contained in Zeit. f. 
wiss. Mik., vi, 3, 1889, p. 330, et seq.) ; Lowit, 8 it zb. k. Akad. 
wiss. Wien, 3, lxxxviii, 1883; xcii, 1885; xcv, 1887; Zieg- 
ler’s Beitr. z. path. Anat., x, 1891, p. 214; Zeit. f. wiss. Mik., 
vi, 1889, pp. 74, 76; viii, 3, 1891, p. 371 ; Arch. f. mik. Anat., 
xxxviii, 1891, p. 524; Zeit. f. Wiss. Mik., ix, 2, 1892, p. 233; 
Studien zur Physiol, u. Path. d. Blutes u. d. Lymphe, Jena, 
Fischer, 1892; Ehrlich, Zeit. f. klin. Medicin, i, 1880, 3, 
p. o 58 ; Zeit. f. wiss. Mik., i, 1884, p. 382, and other papers; 
Muller, Sitzb. k. Acad. Wiss. Wien, xcviii, 3, p. 219; Zeit. 
f. wiss. Mik., ix, 3, 1893, p. 365; Griesbach, Zeit. f. wiss. Mik., 
vii, 3, 1890, p. 326; and many other investigators. 
* Goethe’s Faust, i, 4, line 1387. SOME OTHER HISTOLOGICAL METHODS. 
457 
It is out of the question for me to attempt to abstract these 
memoirs in the space at my disposal, so I must confine myself 
to giving a few methods that may be useful to the general 
student, referring the specialist to the original papers. 
811. Fixing and Preserving Methods.—The time-honoured 
process of drying drops of blood over a flame gives rise to 
great deformation of the elements, and should be abandoned 
as far as possible. It is better to mix the blood at once 
with some fixing and preserving medium, and study it as a 
fluid mount. Some examination liquids and stains for fresh 
blood in the fluid state are given in the next §. 
Most recent authors (Biondi, Mosso, Max Flesch) are 
agreed that by far the most faithful fixing agent for blood- 
corpuscles is osmic acid. A drop or two of blood (Biondi re- 
commends two drops exactly) is mixed with 5 c.c. of osmic 
acid solution, and allowed to remain in it for from one to 
twenty-four hours. The exact degree of concentration of the 
osmium solution is a somewhat important point, and must be 
made out by experiment for each form. As a rule it should 
be strong, 1 to 2 per cent. According to Biondi, 2 per cent, 
is best. Fixed specimens may be preserved for use in acetate 
of potash solution (Max Flesch, Zeit. f. wiss. Mile., v, 1,1888, 
p. 83). 
Griesbach also (op. cit., p. 328) prefers osmic acid, not 
only as being a first-rate fixing agent, but because it can be 
combined with certain stains without decomposing them. He 
mentions methyl green, methyl violet, crystal violet, saf- 
ranin, eosin, Saurefuchsin, rhodamin, and iodine in potassic 
iodide. 
Kossi (Zeit. f. wiss. Mile., vi, 4, 1889, p. 475) advises a mix- 
ture of equal parts of 1 per cent, osmic acid, water, and 
strong solution of methyl green, permanent mounts being 
made by means of glycerin cautiously added. 
The mercurial liquids of Pacini (§ 400) used to be con- 
sidered good. Hayem (op. cit.; see also Zeit. f. wiss. Mik., 
vi, 3, 1889, P- 335) has a similar formula, viz. sublimate 0’5, 
salt 1, sulphate of soda, 5, and water 200. This should be 
mixed with blood in the proportion of about 1:100. Eosin 
may be added to it. Lowit’s formula (Sitzb. le. Alead. Wiss. 
Wien, xcv, 3, p. 129; Zeit. f. wiss. Mile., vi, 1, 1889, p. 75) 458 
CHAPTER XXXIV. 
consists of 5 c.c. cold saturated sublimate solution, 5 grms. 
sulphate of soda, 2 grms. salt, and 300 c.c. water. Mosso 
finds, however, that both of these are too weak in sublimate. 
Of course other well-tried fixing fluids, such as Flemming’s 
solution, or Hermann’s, may also be used for blood. 
Lavdowsky (in a long paper in Zeit. f. wiss. Mik., x, 1, 1893, p. 4) 
describes some remarkable l’esults obtained by fixing with 2 per cent, iodic 
acid, and staining with Neu-Victoriagriin, methyl violet 6 b, or gentian 
violet, a process which is said to reveal the presence of nuclei in elements 
generally considered to be apyrenematous. 
812. Stains for Blood.—Blood prepared as above can be 
satisfactorily stained with many of the usual reagents. 
Eosin stains rose-red all parts of blood-corpuscles that 
contain haemoglobin (see Wissowsky, Arch. f. mih. Anat., 
1876, p. 479); parts that do not contain haemoglobin, such as 
the nucleus, remaining unstained. This suggests double- 
staining with eosin and haematoxylin. 
Wissowsky (1. c.) stains in a solution of equal parts of 
eosin and alum in 200 parts of alcohol, and then with haema- 
toxylin. 
Moore (The Microscope, 1882, p. 73 ; Jo urn. Roy. Mic. Soc,, 
1882, p. /14) stains for three minutes in a similar solution 
without the alum, washes, and stains for two minutes in a 
1 per cent, aqueous solution of methyl green. Red corpuscles, 
red ; nuclei and white corpuscles, bluish green. 
The liquid of Chenzinsky has been given (§ 315). It stains 
nuclei and eosinophilous granules. 
Merkel’s carmine and indigo-carmine stain has been dis- 
cussed above (§ 340). 
Fresh (unfixed) blood is perhaps best treated as follows 
(Bizzozero and Torre, Archivio per le Scienze mediche, vol. iv, 
No. 18, 1880, p. 390):—Dilute a drop of blood with 0‘75 per 
cent, salt solution in which has been dissolved a little methyl 
violet. rlhis liquid in no wise affects the form of the elements, 
stains intensely the nucleus of the red corpuscles, and, in the 
white, stains the nucleus intensely, and the protoplasm less 
intensely. May be used for the study of bone-marrow and 
spleen. 
i or the staining of the blood-plates of Bizzozero, this 
observer (Arch. f. path. Anat. u. Phys. ; Zeit. f. wiss. Mile., SOME OTHER HISTOLOGICAL METHODS. 
459 
1884, p. 389) employs a 0.02 per cent, solution of methyl 
violet in salt solution, or a 1 : 3000 solution of gentian violet. 
Toison {Journ. Sci. med. de Lille, fev., 1885; Zeit. f. wiss. 
Mih., 1885, p. 398) recommends that blood be mixed with the 
following fluid : 
Distilled water . . . .160 c.c. 
Glycerin (neutral, 30° Baume) . 30 „ 
Pure sulphate of sodium . . 8 grammes. 
Pure chloride of sodium . . 1 gramme. 
Methyl violet 5 B . . . 0‘25 ,, 
(The methyl violet is to be dissolved in the glycerin with 
one half of the water added to it; the two salts are to be 
dissolved in the other half of the water, and the two solu- 
tions are to be mixed and filtered.) White blood-cor- 
puscles stain in this medium in five or ten minutes; the 
maximum of coloration is attained in from twenty to thirty 
minutes. White blood-corpuscles, violet; red blood-cor- 
puscles, greenish. 
Ferrier's liquid is said to have a sp. gr. similar to that of 
liquor sanguinis. Fuchsin, 1 grm. ; water, 150 c.c.; rectified 
spirit, 50 c.c.; dissolve, and add glycerin, 200 c.c. (from 
Squire's Methods and Formulae, p. 39). 
Leclerq’s fuchsin followed by malachite green, or Congo followed by 
gentian and eosin, see Bull. Soc. Beige de Mic., xvi, 1890, p. 61; or Journ. 
Boy. Mic. Soc., 1890, p. 675. 
Dekhuysex’s methylen blue and acid fuchsin mixture, see Verhandl. 
Anat. Gesellsch., 1892, p. 90; ox Journ. Boy. Mic. Soc., 1893, p. 116. 
It goes without saying that the Ehrlich-Biondi mixture, 
(§ 306), and Ehrlich’s triacid and acidophilous mixtures, 
(§§ 307 and 323) will be found most valuable reagents in many 
haematological researches. Lowit (Ziegler’s Beitr. z. path. 
Anat., &c., x, 1891, p. 214; Zeit. f. iviss. Mik., viii, 3, 1891, 
p. 371) obtained instructive results by staining sublimate 
preparations for one to two minutes in a concentrated Ehrlich- 
Biondi solution, and examining in water or glycerin. 
For details as to the reactions of the granules of leucocytes and of Ldwit’s 
“ pyrenogenous” corpuscles, see the original paper; also Ehelich’s 
“ Methodologische Beitr. z. Physiol., &c., der Leucocyten,” in Zeit.f. klin. 
Med., i, 1880, 3, p. 558; cf. Zeit. f. wiss. Mik., i, 1884, p. 382, and later 
papers of Lowitz in Anat. Anz., vi, 1891, p. 344, and Arcli.f. mik. Anat., 
xxxviii, 1891, p. 524 (Zeit.f. iviss. Mik., ix, 2, 1892, p. 233). Also, Lovell 460 
CHAPTER XXXIV. 
GtULLand (Journ. of Physiol, xix, 1896, p. 385). For some details con- 
cerning the carrying out of Ehrlich’s cover-glass preparation method, see 
Keinbach, in Arch. f. Min. Chirurg., xlvi, 1893, p. 486 (Zeit.f. wiss. Mih., 
xi, 2,1894, p. 258). For the process employed by Foi (.Festschr. B. Virchow 
gewichn., &c., 1891, i, p. 481; Zeit.f. wiss. Mih., ix, 2, 1892, p. 227) see 
§ 352. 
■fhe elaborate paper of Muller mentioned in § 810 is too rich in detail 
to bear abstracting here. A novelty in it is the impregnation of cover--lass 
preparations with gold chloride by Ranvier’s formic acid process. 
813. Weigert’s Fibrin Stain (.Fortschr. d. Med., v, 1887, 
No. 8, p. 228; Zeit.f. wiss. Mih., iv, 4,1887, p. 512). Sections 
(alcohol material) are stained in a saturated solution of gen- 
tian or methyl violet in anilin water (§ 278). They are brought 
on to a slide and mopped up with blotting-paper, and solution 
of lugol is poured on to them. After this has been allowed 
to act for a sufficient time they are differentiated and cleared 
m anilin oil without previous dehydration with alcohol. They 
are simply mopped up with blotting-paper, and a drop of 
anilin is poured on to them. The anilin soon becomes dark, 
and is then changed for fresh once or twice. The sections 
are bj this means differentiated and cleared at the same time. 
When this has been done, the anilin is thoroughly removed 
by means of xylol, and a drop of balsam and a cover are 
added. This stain may be applied to celloidin sections with- 
out previous removal of the celloidin. Fibrin is sharply 
stained in blue, bacteria and fungi are also stained of a very 
dark blue. See also the modifications of this method by 
Kromayer (§ 665) and Benecke (§ 792). 
814. Demonstration of Blood-plates of Bizzozero (Kemp, Studies 
Jr. the Biol. Lab. Johns Hopkins TJniv., May, 1886, iii, No. 6; 
Nature, 1886, p. 132).—The mere demonstration of the blood- 
plates of Bizzozero is easy enough. A somewhat large drop 
of blood is placed on a slide, and quickly washed with a small 
stream of normal salt solution. The blood-plates are not 
washed away, because they have the property of adhering 
to glass; and on bringing the slide under the microscope 
they will be seen in large numbers. If it be desired to make 
permanent preparations of them, they should first be fixed. 
This is done by putting a drop of osmic acid solution on the 
finger before pricking it. SOME OTHER HISTOLOGICAL METHODS. 
461 
For Bizzozero’s recent methods for the numeration of these elements 
and for the study of their regeneration, see his paper in Festsclir. B. Vir- 
chow gewidm., &c., 1, 1891, p. 459 ; or the report of the methods in Zeit. f. 
wiss. Mik., ix, 2, 1892, p. 229. 
For the application of some digestion methods to the study of blood-plates, 
see Liliexfeld, Arch.f. Anat. u. Physiol., Physiol. Abth., 1892, p. 115; 
or Zeit.f. wiss. Mik., ix, 3, 1893, p. 363. 
For methods for obtaining large quantities of blood-plates, see Drxjebin, 
D ie Herstellung wagharer Mengen von Blutplattchen, Jurjew, 1893 ; Zeit. 
f. wiss. Mik., x, 4, 1893, p. 493. 
815. Biondi’s Section Method for Blood (Arch.fmik. Anat., xxxi, 
1888, p. 103).—A process of imbedding in agar-agar. See last edition, or 
Journ. Boy. Mic. Soc., 1888, pp. 313, 659. Schieffekdecker says that 
celloidin may be employed (see his Gewebelehre, p. 389). 
816. Mucin.—Hoyer, who has made a special study of the 
staining reactions of mucin in tissues (Arch. f. mik. Anat., 
xxxvi, 1890, p. 310; see also Zeit. f. wiss. Mik., viii, 1, 1891, 
p. 67), has the following conclusions : 
Glands. 
The mucin of mucus cells and goblet cells, both of Verte- 
brates and Invertebrates, stains with basic tar colours, but 
not with acid tar colours (see above, § 268). More or less 
specific stains of mucin are obtained, for instance, with 
hydrochlorate or nitrate of rosanilin, commercial fuclisin, 
Griibler’s “neutral” fuchsin (“ n. Unna”), magenta, Magdala 
red, iodine green, methyl green, dahlia, methyl violet, gen- 
tian, iodine violet, crystal violet, Victoria blue. A similar 
reaction is obtained with alum-hsematoxylin solutions, whilst 
carmine behaves like the acid coal-tar dyes, and affords no 
stain. 
Hoyer obtained his best results by means of thionin (violet 
of Lauth), which gives a double stain, the tissues blue, the 
mucin elements ruddy violet. The dye called amethyst, 
prepared by Geigy and Co., of Bale, is a good succedaneum, 
and so are toluidin blue (obtainable from Griibler) or the 
phenylen blue of Oehler in Offenbach, and the p-phenylen 
blue of the Hochst manufactory. Like thionin, all these give 
metachromatic stains. 
Results less brilliant than those given by the above- 
mentioned stains, but nevertheless excellent, are obtained by 
means of methylen blue or Bismarck brown. Methylen green 
and safranin also give good reactions, but are some-what 462 
CHAPTER XXX1Y. 
inconstant in their effects. Methylen blue is particularly 
useful from its power of bringing out the merest traces of 
mucin. 
All of these colours may be used in the same way. Speci- 
mens should be fixed for two to eight hours in 5 per cent, 
sublimate solution, imbedded in paraffin, cut, and the sections 
stained for five to fifteen minutes in a very dilute aqueous 
solution of the dye (two drops of saturated solution to 5 c.c. 
of water). 
It is theoretically interesting to observe that hyaline carti- 
lage, the jelly of Wharton, and the Mastzellen of Ehrlich 
give the same reactions with basic dyes as mucin does, even 
their metachromatic reactions being identical. 
These conclusions had already been in part formulated by 
Sussdorf (.Deutsche Zeit. f. Thiermed., xiv, pp. 345, 349; see 
Zeit.f. wiss.Mik., vi, 2, 1889, p. 205). 
See also the important series of papers by Bizzozero, 
“ Suite ghiandole tubidciri deltubo gastro-enterico,” &c., in the 
Atti R. Accad. di Sci. di Torino, 1889 to 1892 ; reports in 
Zeit. f. wiss. Mik., vii, 1, 1890, p. 61; and ix, 2, 1892, p. 219. 
As regards the safranin reaction, it is well to note that it is 
not obtained with all brands of the dye ; that of Bindschedler 
and Busch, in Bale, gives it, whilst safranin 0 of Grubler 
does not. For the distinctive reactions of old and young 
mucin see the original, or the last-quoted report of the Zeit. 
f. iviss. Mik. 
The subject has recently been carefully investigated by 
Paul Mayer (“Ueber Schleimfarbung,” Mitth. Zool. Stat. 
Neajpel, xii, 2, 1896, p. 303). As regards the haematein reac- 
tion, he establishes the following propositions. 
1. If the staining solution contain free acid, as in Mayer’s 
acid liaemalum or Ehrlich’s haematoxylin, then as a general 
rule the secretion of mucus gland cells does not stain in it. 
2. If it contain a relatively large proportion of alum, say 
5 per cent., as in heemalum, or more, then also as a general 
rule the secretion of mucus gland cells does not stain in it. 
3. If it contain a l’elatively small proportion of alum, but a 
large proportion of haematein, then it stains many sorts of 
mucus, and at the same time stains chromatin strongly. 
As the result of his investigations Mayer gives the follow- 
ing formulae for specific stains of the secretion of mucus cells SOME OTHER HISTOLOGICAL METHODS. 
463 
(the distinction between mucin and mucigen is not taken in 
this paper). 
Mucicarmine.—One gramme of carmine is rubbed up in a 
capsule with 0-5 gramme of aluminium chloride (must be dry, 
not damp and yellow), and 2 c.c. of distilled water. The 
capsule is heated over a small flame for two minutes, until 
the originally light-red mixture has become quite dark. Stir 
thoroughly. The liquid having become thick, add a little 50 
per cent, alcohol, in which the warm mass ought to dissolve 
easily, and rinse the whole with more alcohol into a bottle. 
Make up to 100 c.c. with 50 per cent, alcohol, let it stand for 
at least twenty-four hours, and filter. This gives a stock 
solution, which is as a rule to be diluted for use tenfold with 
distilled or tap water. Exceptionally it may be diluted 
instead five or ten fold with alcohol of 50 per cent, or 70 per- 
cent. The stock solution may be obtained from Griibler 
and Co. Mucicarmine stains in sections or thin membranes 
mucus only. 
Muchsematein. — Hoematein 0’2 g., aluminium chloride 
0T g., glycerin 40 c.c., water 60 c.c. Eub up the heematein 
in a mortar with a few drops of the glycerin, then add the 
other ingredients. If it be desired to avoid employing a 
watery liquid, an alcoholic solution may be made in the same 
way by dissolving the haematein and aluminium chloride in 
100 c.c. of 70 per cent, alcohol, with or without the addition 
of two drops of nitric acid. Either of these solutions is a 
nearly pure mucus stain for sections or thin membranes. 
Mayer’s paper contains such other matter not abstracted 
here, which will well repay careful study. A fairly detailed 
abstract may be found in Zeit. f. wiss. Mik., xiii, 1, 1896, 
p. 38. 
For the methods of Unna for obtaining a specific stain of 
mucin by means of polychromatic methylen blue, see ibid., 
p. 42, or the original, Monatsli. f.prakt. Dermatol., xx, 1895, 
p. 365. 
817. Goblet Cells.—So far as these contain mucin they give 
the reactions above described (see Flemming, Zeit. f. wiss. 
Mik., 1885, p. 519; and Paulsen, ibid., p. 520). But the 
reactions appear to be different for different animals. Thus 
Paneth (Arch. f. mik. Auat., xxxi, 1888, p. 113, et. seq.) 464 
CHAPTER XXXIV. 
found that in the small intestine of the mouse the contents of 
the goblet cells did not stain with Bohmer’s haematoxylin. 
And the goblet cells of the small intestine of man did not 
stain with safranin. 
Ranvier, in a paper too long to be abstracted here (Comptes rend., 1887, 
3, p. 145; see also Zeit.f. iciss. Mik., v, 2, 1888, p. 233), describes a specific 
reation of perruthenic acid (Ru04) on goblet cells. By treating the pharyn- 
geal mucosa of the frog first for ten to twelve hours with vapour of osmium, 
and then for three minutes with vapours of perruthenic acid, the goblet cells 
are brought out with remarkable distinctness. The contained mucigen is 
stained black, but the vacuoles are unstained. Since perruthenic acid is very 
rapidly reduced by organic matter, Ranvier regards this reaction as a proof 
that the vacuoles do not contain any organic substance, but probably only 
water and inorganic salts. 
For detailed instructions for the study of goblet cells, see 
List, in Arch. f. mik. Anat., xxvii, 1886, p. 481. 
818. Salivary Glands.—Solger (TJnters. z. Naturlehre d. 
Mensclien, xv, 5 and 6, pp. 2—15 ; Zeit. f. wiss. Mik., xii, 3, 
1896, p. 374) demonstrates the granules in serous cells and 
half-moons of the submaxillary gland by means of formal- 
dehyde. The gland is hardened for two days or more in a 
10 per cent, solution of formol, and may then either be 
sectioned and examined in the wet way or imbedded in 
paraffin, and the sections stained with haematoxylin of Dela- 
field or of Ehrlich, the granules taking the stain. See 
also § 348. 
819. Liver.—See hereon the important papers of Ranvier, 
“ Les membranes muqueuses et le syst. glandulaire,” in the 
Journ. de Microgr., ix, x, 1885-6; Igacuschi, in Arch.f. path. 
Anat., xcvii, p. 142, or Zeit. f. iviss. MilL\, 1885, p. 243 (gold 
process for study of fibrous networks) ; Kupffer, Sitzb. Ges. 
f. Morph., &c., Miinclien, Juli, 1889, or Zeit. f. wiss. Mik., vi, 
4, 1889, p. 506 (haematoxylin stain for demonstration of ulti- 
mate bile-ducts, and application of Golgi’s silver bichromate 
method to the same object and to the study of fibrous net- 
works) ; Oppel, Anat. Anz., v, 1890, p. 143; vi, 1891, p. 165; 
and Zeit. f. wiss. Mik., vii, 2, 1890, p. 222 ; viii, 2, 1891, p. 224 
(also concerning the application of Golgi’s process to the 
above objects). SOME OTHER HISTOLOGICAL METHODS. 
465 
820. Other Methods for Glandular Structures.—Amongst 
numerous important papers that cannot be quoted here, see 
Ranvier, “ Le mecanisme de la secretion,” in Journ. de Microgr., 
x, 1886-7, and the valuable papers of Heidenhain in Pfliiger’s 
Archiv. 
The peculiar applicability of the Ehrlich-Biondi stain to 
this kind of work hardly needs pointing out. CHAPTER XXXV. 
SOME ZOOLOGICAL METHODS. 
821. Introduction.—It is the purpose of this chapter to 
describe such variations of the usual histological processes as 
are indicated for the study of organisms which offer special 
difficulties. The methods described are all of them such as 
give results applicable to histological study, and no account 
has been taken of such methods as are merely useful for the 
preparation of organisms for museum specimens or for coarse 
dissection. But of course in many cases the methods recom- 
mended for histological work will be found to give admirable 
results for the preparation of show specimens, and may be 
used for that purpose if desired. 
A further word here as to the employment of formaldehyde. 
The introduction of this reagent has singularly facilitated 
museum work. I consider it to be established that as a pre- 
servative agent for delicate organisms, it is in many cases 
greatly superior to alcohol. For as there is not even a partial 
dehydration, all risk of shrinkage on that head is done away 
with. But it is only, I think, as a preservative agent that it 
offers signal advantages. As a fixing agent, even for mere 
museum specimens, it will only, I think, be found to give 
good results in a very few cases. In the vast majority of 
cases, organisms should first be fixed secundum artem, in one 
of the tried and appropriate fixing agents, and then, after 
proper washing if necessary, be transferred to solution of 
formaldehyde for preservation. 
A valuable paper giving an account of a number of the processes employed 
in the Naples Zoological Station for the preservation of marine animals has 
been published by Salvatore Lo Bianco in Mitth. Zool. 8tat. Neapel, ix, 
1890, p. 435. References to the work of S. Lo Bianco in the remainder of 
this chapter are to that paper. An abstract of it is contained in Amer. 
Natural., xxiv, 1890, p. 856, and Journ. Roy. Mic. Soc., 1891, p. 133, and 
a very full account in Zeit.f. wiss. Mile., viii, 1, 1891, p. 54. SOME ZOOLOGICAL METHODS. 
467 
Tunicata. 
822. Fixation of Tunicata.—A method of Salvatore Lo 
Bianco for killing simple Ascidians in an extended state has 
been given above, § 22. In the paper quoted above this plan 
is recommended for Ciona, Ascidia, and Rhopalea. But many 
other forms, such as Clavellina, Perophora, Phallusia, Molgula, 
Cynthia, &c., should first be narcotised by treatment for from 
three to twelve hours with chloral hydrate (1 :1000 in sea 
water), then killed in a mixture containing chromic acid of 1 
per cent. 10 parts, acetic acid 100 parts, and finally hardened 
in 1 per cent, chromic acid. The small proportion of chromic 
acid in the above mixture is stated to be sufficient to neu- 
tralise the swelling action of the acetic acid. 
The compound Ascidians with contractile zooids are difficult 
to manage if one does not go the right way to work. The 
best process known to me is the following (due to van Ben- 
eden, kindly communicated to me by Dr. C. Maurice). Place 
the corms in clean sea water, and leave them alone for a few 
hours, in order that the zooids may become fully extended. 
Seize the corms with your fingers, and plunge them suddenly 
into glacial acetic acid. Leave them there for two, four, or 
six minutes, according to the size of the corms (which, of 
course, you will have taken care to select of as small a size as 
possible). Take them out of the acid with your fingers (or in 
some manner that may dispense with the employment of steel 
instruments, which would blacken the tissues) and bring them 
into 50 per cent, alcohol. Wash them thoroughly in that, and 
then bring them in the usual way through successively stronger 
alcohols. 
I most strongly recommend this process, which gives 
admirably preserved preparations quite free from any opacity 
either in the tissues or the tunic. The acid will not hurt the 
fingers if they be washed immediately. 
S. Lo Bianco recommends for this group the chloral hydrate 
process, followed by fixation with sublimate or chromo-acetic 
acid. 
Small pelagic Tunicates are very easily fixed with osmic 
acid or acid sublimate solution, with the exception of Anchinia. 
The not very numerous preparations I have made of this ex- 
ceedingly delicate form have all been unsatisfactory. And 468 
CHAPTER XXXV. 
some other similar forms may be found difficult. I have had 
a striking failure with Salpa virgula, which I fixed with 
“Flemming/5 and got a very poor preparation. The very 
similar S. pinnata is fixed perfectly in this medium. 
Molluscoida. 
823. Bryozoa.—For some methods of killing and fixing see 
§§ 11, 18, and 19. S. Lo Bianco employs for Pedicellina 
and Loxosoma the chloral hydrate method, fixing with sub- 
limate. For Flustra, Cellepora, Bugula, Zoobothrium, he 
employs the alcohol method of Eisig, § 16. For Cristatella, 
see § 17. 
Mollusca. 
824. Fixation of Mollusca.—Two groups at least amongst 
the Mollusca offer considerable difficulties in the way of fix- 
ation—Lamellibranchiata and Gastropoda. 
If it be attempted to take living and normal Lamellibran- 
chiata from the water they are contained in, in order to throw 
them into a fixing solution, they invariably withdraw their 
siphon and foot, shut their valves, and die in a state of con- 
traction. And if it be attempted to open the shell by force 
after death, the mantle is generally injured, and it is im- 
possible to get the foot and siphon into the extended state. 
De Castellarnau (La Estacion Zoolog. de Napoles, Madrid, 
1885) advises that they be killed by the method of Eisig and 
Andres described for Actiniee in § 16. Before dying, the 
animals protrude largely their feet, siphons, branchiae, and 
tentacles, and die with their shells open. They may be fixed 
as soon as insensibility has supervened, by bringing them into 
picro-sulpliuric acid, or some other rapidly killing fixing 
agent. 
The same methods recommended for Lamellibranchiata 
sometimes give good results with Gastropoda. The asphyxia- 
tion method has been described in § 23. 
S. Lo Bianco advises that Lamellibranchiata, Prosobran- 
chiata, and, amongst the Heteropoda, Atlantidm, be narcotised 
with 70 per cent, alcohol, § 16. Opisthobranchiata ought not 
to give much trouble, and I recommend sudden killing with 
liquid of Perenyi, or the acetic acid method, § 822. Aplysia SOME ZOOLOGICAL METHODS. 
469 
may first be narcotised by subcutaneous injection of about 
1 c.c. of a 5 to 10 per cent, solution of hydrochlorate of 
cocain (Robert, Bull. Sclent. de la France, &c., 1890, p. 449 ; 
Zeit. f. iviss. Mik., ix, 2, 1892, p. 216). 
For Ancylus, Andre (Rev. Suisse de Zool., 1, 1893, p. 429) 
recommends boiling water. 
For Pteropoda in general, liquid of Perenyi. Creseis is a 
difficult form. S. Lo Bianco advises the alcohol method, 
§ 16. 
Note the hydroxylamin method of Hoper, § 20. 
For preservation it may be noted that for Heteropoda and Pteropoda, 
formaldehyde (preceded by due fixation in a chromic or sublimate solution) 
is an admirable medium, so far at least as macroscopic appearances are con- 
cerned, and for this purpose superior to alcohol. 
825. Terrestrial Gastropods.—The asphyxiation method has 
been described in § 23. 
The quantity of mucus that exists in the integument of Gastropoda is 
often a serious obstacle in the way of preparation. Marchi {Arch.f. mik. 
Anat., 1867, p. 204) finds that if a living Limax be thrown into moderately 
concentrated salt solution it will throw off enormous quantities of mucus, 
and die in a few hours. The epidermis will be found well preserved. If 
the animal be thrown into osmic acid or Muller’s solution, if I understand 
the writer justly, no secretion of mucus will occur. 
826. Eyes of Gastropoda (Flemming, Arch.f. mik. Anat., 1870, 
p. 441).—The first difficulty here is to obtain the excision of 
an exserted eye. It is impossible to sever the exserted 
peduncle in a living- animal without its retracting at least 
partially before the cut is completed. Never mind that; make 
a rapid cut at the base, and throw the organ into very dilute 
chromic acid, or 4 per cent, bichromate; after a short time 
it will evaginate, and remain as completely erect as if alive. 
Harden in 1 per cent, osmium, in alcohol, or in bichromate. 
Carriere (Zool. Anz., 1886, p. 221) gives the following in- 
structions :—Remove the eye, together with a portion of the 
tentacle, and fix it by exposing it for some minutes to vapour 
of osmium. Make sections according to the usual methods 
and fix them on a slide with Schallibaum’s collodion. Stain 
them with picro-carmine; or first depigment them by very 
careful treatment with very dilute eau de Javelle, and then 
stain with picro-carmine. Mount in dammar. Successful 470 
CHAPTER XXXV. 
preparations show the tissues perfectly preserved; but Car- 
riere has only been able to make the depigmentation process 
succeed with Helix pomatia; with Prosobranchiata he failed. 
827. Eyes of Cephalopoda and Heteropoda (Geenachee, Abh. 
naturf. Ges. Ealle-a.-S., Bd. xvi; Zeit. f. wiss. Mile., 1885, 
p. 244).—Fix in picro-sulphuric acid, or in a saturated solu- 
tion of corrosive sublimate in picro-sulphuric acid (this mix- 
ture is especially useful for Octopus, Eledone, and Sepia, but 
does not succeed with the pelagic forms, such as Loligo, 
Ommatostrephes, and Rossia). Depigmient the specimens with 
hydrochloric acid (in preference to the nitric acid used by 
Grenacher in former researches). The mixture § 599 may also 
be used. The operation of depigmentation may be combined 
with that of staining; if you stain with borax-carmine and 
wash out in the last-mentioned mixture the pig’ment will be 
found to be removed quicker than the stain is washed out. 
But this process is delicate, and requh’es a practised hand. 
The operation of depigmentation may be carried out on sec- 
tions, but it is better to use portions of retina of 2 to 5 mm. 
in thickness. Grenacher mounted his preparations in castor 
oil, see § 447. 
Similar methods are recommended by the same author for 
the eyes of Heteropoda (see Abh. naturf. Ges. Ealle-a.-S., 1886; 
Zeit. f. wiss. Mih., 1886, p. 243). 
828. Eyes of Chitonidse (Moseley, Quart. Journ. Mic. Sci., 
1885, p. 40).—Moseley worked by decalcifying the shell with 
nitric acid of 3 to 4 per cent, and making sections. 
829. Eyes of Pecten and other Forms, see Patten, in Mitth. 
Zool. Stat. Neapel, vi, 4, 1886, p. 733. 
830. Shell.—Sections of non-decalcified shell are easily 
obtained by the usual methods of grinding, or, which is often 
a better plan, by the methods of v. Koch or Ehrenbaum, 
§§ 174, 175. For sections of decalcified shell, Moseley, who 
has had great experience of this kind of work, particularly 
recommends the method of decalcification given above, 
§ 828. SOME ZOOLOGICAL METHODS. 
471 
831. Injection of Acephala (Flemming, Arch.f. mik. Anat., 
1878, p. 252).—To kill the animals freeze them in a salt-and- 
ice mixture, and throw them for half an hour into lukewarm 
water. They will be found dead, and in a fit state for in- 
jection. Chloroform and ether are useless (but see § 20). 
The injection-pipe may be tied in the heart; but when this 
has been accomplished there remains the problem of occlud- 
ing cut vessels that it is impossible to tie. To this end, after 
the pipe has been tied, the entire animal is filled and covered 
up with plaster of Paris. As soon as the plaster has hardened 
the injection may be proceeded with. 
832. Maceration Methods for Epithelium of Mollusca.—For the 
study of ciliated epithelium the following methods are recom- 
mended by Engelmann {Pfinger’s Arch., xxiii, 1880, p. 505) : 
Cyclas Cornea (intestine), maceration in osmic acid of 0‘2 
per cent, (after having warmed the animal for a short time to 
45° to 50° C.). Also, concentrated boracic acid solution. 
The Intra-cellular Processes of the Cilia.—The entire intra- 
cellular fibre apparatus may be isolated by teasing fresh epi- 
the liumfrom the intestine of a Lamellibranch (e. g. Anodonta) 
in either bichromate of potash of 4 per cent., or salt solution 
of 10 per cent. To get good views of the apparatus in situ 
in the body of the cell, macerate for not more than an hour 
in concentrated solution of boracic or salicylic acid. Very 
dilute osmic acid (e.g. 0T per cent.) gives also good results. 
The “ lateral cells ” of the gills are best treated with strong 
boracic acid solution (5 parts cold saturated aqueous solution 
to 1 part water). 
Bela Haller’s Mixture, see § 554. 
Brock’s Medium, § 549. 
Mobius’s Media, § 550 ; the second of these is much recom- 
mended by Droost (Morphol. Jahrh., xii, 2, 1866, p. 163) for 
Cardium and My a. 
See also the media recommended by Patten (.Mitth. Zool. 
Stat. Neapel, vi, 4, 1886, p. 736). Sulphuric acid, 40 drops 
to 50 grammes of water, is here recommended as a most 
valuable macerating and preservative agent. Entire molluscs, 
without the shell, may be kept in it for months. 472 
CHAPTER XXXV. 
Arthropoda. 
833. General Methods for Arthropoda.—It may safely be 
stated that, as general methods for the study of chitinous 
structures, the methods worked out by Paul Mayer (see §§ 7 
and 9, and also 75, 240, and 241) are superior to all others. 
It is absolutely necessary that all pi-ocesses of fixation, wash- 
ing, and staining should be done with fluids possessing great 
penetrating power. Hence picric acid combinations should 
in general be used for fixing, and alcoholic fluids for washing 
and staining. Concentrated picro-sulphuric acid is the most 
generally useful fixative, 70 per cent, alcohol is the most 
useful strength for washing out, and tincture of cochineal in 
alcohol of 70 per cent. (§ 241) is a very generally useful stain- 
ing fluid. Mayer’s hmmacalcium (§ 257) may sometimes be 
preferable, and alcoholic carmine and borax-carmine will 
occasionally give more satisfactory results. 
Alcoholic picro-sulphuric acid may be indicated for fixing- 
in some cases. 
Some forms are very satisfactorily fixed with sublimate. 
Such are the Copepoda and the larvie of Decapoda. It is 
sometimes indicated to use the sublimate in alcoholic solution. 
Some Copepoda, however (Copilia, Sapphirina), are better 
preserved by means of weak osmic acid, and so are the Ostra- 
coda. In many cases the osmic acid will produce a sufficient 
differentiation of the tissues, so that further staining may 
be dispensed with; Copilia and Phyllosoma are examples of 
forms that may be prepared in this simple manner. The 
pyrogallic process (§ 377) may often prove helpful in the 
study of such forms. 
For Ostracoda, Muller {Fauna u. Flora d. Golfes von Neapel, 
xxi (Ostracoda) 1894, p. 8) recommends fixing in a mixture 
of 5 parts of ether and 1 of absolute alcohol, followed by 70 
per cent, alcohol. 
834. Methods for Clearing and Softening Chitin.—The employ- 
ment of eau de Javelle or eau de Labarraque, as suggested 
by Looss, for making chitin transparent and permeable to 
reagents has been described above, § 570. 
List [Zeit. f. wiss. Mik., 1886, p. 212) has obtained good 
results with Coccidse by treating them (after hardening) fo* SOME ZOOLOGICAL METHODS. 
473 
eighteen to twenty-four hours with eau de -Javelle, diluted 
with four volumes of water. After washing out with water, 
the objects may be dehydrated with alcohol and imbedded in 
paraffin, the chitin being sufficiently softened to allow of their 
being penetrated and good sections being obtained. You may 
stain before imbedding, with alum-carmine or picro-carmine 
(five to six days). 
The same methods are applicable to the preparation of the 
ova of Insecta—for instance, Periplaneta (see Morgan, Am. 
Mon. Mic. Journ., ix, 1888, p. 234). 
835. Other Depigmentation Methods.—Besides the depig- 
mentation processes discussed in Chap. XXYI, the following 
methods are available. 
Sazepin’s Method for Antennse of Chilognatha [Mem. Acad. 
Imp. St. Petersb., xxxii, 9, 1884, pp. 11, 12).—Sazepin 
treated antennae that have been dehydrated with alcohol by 
steeping them in chloroform. The reaction is slow, the chitin 
becomes gradually less opaque, but the pigment does not 
entirely disappear. In order to remove the last trace of 
it, it will be sufficient if a drop of fuming nitric acid be 
now added to the chloroform. The mixture must be occa- 
sionally agitated, in order to prevent the acid from floating 
on the chloroform. The reaction is complete in twenty-four 
hours. 
Employed in this manner, nitric acid has no injurious 
action on tissues. 
836. Eyes of Arthropods.—Lankester and Bourne [Quart. 
Journ. Mic. Sci., 1883, p. 180) prepared the eyes of Limulus 
as follows:—Alcohol, turpentine, paraffin; sections made and 
carefully depigmented under the microscope with nitric acid 
of 5 to 10 per cent., then mounted in balsam, some after 
staining with borax-carmine, others unstained. Non-depig- 
mented sections also mounted in the same manner. 
Hickson [ibid., 1885, p. 243) prepared the eye of a fly as 
follows :—Remove the posterior wall of the head, and expose 
the rest, with the eyes in situ, for twenty minutes to vapour 
of osmium. Wash for a few minutes in 60 per cent, alcohol. 
Harden in absolute alcohol. Make sections. To depigment 
them, mount them on a slide with Mayer’s albumen, remove 474 
CHAPTER XXXV. 
the paraffin with turpentine, treat them with absolute alcohol, 
and invert the slide over a capsule containing 90 per cent, 
alcohol to which a few drops of strong nitric acid have been 
added. Nitrous vapours are freely given off, and the pigment 
dissolves. The reaction may be stopped at any moment by 
washing with pure alcohol. 
For dissociation preparations, put the eye or the optic 
nerve for twenty-four hours into 5 per cent, solution of chloral 
hydrate, tease, and mount in glycerin. If the elements of the 
teased tissues be fixed to the slide by means of Mayer’s 
albumen, they may be washed with alcohol and stained in situ, 
or they may be depigmented before staining. 
The methods of Parker for eyes of scorpions have been 
given, § 593. 
For the eye of Homarus see Bull. Mus. Comp. ZooL, Cam- 
bridge, U.S.A., xx, 1890; p. 1 (Zeit.f. wiss. MiJc.,viii, 1, p. 82). 
In a later paper (Mitth. Zool. 8tat. Neapel, xii, 1895, p. 1 : 
Zeit. f. wiss. Mik., xii, 4, 1896; p. 496) Parker describes the 
application of the methylen-blue method to the study of the 
retina and optic ganglia in Decapods, especially in Astacus. 
He injected 0T c.c. of a 0‘2 per cent, solution into the ventral 
sinus. After twelve to fifteen hoars the animals were killed; 
the ganglia quickly dissected out and brought into aqueous 
solution of corrosive sublimate; which fixes the stain (see § 294). 
After fixation the preparations were either dehydrated by the 
methylal method; described in § 294, or in a modification of 
that process without the methylal. A stock of 8 per cent, 
solution of corrosive sublimate in absolute alcohol is prepared, 
and from this are prepared weaker grades of sublimate- 
alcohol, by dilution with saturated aqueous sublimate solution. 
For instance, to make 30 per oent. sublimate-alcohol you take 
30 c.c. of the absolute alcohol solution and 70 c.c. of the 
aqueous solution, and so on. The preparations are dehydrated 
by passing them through a succession of grades of sublimate- 
alcohol thus prepared, and of the strengths of 30 per cent., 
50 per cent., 70 per cent., and 95 per cent., remaining in each 
for a quarter of an hour. They then come into the 8 per 
cent, solution in absolute alcohol for an hour, then into a 
mixture of one part of this solution with one of xylol, remain- 
ing there also for an hour, and finally into pure xylol, in 
which they may remain till imbedded. SOME ZOOLOGICAL METHODS. 
475 
For the methods of Purcell for the eyes of Phalangida, see 
Zeit. f. wiss. Zool., lviii, 1894, p. 1; Zeit. f. wiss. Mile., xii, 1, 
1895, p. 44. He has the following’ stain. The objects are 
brought for twenty minutes into 50 per cent, alcohol warmed 
to 45° or 50° C., and saturated with picric acid. The pigment 
dissolves in this solution and stains the nuclei and some 
other parts of the rhabdoms, so that no further stain is 
required. 
See also the methods of Yiallanes (Ann. d. Sci. Nat., xiii, 
1892, p. 354; Journ. Boy. Mic» Soc., 1893, p. 260). 
837. Nerve and Muscle of Arctiscoida (Doyeee, Arch. f. mik. 
Anat., 1865, p. 105).—A score or so of Milnesium tardigradum are collected 
(it is well to have a large number, as the process hj no means succeeds with 
all individuals) and put into a test-tube with water that has been deprived 
of its air by boiling. A drop of oil is run on to the surface of the watei-, so 
as thoroughly to exclude the air. After twenty-four to forty-eight hours 
the animals will be found, not dead, but fixed and extended in a cataleptic 
state ; the circulation of the perivisceral fluid has ceased, the pigment of the 
cuticle has disappeared or collected into patches that are no hindrance to 
observation, the entire animal has gained in transparency, and the nervous 
and muscular systems stand boldly out. The animals are examined in boiled 
water, unless it be wished to study the phenomena of resuscitation, in which 
case spring water should be used. 
838. Phalangida (Eosslee ; see previous editions, or Zeit.f. wiss. Zool., 
xxxvi, 1882, p. 672). 
839. Macrotoma plumbea (Somheb ; see previous editions, or Inaug. 
Diss., 1884, p. 4; Zeit. f. wiss. Mile., 1885, p. 234). 
840. Bethe’s Stain for Chitin (Zool. Jahrb., Abth. f. Anat., 
viii, 1895, p. 544; Zeit. f. wiss. Mik., xii, 4, 1896, p. 498). 
Desirous of staining the chitinous hairs and plates of the 
otocyst of Mysis, Bethe found advantage in employing a 
process, borrowed from industrial dyeing, in which anilin 
black is produced on the tissue itself. Anilin black is a pro- 
duct of the oxidation of anilin hydrochloride. Bethe 
proceeds as follows :—Series of sections mounted on a slide 
are put for three or four minutes into a freshly prepared 10 
per cent, solution of anilin hydrochloride, to which has been 
added one drop of hydrochloric acid for every 10 c.c. They 
are then rinsed in water, and the slide is put with the sec- 
tions downwards into 10 per cent, solution of bichromate of 476 
CHAPTER XXXV. 
potash. The stain quickly begins to show itself, but is at 
first in general not sufficiently intense. The process is then 
repeated until the desired intensity of stain is obtained, 
care being taken to rinse the sections well with water after 
each of the operations, in order to avoid the formation of 
precipitates. The stain is at first green, but becomes blue 
in tap water or alcohol containing ammonia. 
The same paper contains a hint concerning the preparation 
of telsons for section cutting. They are put for eight to 
fourteen days into 40 per cent, alcohol, to which nitric acid 
is gradually added, so that by the end of that time they 
have been brought into alcohol containing 20 per cent, of 
the acid. This softens the chitin, and somewhat breaks 
down the structure of the otolith, so that good sections 
through it are occasionally obtained. 
Vermes. 
841. Cestodes.—This group must of course be chiefly studied 
by the usual section methods. As pointed out by Vogt and 
Yung (Traite d’Anat. comp, prat., p. 204), the observation of 
the living animal may be of service, especially in the study 
of the excretory system. And, as shown by Pintner, taeniae 
may be preserved alive for several days in common water to 
which a little white of egg has been added. 
Lonnberg (Centralb. f. Bakteriol. u. Parasitenk., xi, 1892, 
p. 89; Journ. Boy. Mic. Soc., 1892, p. 281) has kept Triseno- 
phorus nodulosus, a parasite of the pike, alive for a month in 
a slightly acid pepsin-peptone solution containing from 3 to 
4 per cent, of nutritive matter, and less than 1 per cent, of 
XaCl. 
Zeknecee (Zool. Jahrb., Abth. f. Anat., ix,1895, p. 92; Zeit.f. wiss. Mile., 
xii, 4, 1896, p. 494) has employed with success the bichromate of silver im- 
pregnation of Golgi. He kills Ligula in the osmio-bichromic mixture 
(4:1), impregnates as usual, makes sections in liver, and treats them by the 
hydroquinon process of Ivallius. Besides the peripheral and central nervous 
system, muscle-fibres, parenchyma cells, and the excretory vascular system 
are impregnated. 
He has also obtained good results by the methylen-blue method. 
842. Trematodes (Fischer, Zeit.f. wiss. Zool., 1884, p. 1).— 
Opisthotrema cochleare may be mounted entire in balsam. 
For sectioning, Fischer recommends imbedding in a mass SOME ZOOLOGICAL METHODS. 
477 
made by dissolving 15 parts of soap in 17*5 parts of 96 per 
cent, alcohol. This mass melts at about 60° C., penetrates 
very rapidly, and solidifies very quickly. The sections should 
be studied in glycerin. 
Wright and Macallum (Journ. of Morph., i, 1887, p. 1) find 
that Sphyranura is for most purposes best fixed in liquid of 
Flemming, and stained with alum-cochineal. 
Cercarise.—Schwarze {Zeit. f. wiss. Zool., xliii, 1886, p. 45) 
found that the only fixing agent that would preserve the his- 
tological detail of these forms was cold saturated sublimate 
solution warmed to 35°—40° C. 
843. Turbellaria.—Methylen blue will in some cases be 
found useful for the study of living specimens. 
For Rhabdocoela, Braun {Zeit.f.wiss. Mik., iii, 1886,p. 398) 
proceeds as follows :—For preparing entire animals, the 
specimens are got on to a slide, lightly flattened out with a 
cover, and killed by running under the cover a mixtui*e of 
three partsof liquid of Lang with one of 1 per cent, osmic acid 
solution. Other fixing media than that described were not 
satisfactory. (Bohmig, however, commenting on this, says 
that for some of the tissues, such as muscle and body paren- 
chyma, nitric acid and picro-sulphuric acid are very useful.) 
Sections may be made by the usual paraffin method. 
Fixing is difficult, and generally unsatisfactory. 
Delage (Arch, de Zool. exp. et gen., iv, 2, 1886; Zeit.f. wiss. Mile., iii, 
2, 1886, p. 239 ; Journ. Boy. Mic. Soc., 1886, p. 1073) strongly recommends 
fixation (of Rhabdocoela Accela) by tbe osmium-carmine mixture, § 237. 
Concentrated solution of sulphate of iron is also an excellent fixing medium. 
Tbe animals (Convoluta) die in it fully extended. Liquid of Lang was not 
successful. 
For staining, he recommends either the osmium-carmine stain or impreg- 
nation with gold formic acid, two minutes; 1 per cent, gold chloride, ten 
minutes ; 2 per cent, formic acid, two or three days in the dark. It is well 
to allow an excessive reduction to take place, and then lighten the stain by 
means of 1 per cent, solution of cyanide of potassium). 
Bohmig, commenting on the above, says that he has obtained very in- 
structive images with Plagiostomidse fixed with sublimate and stained with 
osmium-carmine. 
Von Graff (Die Organisation d. Turbellaria Acoela, Leipzig, 
1891; see Zeit. f. wiss. Mile., ix, 1, 1892, p. 76) has the 478 
CHAPTER XXXV. 
following remarks : Chromo-aceto-osmic acid, followed by 
hmmatoxylin, is good for the skin; but even this method will 
not afford a satisfactory preservation of the Rhabdites, which 
in Acoela and Alloiocoela seem to be destroyed by swelling, 
whilst in terrestrial and fresh-water Planaria, Polyclada, 
and most Rhabdoccela they are better preserved. The same 
method is also good for the parenchyma of Amphichoerus- 
cinereus, Convoluta paradoxa, and C. sordida. Sublimate is 
not good for these forms, but it is good for Convoluta 
Roscojfensis. For some forms it is important to avoid picro- 
carmine, which destroys the central parenchyma. The ner- 
vous system may be investigated by the methods of Delage. 
For Dendrocoela sublimate solutions, sometimes hot, appear 
indicated for fixing. Chichkoff (Arch, de Biol., xii, 1892, 
p. 438; Journ. Roy. Mic. Soc., 1893, p. 262) recommends the 
following for fresh-water Dendrocoela :—2 per cent, sublimate 
solution, 6 parts; 15 per cent, acetic acid, 4 parts; pure 
nitric acid, 2 parts; 14 per cent, chloride of sodium, 8 parts; 
and 2 per cent, alum, 1 part. The animals are said to die in 
it without contraction. Note also the mixtures of Lang, 
§ 58. Mayer’s tincture of cochineal, § 241, may be found 
useful for the study of glands, for which purpose the Ehrlich- 
Biondi stain may also be employed. 
844. Nemertina.—After considerable experience of this 
difficult group I have to say that I know of no method of 
fixation that will certainly give good results. My best results 
have always been obtained with cold saturated sublimate 
solution, acidified with acetic acid. I have tried most of the 
energetically hardening fixing agents, such as the osmic and 
chromic mixtures, and do not recommend them for this 
group, for they seem (the chromic mixtures and perchloride 
of iron in particular) to act as irritants, and provoke such 
violent muscular contractions that the whole of the tissues 
are crushed out of shape by them. And, besides, they do 
not kill as quickly as sublimate. 
I have found it a good plan to decapitate the animals (in 
the larger forms), cut them up quickly into lengths (not too 
long), and throw these sharply into the sublimate, the mus- 
cular contractions being less energetic in segments that are 
no longer in connection with the cerebral ganglia. SOME ZOOLOGICAL METHODS. 
479 
Perliaps a better method than this will be found in the 
simple process, suggested to me by Prof, du Plessis, of fixing 
with hot (almost boiling) water. On the few occasions on 
which I have tried it the animals have died in extension, 
without vomiting their proboscis ; and I think it is certainly 
worth trial, especially for the larger forms. 
I have tried Foettinger’s chloral hydrate method (§ 18). 
My specimens died fairly extended, but vomited their pro- 
boscides. According to S. Lo Bianco narcotisation with a 
solution of 0T to 0’2 per cent, in sea water is found successful 
at Naples. 
De Castellarnau (Estacion Zool. de Napoles, p. 137) says 
that Nemerteans can be successfully narcotised by Eisig’s 
alcohol method, described § 16, and I think the process may 
be a good one for some of the larger forms. 
Dendy (see Journ. Boy. Mic. Soc., 1893, p. 116) has suc- 
ceeded with Geonemertes by exposing it for half a minute to 
the vapour of chloroform. 
Intra vitam staining with methylen blue may be found 
useful in some cases. For the application of the methylen- 
blue method to the study of the nervous system see § 290. 
For staining fixed specimens in toto I hold that it is well- 
nigh necessary to employ alcoholic stains, for even the most 
delicate species are not satisfactorily penetrated by watery 
stains in any reasonable lapse of time. Borax-carmine or 
Mayer’s alcoholic carmine may be recommended; not so 
cochineal or hsematoxylin stains, on account of the energy 
with which they are held by the mucin which in general 
exists in such great abundance in the skin of these 
animals. 
Sections by the paraffin method, after penetration with oil 
of cedar (chloroform will fail to penetrate sometimes after the 
lapse of weeks). 
845. Nematodes.—The extremely impermeable cuticle of 
these animals is a great obstacle to preparation. According 
to Looss (Zool. Anz., 1885, p. 318) this difficulty may be over- 
come by treating the animals (or their ova, which are in the 
same case) with eau de Javelle or eau de Labarraque, in the 
manner described in § 570. 
For fixing, most recent authors recommend sublimate solu- 480 
CHAPTER XXXV. 
tions; chromic solutions seem to have a tendency to make the 
worms brittle. 
But according to zur Strassen {Zeit. f. wiss. Zool., liv, p. 
655), Bradynema, rigidum, a parasite of Aphodius fimetarius, 
ought to be fixed for at least twelve hours in mixture of 
Flemming. 
Augstein {Arch. f. Naturg., Jahrg. lx, 1, 1894, p. 255 ; 
Zeit. f. wiss. Mik., xii, 2, 1895, p. 227) found that for Stron- 
gylus filo-ria the best fixing agent was Mayer’s picro-nitric 
acid. 
Staining is frequently difficult, and sometimes alcoholic 
carmine, § 240, is the only thing that will give fair results. 
Braun (see Journ. Boy. Mic. Soc., 1885, p. 897) recommends that small 
unstained Nematodes be mounted in a mixture of 20 parts gelatin, 100 
parts glycerin, 120 parts water, and 2 parts carbolic acid, which is melted at 
the moment of using. Canada balsam, curiously enough, is said to some- 
times make Nematodes opaque. 
846. Demonstration of Living Trichinae (Barnes, Amer. Mon. Mic. 
Journ., xiv, 1893, p. 104 ; Journ. Roy. Mic. Soc., 1893, p. 406).—A piece 
of trichinised muscle of the size of a pea should be placed in a bottle in a 
mixture of 3 gr. of pepsin, 2 dr. of water, and 2 minims of hydrochloric 
acid. The whole should be kept at body temperature for about three hours 
with occasional shaking. The flesh and cysts being dissolved, the fluid is 
poured into a conical glass, and allowed to settle ; the trichinae are drawn 
off from the bottom with a pipette, got on to a slide with water, and exa- 
mined on a hot stage. 
847. Acanthocephali.—Jt is very difficult to kill Echino- 
rhynci so as to have the animals duly extended and the tissues 
well preserved. Neither corrosive sublimate nor strong osmic 
acid will, as a rule, attain this end, even after preliminary 
intoxication with tobacco smoke or chloroform, the animal 
thus treated dying contracted. 
Hamann, however {.Jen. Zeit. f. Naturw., xxv, 1890, p. 113; 
Zeit. f. wiss. Mik., viii, 2, 1891, p. 209), has succeeded with 
sublimate, and also with alcohol containing a little platinum 
chloride. 
Saefftigen (Morphol. Jcihrb., x, 1884, 120; Journ. Roy. 
Mic. Soc. [N.S.], v, 1885, p. 147) obtained the best results 
by killing gradually with 0‘1 per cent, osmic acid; the 
animals placed in this contract during the first hours, but 
stretch out again and die fully extended. 
Another method of killing is treatment with 0*1 per cent. SOME ZOOLOGICAL METHODS. 
481 
chromic acid; Echinorhynci live for days in it, but eventually 
die fully extended. 
Keiser (Biblioth. Zool., H. vii, 1 Halfte, 1891; Zeit. f. wiss. 
Mik., viii, 3, 1891, p. 363), found that a saturated aqueous 
solution of cyanide of mercury, warmed to 45° to 50° C., and 
allowed to act for from fifteen to sixty minutes, and then 
washed out with 70 per cent, alcohol, was the best of all 
fixing media for Acanthocephali. 
He also found the following’ mixture excellent : 
Picric acid ... 1 gramme. 
Cone, sulphuric acid . . 10 grammes. 
Chromic acid ... 1 gramme. 
Water .... 1000 grammes. 
To be warmed to 55° C., allowed to act for fifteen to twenty 
minutes, washed out for five to ten minutes with hot water, 
and afterwards for some days in 60 per cent, alcohol. 
848. Gephyrea.—Yogt and Yung {Anat. comp, prat., p. 373) 
direct that Siphunculus nudus be kept for some days in per- 
fectly clean basins of sea water, in order that the intestine of 
the animals may be got free from sand, which would be an 
obstacle to section cutting, and then anaesthetised with chloro- 
form, under which treatment they die extended, and may be 
fixed as desired. 
Ward {Bull. Mus. Comp. Zool., Cambridge, Harvard Coll., 
xxi, 3, p. 144) found the best plan was to put the anijnals into 
a shallow dish with sea water and pour 5 per cent, alcohol in 
a thin film on to the surface of the water. After four to eight 
hours, if the animals make no contractions on being stimu- 
lated, they may be removed to 50 per cent, alcohol. 
S. Lo Bianco says killing with 0’5 per cent, chromic acid 
or with 0’1 per cent, chloral hydrate in sea water may be 
tried, but either method is uncertain. Phascolosoma and 
Phoronis should be treated by the alcohol method. 
Apel (Zeit. f. wiss. Zool., xlii, 1885, p. 461) says that 
Priapulus and Halicryptus can only be satisfactorily killed by 
heat. The animals may either be put into a vessel with sea 
water and be heated on a water-bath to 40° C.; or they may 
be thrown as rapidly as possibly into boiling water, which 
paralyses them so that they can be quickly cut open and 482 
CHAPTER XXXV. 
thrown into one third per cent, chromic acid, or picro- 
sulphuric acid. 
849. Rotatoria.—By far the most important method for the 
study of this group consists in the observation of the living 
animals. Great difficulty exists in the way of getting them 
to keep sufficiently quiet. Vogt and Yung {Anat. com. prat., 
p. 420) say that a drop of solution of any of the soluble salts 
of strychnin run under the cover sometimes renders service. 
Weber {Arch, de Biol., viii, 4, 1888, p. 713) finds that 
strychnin, prussic acid, and curare act too strongly; of all 
the reagents he tried, 2 per cent, solution of hydrochlorate 
of cocain gave the best results. Warm water gave him good 
results for large species, such as those of Hydatina and 
Brachionus. 
Hardy {Journ. Boy. Mic. Soc., 1889, p. 475) recommends 
thick syrup added drop by drop to the water. Hudson {ibid., 
p. 476) mentions weak solution of salicylic acid. 
Hoeee’s hydroxylamin method has been given, § 20, and 
Tullberg’s chloride of magnesium method, §21; the pro- 
cesses of Eismond and Jensen, § 872, may be tried. Methylen 
blue, § 288, may be found useful. 
Permanent preservation of Rotifers has, until lately, been 
considered by those who have tried it to be well-nigh impos- 
sible. Now, however, thanks to the patient experimentation 
of Rousselet, this difficult problem may be considered to be 
fairly conquered. Rousselet now proceeds as follows (Journ. 
Quehett Mic. Club, v, March, 1895, p. 1) : The animals are 
got together in a watch-glass. They are narcotised by adding 
to the water at intervals a few drops of the following 
mixture— 
Hydrochlorate of coca'in, 2 per cent, solution . 3 parts. 
Methylated spirit . . . .1 part. 
Water . . . . . .6 parts. 
They are watched under a dissecting microscope, and at the 
moment when the cilia have ceased to beat, or are seen to be 
on the point of ceasing to beat, they are fixed by adding a 
drop of liquid of Flemming or of £ per cent, osmic acid. The 
fixing agent is allowed to act for half a minute or less, after 
which the animals are taken out with a pipette, and thoroughly SOME ZOOLOGICAL METHODS. 
483 
Avashed by passing' them through two or three Avatch-glasses 
of distilled water. They are then definitely mounted in 2 4 
per cent, solution of formaldehyde (formol 24 parts, distilled 
Avater 374 parts). 
For some details concerning variations of this method 
adapted to the preservation of the different forms, see the 
paper quoted. Mr. Rousselet has been good enough to show 
me a large series of preparations made by this method, and I 
can testify that they are most beautiful. 
Annelida. 
850. Cleansing Intestine of Lumbricus (Kukenthal, Journ. 
Roy. Mic. Soc., 1888; p. 1044).—Pat the animals into a tall 
glass vessel which has been filled up with bits of moistened 
blotting-paper. They gradually evacuate the earthy particles 
from the gut, and fill it instead with paper. 
Vogt and Yung (Trciite d’Anat. Comp. Prat., v) recommend 
coffee-grounds instead of paper; paper becomes rather hard 
when imbedded, whereas coffee-grounds cut fairly well. 
851. Chaetopoda.—Lumbricus maj be anaesthetised by putting 
the animals into water with a few drops of chloroform. Perrier 
has pointed out that it is better not to let the chloroform 
act directly in solution on the animals, but to put them into 
water in a shallow dish, set up a watch-glass with chloroform 
in the corner of it, and cover the whole. In half an hour the 
worms will be more or less narcotised, and if allowed to 
remain will die in a state of extension. 
Cereontaine (Arch, de Biol., x, 1890, p. 327; Zeit. f. wiss. 
Mik., viii, 2, 1891, p. 210) much recommends curare, ad- 
ministered by interstitial injection of a dose of about 2 c.c. of 
a 1 : 500 solution. The animal should afterwards be put into 
water, and after a quarter of an hour will be found dead. 
In order to kill Criodrilus lacuum, Collin (Zeit. f. toiss. 
Zool., xlvi, 1888, p. 474) puts tlie animals into a closed vessel 
with a little water, and hangs up in it a strip of blotting- 
paper soaked in chloroform. Kukenthal (Die mik. Technik, 
1885; Zeit.f. wiss. Mik., 1886, p. 61) puts Annelids into a glass 
cylinder filled with water to the height of 10 centimetres, and 
then pours 70 per cent, alcohol to a depth of 1 to 2 centi- 
metres on to the water. The animals will be found suffi- 484 
CHAPTER XXXV. 
ciently narcotised for fixation in from four to eight hours. 
For Opheliadse he also employs 0-l per cent, of chloral hydrate 
in sea water. 
Many marine Chaetopoda may be successfully narcotised 
(S. Lo Bianco) in sea water containing 5 per cent, of alcohol, 
or by means of the mixture, § 16. 
The Polychseta sedentaria offer the difficulty of a complex 
and very contractile branchial apparatus. They may some- 
times be satisfactorily fixed by bringing them rapidly into 
corrosive sublimate. Cold, not hot solutions should be taken, 
as heat frequently shrivels up the branchiae. The species of 
Polychseta errantia that offer a contractile branchial appara- 
tus, as Eunice and Onuphis, may be treated in the same way. 
S. Lo Bianco advises killing Chaetopteridse, Sternaspidie, 
Spirographis, Protula, by putting them for half an hour into 
1 per cent, chromic acid. I have satisfied myself that good 
show specimens can be obtained in this way; but I doubt the 
histological preservation of the parts being so good as with 
sublimate specimens. Some of the sedentaria may be got 
protruded from their tubes by leaving them for some hours 
in 0-l per cent, chloral hydrate in sea water (S. Lo Bianco). 
See also the methods §§ 18 to 23. 
Note also the liquid of Ehlers, § 43. 
Staining.—For the staining of small Annelids entire, I find 
carmalum gives very good results, I think better than borax- 
carmine or paracarmine. 
852. Blood-vessels of Annelids (Kukenthal, Zeit. f. wiss. 
Mik., 1886, p. 61).—The animals should be laid open and put 
for two or three hours into aqua regia (4 parts of nitric acid 
to 2 of hydrochloric acid). The ramifications of the vessels 
will then be found to be stained black, the rest of the 
preparation yellow. 
853. Nerves of Annelids.—The methylen-blue method and 
the bichromate of silver method of Golgi (the rapid method). 
For the latter see v. Lenhossek (Arch. f. mik. Anat., xxxix, 
p. 102 ; Zeit. f. wiss. Mik., ix, 3, 1893, p. 432). 
854. Hirudinea.—For the methods of killing see those given 
for Lumbricus in § 851, also §§ 18 to 23. 
Whitman (Meth. in mic. Anat., p. 27) recommends that they SOME ZOOLOGICAL METHODS. 
485 
be killed with sublimate. This reagent is said to kill leeches 
with such rapidity that they die in general without having 
time to change the attitude in which they were found at the 
moment when the liquid came into contact with them. 
I have obtained better results myself by narcotising with 
carbonic acid (§ 24), and fixing with liquid of Flemming. 
Small specimens of Nephelis are narcotised in a few minutes; 
large ones will require several hours. I have also found that 
lemon juice kills them in a state of very fair extension. 
Carmalum I find excellent for staining entire animals; 
curiously enough, I have found it to have a better penetration 
than borax-carmine or paracarmine. Ehrlich-Biondi mix- 
ture sometimes gives fine results with sections. 
Graf (Jen. Zeit., 1893, p. 165) states that he has obtained 
good results by narcotising with decoction of tobacco. 
Injection.—Whitman (Amer. Natural., 1886, p. 318) states 
that very perfect natural injections may often be obtained 
from leeches that have been hardened in weak chromic acid 
or other chromic liquid. He considers that these injections 
are the best for the purpose of the study of the circulatory 
system by means of sections. 
Of course Hirudinea (or any other Annelids) on which it is 
desired to make artificial injections must be killed by some 
procedure that leaves the tissues in a state that will allow the 
injection to run freely. 
Jacquet (Mitth. Zool. Stat. Neapel, 1885, p. 298) advises 
that leeches be put into water with a very small quantity of 
chloroform; they soon fall to the bottom of the vessel and 
remain motionless. They should be allowed to remain a day 
or two in the water before injecting them. 
Echinodermata. 
855. Holothurioidea.—These animals are difficult to fix on 
account of their contracting with such violence under the in- 
fluence of irritating reagents as to expel their viscera through 
the oral or cloacal aperture. 
S. Lo Bianco puts Holothurids into pure sea water until 
they have expanded their tentacles, then seizes them with 
forceps behind the tentacles, so as to mechanically render 
impossible their withdrawal, and immerses the anterior part 486 
CHAPTER XXXV. 
of the body in acetic acid, whilst at the same time an assistant 
injects 90 per cent, alcohol through the anus. 
Vogt and Yung (Anat. Comp. Prat., p. 641) say that Cucu- 
maria Planci (C. doliolum, Marenzeller) is free from the vice 
of expelling its intestines under irritation ; but they recom- 
mend that it be killed with fresh water, or by slow intoxi- 
cation with alcohol, chromic acid, or sublimate added to the 
sea water in which it is contained. 
Synapta may be allowed to die in a mixture of equal parts 
of sea water and ether or chloroform (S. Lo Bianco). 
Holothurids, Dr. Weber informs me, are admirably pre- 
served in formaldehyde; a weak solution is sufficient. 
856. Asteroidea.—Hamann (JBeitr. z. Hist. d. Echinodermen, 
ii, 1885, p. 2) finds it best to inject the living animal with a 
fixing liquid. The cannula should be introduced under the 
integument at the extremity of a ray, and the liquid injected 
into the body-cavity. The ambulacral feet and the branchim 
are soon distended by the fluid, and as soon as it seems to 
have penetrated sufficiently the animal is thrown into a quan- 
tity of the same reagent. 
The study of the eyes presents points of special difficulty. 
In order to study them in sections, with the pigment preserved 
in situ, the eye should be removed by dissection, should be 
hardened in a mixture of equal parts of 1 per cent, osmic acid 
and 1 per cent, acetic acid, and imbedded in a glycerin gum 
mass, or some other mass that does not necessitate treatment 
with alcohol (which dissolves out the pigment, leaving the 
pigmented cells perfectly hyaline). For maceration use one- 
third alcohbl, the aceto-osmic mixture failing to preserve the 
rods of the pigmented cells. 
Formaldehyde is not to be recommended for the preser 
vation of Asteroidea (Weber). 
857. Ophiuridea.—Should be killed in fresh water if it be 
desired to avoid rupture of the rays (De Castellarnau, La 
Est. Zool. de Napoles, p. 185). 
858. Echinoidea.—I advise that they be killed by injection 
of some fixing liquid. For preservation, formaldehyde has 
proved admirable in all respects, and greatly superior to 
alcohol (Weber). SOME ZOOLOGICAL METHODS. 
487 
859. Larvae of Echinodermata.—I am greatly obliged to my 
able friend Dr. Barrois for kindly writing down for me (for 
the Traite des Meth. techn., from which they are translated) 
the following instructions, which are the outcome of a pro- 
longed and minute study of the metamorphoses of the Echino- 
dermata. 
Pluteus.—In order to a fruitful study of the metamorphoses 
of the Echinoidea and Ophiuridea it is necessary to obtain 
preparations that offer the advantages presented by the study 
of the living larvae; and especially such as give distinct 
images of the different organs, and show the calcareous 
skeleton preserved intact (a point of considerable importance, 
since this skeleton frequently affords landmarks of the greatest 
value). These preparations should further possess the fol- 
lowing points :—They should give clear views of the region 
of formation of the young Echinoderm (which is generally 
opaque in the living larva). And they should possess suffi- 
cient stiffness to allow of the larva being turned about in 
any desired way, and placed in any position under the micro- 
scope. 
It is not very easy to obtain preparation fulfilling these 
conditions, on account of the difficulty of obtaining a selective 
stain whilst preserving the integrity of the calcareous skeleton. 
The following method is recommended :—Pluteus larvae are 
fixed in a cold saturated solution of corrosive sublimate, in 
which they remain not more than two or three minutes. They 
are then washed with water, and brought into dilute Mayer’s 
cochineal (§ 241). This should be so dilute as to possess a 
barely perceptible tinge of colour. The objects should remain 
in the stain for from twelve to twenty-four hours, being care- 
fully watched the while, and removed from the stain at the 
right moment and mounted in balsam, or, which is frequently 
better, in oil of cloves or cedar-wood. This method is per- 
fectly satisfactory for the study of the chief phases of meta- 
morphosis. 
Auriculctria and Bipinnaria.—The method described above 
is equally applicable to these forms, and seems to be altogether 
the best method for the study of the metamorphosis of Bipin- 
naria. The earlier stages of the metamorphosis of Auricularia 
are better studied by fixing with osmic acid, staining with 
Beale’s carmine, and mounting in glycerin. 488 
CHAPTER XXXV. 
Larvae of Gomatula.—The best method for the study of the 
embryonal development of Gomatula consists in fixing with 
liquid of Lang, and staining with dilute borax-carmine. It 
is important (for preparations that are not destined to be sec- 
tioned) to use only dilute borax-carmine, as the strong solution 
produces an over-stain that cannot easily be reduced. 
Narcotisation by chloral hydrate before fixing is useful, 
especially for the study of Pentacrinus larvae and of the young 
Synaptae formed from Auricularia. Without this precaution 
you generally get preparations of larvae either shut up 
(Pentacrinus), or entirely deformed by contraction (young 
Synaptae). 
See also MacBride on the development of Amphiura squamata, Quart. 
Journ. Mic. Sci., xxxiv, 1892, p. 131; Journ. Roy. Mic. Soc., 1893, p. 117 
(osmic acid followed by liquid of Muller and alcohol; decalcification with 
nitric acid in alcohol ; staining with Mayer’s paracarmine or hsemalum); and 
Seeliger on the development of Antedon, Zool. Jahrb., Abth. f. Anat., vi, 
1892, p. 161; Zeit.f. wiss. Mik., x, 2, 1893, p. 229. 
Goelenterata. 
860. Actinida.—Narcotisation.—For suitable narcotisation 
methods see §§ 13 to 23. 
Fixation.—In Le Attinie, Fauna u. Flora d. Golfes v. Neapel, 
Andres gives the following hints :—Hot corrosive sublimate 
often gives good results. In the case of the larger forms the 
solution should be injected into the gastric cavity, and a 
further quantity of the liquid be poured over the animals. 
Freezing sometimes gives good results. A vessel contain- 
ing Actiniae is put into a recipient containing an ice-and-salt 
freezing mixture and surrounded by cotton wool. After 
freezing, the block of ice containing the animals is thawed in 
alcohol or some other fixing liquid. 
Maceration.—For the Hertwigs’ well-known method (Jen. 
Zeit., 1879, p. 457) see § 553. The tissues should be left to 
macerate in the acetic acid for at least a day, and may then 
be teased in glycerin. 
List (Zeit. f. wiss. Mik., iv, 2, 1887, p. 211) recommends 
dilute liquid of Flemming. Tentacles of Anthea cereus and 
Sagartia 'parasitica treated for ten minutes with a mixture of 
100 c.c. of sea water with 30 c.c. of Flemming’s liquid (the 
strong solution, § 47), then washed out for two or three hours SOME ZOOLOGICAL METHODS. 
489 
in 0-2 per cent, acetic acid, and teased in dilute glycerin, give 
fine dissociations of the connective, sensory, and urticant cells 
of the ectoderm, and after removal of the epidermis allow of 
the demonstration of ganglion-cells and the supporting 
lamella. Picro-carmine may be used for staining. 
861. Zoantharia with Calcareous Skeletons are difficult to 
deal with on account of the great contractility of the polyps. 
Sublimate solution, which ought very often to be taken boil- 
ing, sometimes gives good results. De Castellarnau (La Est. 
Zool. de Napoles, p. 132) says that this process succeeds well 
with Dendrophyllia, Antipathes, Astroides, Cladocora and 
Carifophyllia. 
Sections.—For preparing sections, besides the usual methods 
for sectioning decalcified specimens, we have the valuable 
methods of von Koch aud Ehi-enbaum, §§ 174 and 175, which, 
being applicable to undecalcified specimens and furnishing- 
preparations showing atone and the same time soft parts and 
hard parts in situ, render most inestimable services. 
862. The Alcyonaria have also extremely contractile polyps. 
In a former edition I suggested for their fixation either hot 
sublimate solution or glacial acetic acid (§ 68). S. Lo Bianco 
has since recommended essentially similar processes. G-arbini 
(Manuals, p. 151) says that the polyps may be fixed in the 
state of extension by drenching- them with ether, and then 
bringing them into strong alcohol. 
Wilson (Mitth. Zool. Stcit. Neapel, 1884, p. 3) kills Alcyo- 
naria with a mixture of 1 part of strong acetic acid and 2 
parts of concentrated solution of corrosive sublimate, the 
animals being removed as soon as dead and hardened for two 
or three hours in concentrated sublimate solution. 
Schultze [Biol. Centralb., 1887, p. 760) says that for Pen- 
natulidse with large polyps the gradual addition of fresh 
water is a good plan. 
863. Zoantharia and Alcyonaria.—Braun (Zool. Anz., 1886, 
p. 458) recommends that for both Zoantharia and Alcyo- 
naria a little osmic acid be added to the sublimate employed 
for fixation. For Alcyonium palmatum, Sympodium coral- 
loides, Gorgonia verrucosa, Garyophyllia cyathus, and Palythoa 490 
CHAPTER XXXV. 
axinellse he proceeds as follows :—The animals are left for a 
day or two in a glass vessel, so that the polyps may become 
thoroughly extended. They are then suddenly drenched 
with a mixture of 20 to 25 c.c. of concentrated solution of 
sublimate in sea water with four to five drops of 1 per cent, 
osmic acid. This is allowed to act for five minutes. 
(This method also gives good results with Hydra and some 
Bryozoa and Rotifers.) 
864. Hydroidea, Polypoid Forms.—For suitable narcotisation 
methods see those quoted in last section. 
Fixation.—In general the polyps may be very well killed 
in saturated sublimate solution, in which they should be 
plunged for an instant merely, and be brought into alcohol. 
The solution should be employed cold in general for Gymno- 
blastea, hot for most Calyptoblastea. 
Ether attentively administered gives good results with Cam- 
panulai’idae. Hydra is very easily killed by treatment with a 
drop of osmicaoid on a slide. Breckenfeld (Anxer. Mon. Mic. 
Journ., 1884, p. 49) obtains good results by heating the 
animals in a drop of water on a slide for from three to five 
seconds over a petroleum lamp. The methods for sections are 
the usual ones. 
The methylen-blue method of intra vitam staining is appli- 
cable to this group, see Zoja, 1. c., § 288. 
865. Medusae : Fixation.—There is some difficulty in properly 
fixing the forms with, contractile tentacles, which easily roll 
up on contact with reagents. The best results I have had 
with these forms have been obtained by means of van 
Beneden’s acetic acid method, § 68, followed by alcohol. 
The secret of success with the long-tentacled forms lies in a 
trick of manipulation. Put sufficient acetic acid into a 
deepish dish, hold it in your left hand (or, better, in both 
hands if you have an assistant), and keep it moving in a 
circle so as to communicate a vortex motion to the liquid. 
Take up a medusa in a spoon with as little sea water as 
possible, and throw it into the moving liquid, and keep the 
liquid steadily swirling round so as to cause the tentacles to 
trail out at full length behind the animal until it is thoroughly 
fixed, then pass carefully into alcohol. Do not, unless you SOME ZOOLOGICAL METHODS. 
491 
are very expert, try to fix more than one medusa at a time; 
it is also better to keep the specimens separate, even in the 
alcohol, as, if several are together, it generally happens that 
their tentacles become entangled. The method is due to 
S. Lo Bianco. Oceania conica and Tiara may usefully, 
according to S. Lo Bianco, be narcotised with 3 per cent, 
alcohol in sea water before fixation. Liquid of Kleinenberg, 
which I have seen much used for the fixation of these and 
similar forms, is, in my opinion, histologically a very objec- 
tionable reagent for the purpose. 
Trachymedusm and Acalephaa may be fixed in the usual 
way in chromic or osmic mixtures. Osmic acid may conve- 
niently in some cases be added to the sea water containing 
the animals, which should be removed to fresh water as soon 
as they begin to turn brown. Cassiopeia borbonica, according 
to de Castellarnau, ought to be treated with osmic acid as 
described, and then put for two or three days into 5 per cent, 
solution of bichromate of potash. I have tried this process, 
with good results. 
866. Medusae : Sections.—I am not acquainted with any per- 
fectly satisfactory method of sectioning these extremely 
watery organisms. Paraffin and collodion will afford good 
sections of some organs, but are certainly not satisfactory as 
all-round methods for this group. Some modification of the 
method employed by the Heetwigs (New ensy stem der Medusen, 
1878, p. 5) might be successful. They imbedded in liver with 
the aid of glycerin gum, and hardened the objects and 
the mass in alcohol. I should think better results would be 
obtained by one of the freezing methods given in §§ 177 to 
184. 
867. Medusae: Maceration.—The methods of the Hertwigs, 
§ 553, have deservedly become classical for the study of the 
tissues of this group. 
Amongst other advantages of this process it should be 
noted that the reduction of osmic acid by albuminates is 
greatly hastened by the presence of acetic acid, which in the 
case of animals so transparent and poor in cells as Medusae is 
an advantage for the study of the nervous system ; for gan- 
glion-cells and nerve-fibrils reduce osmic acid quicker than 492 
CHAPTER XXXV. 
common epithelium-cells. They become greenish brown, and 
are easily distinguished from surrounding tissues. Doubtless 
in many cases the pyrogallic acid reaction, § 377, would give 
better results. 
The isolation of the elements of the macerated tissues is 
best done by gently tapping the cover-glass (which may be 
supported on wax feet). This gives far better results than 
teasing with needles. A camel-hair pencil also sometimes 
renders good service. 
868. Siphonophora.—This group contains some of the most 
difficult forms to preserve that are to be found in the whole 
range of the animal kingdom. You have not only to deal 
with the very great contractility of the zooids, but with the 
tendency to general disarticulation of the swimming-bells and 
prehensile polyps. 
The cupric sulphate method of Bedot (Arch. d. Sci. pliys. et 
nat., Juin, 1889, t. xxi, p. 556) recommended for the prepara- 
tion of Siphonophora and other delicate pelagic animals, is as 
follows :—Bedot directs that a large quantity of 15 to 20 per 
cent, solution of the salt be suddenly added to the sea water 
containing the animals. As soon as they are fixed (which 
happens in a few minutes) a few drops of nitric acid are to 
be added and mixed in (this is in order to prevent the forma- 
tion of precipitates), and the whole is left for four to five 
hours. The specimens are then to be hardened before bring- 
ing them into alcohol. Bedot recommends that this be done 
with solution of Flemming. The strong solution is the one 
that should be taken, and it should be added to the solution 
of sulphate containing the Siphonophore, about two volumes 
of it being taken for one of the sulphate solution. The whole 
should be left for at least twenty-four hours. After harden- 
ing in the mixture a few drops of 25 per cent, alcohol should 
be added to the fluid with a pipette, being dropped in as far 
as possible from the colony, which should be disturbed as 
little as possible; and further alcohol, of gradually increasing 
strength, should be added so gradually that the strength of 
70 per cent, be not attained under fifteen days at least. 
Isiuety per cent, alcohol should be used for definite pre- 
servation. 
I have tested this method. I do not find that the his- SOME ZOOLOGICAL METHODS. 
493 
tological preservation is superior to that obtained by means 
of the usual processes; but the method is certainly a valuable 
one in so far as it enables one to preserve specimens with all 
their swirnmitig-bells and polyps in situ, a result which is not 
obtained by means of the usual methods. 
Friedlaender (Biol. Centralbl., x, 1890, p. 483 ; Journ. Boy. 
Mic. Soc., 1890, p. 804) preserves this class of objects by 
inundating them with a mixture of 125 parts cupric sulphate, 
125 parts zinc sulphate, and 1000 parts water. 
S. Lo Bianco employs for the majority of Siphonophora a 
mixture of 10 c.c. of saturated solution of corrosive sublimate 
with 100 c.c. of 10 per cent, solution of copper sulphate. This 
is used as in Bedot’s process. Diphyes, Rhizophysa, and 
Physalia, however, are killed with sublimate solutions; Velella 
with chromo-picric acid, or a mixture of 100 c.c. of sublimate 
solution with 50 c.c. of 1 per cent, chromic acid; Porpita by 
poisoning with liquid of Kleinenberg*. 
Korotneff’s method of paralysing* with chloroform has 
been given in § 15. I would only add that I have seen 
Physophora very successfully killed by the careful adminis- 
tration of ether. 
Preservation, after fixation and washing, is greatly sim- 
plified by the use of formaldehyde instead of alcohol. Dr. 
Weber has prepared some beautiful specimens at Yillefranche 
by this method. 
869. Ctenophora : Fixation.—The small forms are very easily 
prepared by means of osmic acid. For the large forms see 
the paper of S. Lo Bianco, quoted § 821. 
Samassa has succeeded in making sections of Ctenophora 
by means of the double-imbedding method, § 169 (see Arch, 
f. mik. Anat., xl, 1892, p. 157; Zeit. f. wiss. Mik., 1893, 
p. 340). 
Porifera. 
870. Spongise : Fixation.—The smaller forms (Calcispongias) 
can be fairly well fixed by the usual reagents, osmic acid being 
one of the best. For the larger forms no satisfactory fixing 
agent has yet been discovered, so far as I can ascertain. 
The tissues of this group are very watery, very delicate, very 
friable after hardening, and macerate with the greatest 
facility. For all but very small specimens, absolute alcohol 494 
CHAPTER XXXV. 
is apparently the best fixing agent. If any watery fluid be 
preferred, care should at all events be taken to get the sponges 
into strong alcohol as soon as possible after fixation, on account 
of the rapidity with which maceration sets in in watery fluids. 
Fiedler (Zeit.f. wiss. Zool., xlvii, 1888, p. 87) has been using 
(for Sponyilla), besides absolute alcohol, an alcoholic subli- 
mate solution, and the liquids of Kleinenberg and Flemming 
with good effect. 
Staining.—On account of the great tendency to maceration 
above referred to, I hold (notwithstanding many recommen- 
dations of watery stains that are to be found in the literature 
of the subject) that alcoholic stains be alone employed for 
staining sponges, and I particularly recommend Mayer’s tinc- 
ture of cochineal as giving the best results personally known 
to me. Von Lendenfeld (Zeit. f. wiss. Mik., xi, 1, 1894, p. 
22) uses aqueous solutions of Congo red and anilin blue for 
the coloration of collar-cells. 
Sectioning.—Calcareous sponges may be decalcified in alco- 
hol slightly acidified with hydrochloric acid, and then im- 
bedded in the usual way. Siliceous sponges may be desi- 
licified by Mayer’s hydrofluoric acid method, see § 588. 
Preparation of Hard Parts.—Siliceous spicules are easily 
cleaned for mounting by treating them on a slide with hot 
concentrated nitric or hydrochloric acid, or solution of potash 
or soda. The acids mentioned are very efficient, but it must 
be pointed out that they will attack the silex of some delicate 
spicules. Thus Dezso found that the small stellate spicules 
of the cortex of Tethya lyncurium are completely dissolved 
by boiling hydrochloric acid. Potash solution is therefore 
frequently to be preferred, notwithstanding that, in my expe- 
rience, it does not give such clean preparations. 
According to Noll, eau de Javelle is preferable to any of 
these reagents (see § 569). 
Impregnation with Silver (see § 363). 
Larvae of Spongise.—Schultze (Zeit. f. wiss. ZooL, xxxi, 
p. 295) places the ova and larvae of Sycandra raphamts in 
hanging-drop moist chambers, oxygenated by means of a few 
fronds of green algae. He also (ibid, xxxiv, 1880, p. 416) 
found that the best sections of the more advanced sessile 
larvae of Plakina were obtained by selecting larvae that had 
settled down on thin fronds of algae, and treating them, SOME ZOOLOGICAL METHODS. 
495 
together with the fronds, with osmic acid, staining with alum- 
carmine, and bringing into paraffin in the usual way. 
Protozoa. 
871. Introductory.—Since the Protozoa may be considered 
as free cells, and their peculiar organs known as “ nucleus ” 
and “ nucleolus,” “ macronucleus ” and “ micronucleus,” &c., 
present in the main the same reactions as cell-nuclei, it is 
evident that the reagents and methods of c}7tology are in great 
part applicable to this group. One of the most generally 
useful of these reagents will be found in the acid solution of 
methyl green; it is the reagent that allows of the readiest and 
best demonstration of the presence and form of the nucleus 
and nucleolus (Balbiani et Henneguy, Comjpt. rend. Soc. de 
Biol., 1881, p. 131). 
Amongst useful reagents not mentioned in the following 
descriptions of the methods employed by different authors, I 
call attention to the weak solutions of alum, potash, and borax, 
which serve to demonstrate the striations of the cuticle and 
the insertions of the cilia of Infusoria. 
872. Methods for quieting Infusoria.—The narcotisation 
methods, §§ 18 to 22, are available for this purpose. 
According to Schurmayer (Jen. Zeit., xxiv, 1890, pp. 402 — 
470; Zeit. f. miss. Alik., vii, 4, 1891, p. 493) nitrate of 
strychnin in weak solution, 0‘01 per cent, or less, gives good 
results with some forms, amongst which are Stentor and 
Carchesium. Antipyrin in concentrated solution (0T per 
cent.) or cocain of 0'01 per cent, seems only to have given 
good results as regards the extension of the stalk in stalked 
forms. 
Eismond (Zool. Anz., No. 352, Dec., 1890, p. 723) has pro- 
posed a mechanical means of slowing the movements of small 
organisms (small worms and Crustacea as well as Ciliata). 
He directs that a drop of thick aqueous solution of cherry- 
tree gum be added to the water containing the organisms 
(gum arabic and the like, it is stated, will not do). The 
objects remain tixed in their places, with cilia actively moving, 
and all vital processes retaining their full activity. I am 
greatly obliged to Dr. G-rubler for having been at much pains 
in making inquiry for me concerning the cherry-tree gum 496 
CHAPTER XXXV. 
that should be used. It appears that this gum is a somewhat 
insoluble one, and it is difficult to get hold of a sample that 
will give a good solution. Further, the solutions will not 
keep, and must be made up fresh every day. In the face of 
these difficulties it would seem that the method is at present 
a far from perfect one. It should, however, be stated that 
Certes (Bull. Soc. Zool. France, xvi, 1891, p. 93; Journ. Boy. 
Mic. Soc., 1891, p. 828) has found that the method gives 
excellent results. He has also found that an intra vitam 
stain may be obtained by adding methyl blue or “ violet 
dahlia No. 170,” to the gum solution. 
A similar process of inhibiting movements whilst preserving 
life has been worked out by Jensen (after Stahl; see Biol. 
Centralb., xii, 1892, 18, 19, p. 556 : Zeit. f. tviss. Mik., ix, 4, 
1893, p. 483; Journ. Roy.Mic. Soc., 1892, p. 891). A solution 
of 3 grms. of gelatin in 100 c.c. of ordinary water is made by 
the aid of heat. This makes a jelly at the normal tempera- 
ture. It is slightly warmed, and a drop of it is mixed in a 
watch-glass with a drop of water containing the organisms. 
This plan is said to afford great facilities for the vivisection 
of Infusoria. 
873. Staining intra vitavi.—The possibility of staining In- 
fusoria intra vitam was discovered independently and almost 
simultaneously by Brandt (Verh. d.physiol. Ges. Berlin, 1878), 
by Certes (Soc. Zool., 25 janv., 1881), and by Henneguy (Soc. 
philom., 12 fev., 1881). See on this subject § 213. 
Certes found that living Infusoria stain, while continuing- 
in life for a certain time, in weak solutions of cyanin, Bismarck 
brown, dahlia, violet 5 B, chrysoidin, nigrosin, methylen blue, 
malachite green, iodine green, and other tar colours, and 
liEematoxylin. The solutions should be made with the liquid 
that constitutes the natural habitat of the organisms. They 
should be very weak, that is, of strengths varying between 
1 : 10,000 and 1 : 100,000. For cyanin, 1 : 500,000 is strong 
enough. 
The “nucleus” may be stained in the living organism by 
dahlia and malachite green. Bismarck brown only colours 
the “ nucleus ” of certain species (Nyctotherus, Opalinct—Hen- 
neguy). The “nucleus” frequently behaves differently in 
allied species. SOME ZOOLOGICAL METHODS. 
497 
A double stain of the nucleus (green) and protoplasm 
(violet) may be obtained by the simultaneous employment of 
dahlia and malachite green. 
Examination in a coloured medium in which the organisms 
do not stain, but show up on a coloured background in a 
manner that produces somewhat the effect of dark-ground 
illumination, is sometimes helpful. Certes {Bull. Soc. Zool. 
de France, xiii, 1888, p. 230) recommends solution of anilin 
black for this purpose; Infusoria will live in it for weeks. 
Fabre-Dohergue {Ann. de Microgr., ii, 1889, p. 545; Journ. 
Boy. Mic. Soc., 1889, p. 832) recommends concentrated solu- 
tion of diphenylamin blue. 
874. Fixing and Preserving.—Pfixzner (Morph. Jahrb., xi, 
1885, p. 454) used concentrated solution of picric acid run in 
under the cover. 
Blanc (Zool. Anz., 1882, p. 22) advises liquid of Kleinen- 
berg diluted with about a volume of water, and acidified 
with acetic acid. 
Korschelt (Zool. Anz., 1882, p. 217) recommends 1 per 
cent, osmic acid, or for Amoebae, 2 per cent, chromic acid. 
Lansberg (ibid., p. 336) advises the same reagents, but 
recommends bringing the organisms into the fixing liquid 
with a pipette, instead of running in the fixing liquid under 
the cover. 
Saville Kent and Berthold (Manual of the Infusoria; 
Journ Roy. Mic. Soc., 1883, p. 451) prefer a brownish-yellow 
solution of potassium iodide to osmic acid for fixing. See § 73. 
The employment of vapour of iodine has been described, § 73. 
Cattaneo (Bollettino Scientifico, iii and iv; Journ. Roy. 
Mic. Soc., 1885, p. 538) recommends fixing for a few minutes 
with i per cent, aqueous solution of chloride of palladium. 
This is said to be the best fixing agent, as it hardens in a few 
minutes without blackening the structures. 
875. Methods of Brass (Zeit. f. wiss. Mik., i, 1884, p. 39).— 
He employs for fixing unicellular organisms the following 
liquid : 
Chromic acid . . . . .1 part. 
Platinum chloride . . . . 1 ,, 
Acetic acid. . . . . . 1 „ 
Water .... 400 to 1000 parts. 498 
CHAPTER XXXV. 
For protozoa that are opaque through accumulation of nutritive material, 
he proceeds as follows :—The organisms are treated for three or four minutes 
with liquid of Kleinenberg, and then for some time with boiling water. 
They are then brought into water containing a small proportion of ammonia, 
in which they reassume their natural forms and dimensions. The ammonia 
is then neutralised by addition of a little acetic acid, and the preparation 
is stained with borax-carmine or ammonia-carmine. After washing, the 
objects are mounted in dilute glycerin. This treatment is said to afford 
extremely transparent preparations. 
Brass also obtained good results with sublimate solution. 
876. Certes (Comptes rend., 1879, 1 sem., p. 433) makes 
permanent preparations as follows :—Fix with osmic acid of 
2 per cent. (In the case of very contractile Infusoria, place 
a drop of the solution on the cover-glass, and place it on the 
drop of water that contains them. But generally speaking 
it is best to employ only the vapour of the solution, exposing 
the organisms to its action for not more than from ten to 
thirty minutes.) 
The objects having been covered, the excess of liquid is 
removed by means of blotting-paper, and the following stain 
is allowed to flow in : 
Glycerin . . .' . .1 part. 
Water . . . . . . 1 ,, 
Picro-carmine . . . . . 1 ,, 
(Eosin may also be used. Soluble anilin-blue does not give 
such good results.) The stain should be placed at the edge 
of the cover, and the slide put away in a moist chamber, in 
order that the water may evaporate very slowly and be 
changed very gradually for the glycerin mixture; if this 
precaution is not taken, shrinkage may occur. When the 
exchange has taken place, strong glycerin may be added, and 
gradually substituted for the dilute glycerin. 
Certes states that the organisms thus prepared are fixed 
perfectly in their natural form, and allow of the study of the 
minutest detail of cilia, flagella, and the like, with the highest 
powers; the green coloration of Euglenaa and Parainecia is 
preserved. The nuclear structures are sharply brought out by 
the picro-carmine. 
877. The Method of Geza Entz (Zool. Anz., No. 96, 1881, 
p. 575).—A few drops of liquid of Kleinenberg are added to a SOME ZOOLOGICAL METHODS 
499 
watch-glass of water, containing the organisms. After one or 
two minutes, the liquid is drawn off and the preparation is 
washed for half an hour with alcohol of medium strength. 
The objects are then stained for ten to twenty minutes in 
picro-carmine, washed with water till the picric acid is 
removed, and mounted in a mixture of equal parts of glycerin 
and water. 
878. Other General Methods.—Du Plessis (Yogt et Yung, Trait. 
Anat. Comp. Prat., p. 92) recommends fixation with 02 per cent, solution 
of corrosive sublimate. Let the preparation dry up, and if the organisms 
have preserved their shape, stain and mount in balsam. This seemingly 
barbarous mode of procedure is said to give very fine preparations when 
successful. 
Fol (Lehrb., p. 102) fixes delicate marine Infusoria (Tintinnodea) with 
the perchloride of iron solution (§ 67), added to the water containing them, 
and stains with gallic acid as directed (§ 379), and states that this is the 
only method that has given him good results, especially as regards the pre- 
servation of cilia. 
See also the methods of Fabre-Domeegue, Ann. de Microgr., ii, 1889, 
p. 545 ; Schewiakoff, Biblioth. Zool., v, 1889, p. 5; Journ. Boy. Mic. 
Soc., 1889, pp. 832, 833 ; Zoja, Boll. Sci. Pavia, 1892; Zeit.f. iviss Mik., 
ix, 4,1893, p. 485 ; Longhi, Bull. Mas. Zool. Univ. Genova, 4, 1892; Zeit. 
f. wiss. Mik., ix, 4, 1893, p. 483; Brandt, Fauna u. Flora d. Golfes v. 
Neapel, 1885; Journ. Boy. Mic. Soc., 1888, p. 665 (for Sphserozoa). 
879. Demonstration of Cilia (WAddington, Journ. Roy. Mic. 
Soc., 1883, p. 185).—Solution of tannin, or a trace of alcoholic 
solution of sulphurous acid. 
880. Stains for Flagella.—The celebrated method of Loffler 
has run through several forms (Gentralb. /. Bacteriol., vi, 
1889, p. 209 ; vii, 1890, p. 625; Zeit.f. wiss. Mik., vi, 3, 1889, 
p. 359; vii, 3, 1890, p. 368; Journ. Roy. Mic. Soc., 1889, 
p. 711; 1890, p. 678), of which that given here is the latest. 
To 10 c.c. of a 20 per cent, solution of tannin are added 5 c.c. 
of cold saturated solution of ferrous sulphate and 1 c.c. of 
(either aqueous or alcoholic) solution of fuchsin, methyl violet, 
or “ Wollschwarz.” The mixture will require for some forms 
the addition of a few drops of 1 per cent, solution of caustic 
soda ; e.g. for typhoid bacilli, 1 c.c.; for Bacillus subtilis, 28 to 
30 drops; for bacilli of malignant cedema, 36 to 37 drops. 
Some other forms will require besides the addition of a trace 
of sulphuric acid to the soda solution : so for cholera bacteria, 
half a drop to 1 drop; for Spirillum rubrum, 9 drops. 500 
CHAPTER XXXV. 
Cover-glass preparations are made and fixed in a flame in 
the usual way, special care being taken not to over-heat. 
Whilst still warm the preparation is treated with mordant 
(i.e. the above-described mixture), and is heated for half a 
minute until the liquid begins to vaporise, after which it is 
washed in distilled water and then in alcohol. It is then 
treated in a similar manner with the stain, which consists of 
a saturated solution of fuchsin in anilin water, the solution 
being preferably neutralised to the point of precipitation by 
cautious addition of 0T per cent, soda solution. 
The modifications of this method by Bunge are as follows: 
—Firstly (see Journ. Roy. Mic. Soc., 1894, p. 640, or Zeit.f. 
wiss. Mik., xiii, 1, 1896, p. 96), he modifies the mordant by 
taking Liquor Ferri Sesquichlorati instead of the sulphate. He 
dilutes the Liquor Ferri Sesquichlor. with twenty vols. of dis- 
tilled water, takes three parts of the tannin solution and one 
part of the dilute iron solution, and adds to 10 c.c. of the 
mixture 1 c.c. of saturated aqueous solution of fuchsin. The 
mordant should be allowed to ripen exposed to the air for 
some days or weeks. In a later paper (see Journ. Roy. Mic. 
Soc., 1895, pp. 129 and 248) he recommends adding to the 
ripened mordant a few drops of peroxide of hydrogen, until 
it becomes of a red-brown hue. It is then shaken up and 
filtered on to the prepared cover-glass, on which it is allowed 
to act for about a minute. The cover-glass is then mopped 
up, dried, and stained, preferably with carbol-gentian. 
Tkenkmann (Centralb., vi, 1889, p. 433 ; Zeit.f.wiss. Mik., vii, 1, 1890, 
p. 79) mordants for several hours at the normal temperature in a 1 percent, 
solution of tannin in 05 per cent, hydrochloric acid, and stains for several 
hours in carbolic fuchsin ; and gives also two other similar methods. 
Bkown (The Observer, iii, 1892, p. 298 ; Journ. Boy. Mic. Soc., 1893, 
p. 268) mordants for several hours in a mixture of 30 gr. tannin, 12 drops 
anilin oil, and 1 fl. oz. of alcohol, which may, if required, be alkalised by 
addition of a trace of caustic soda (so for Spirillum undula and Bacillus 
ulna), or be acidified for some forms with a little hydrochloric acid. The 
cover is stained by the process of heating over a flame for a few minutes with 
any anilin-water solution of fuchsin, methyl violet, dahlia, methyl green, 
&c., neutralised with caustic soda as in Loeffler’s process, or witli a solution 
of rosanilin in anilin water. 
See also Julien, quoted ibid., 1894, p. 403; van Erjiengem, ibid., 
p. 405 ; Sclavo, quoted Zeit.f. wiss. Mik., xiii, 1, 1896, p. 96 ; and Hessert, 
ibid., p. 98. APPENDIX. 
881. The Usual Alcohols.—The following, or a similarly- 
spaced series of alcohols, should be kept on the table. 
Absolute Alcohol.—See § 84. The so-called “absolute 
alcohol ” of commerce is generally of about 98 per cent, 
strength. This grade is convenient, but not necessary for 
ordinary work. 
95 per cent. Alcohol.—This is the average strength of the 
common strong commercial alcohol, which ranges in general 
from 94 per cent, to 96 per cent, according to temperature. 
The strength of this, or of the following, should be deter- 
mined by means of an areometer (Gay Lussac’s being very 
convenient), so as to form a starting-point for the following 
mixtures, which may be made by means of the subjoined 
table. This is the usual grade for dehydrating before 
clearing. It is the highest grade that should be used for de- 
hydrating celloidin sections. 
90 per cent. Alcohol.—May be made by taking 100 vols. 95 
per cent, alcohol, and 5'5 vols. water. This is the usual 
strength of the strongest commercial Methylated Spirit, which 
(if free from mineral naphtha) may be taken instead of pure 
alcohol for common work. If naphtha be present the alcohol 
becomes turbid on the addition of water. Oil of bergamot 
will clear from this grade. 
85 per cent. Alcohol.—Rectified Spirit, B.P., is a little weaker 
than this, viz. 84'5 per cent. 
70 per cent. Alcohol.—Only exceptionally powerful clearers, 
such as anilin oil, will clear from this grade; see § 125. This 
is the proper grade in general for preserving organisms and 
tissues in (but see the remarks on pp. 4 and 5); higher grades 
should not generally be used unless it is desired to harden. 
This is the proper grade for washing out borax-carmine stains, 
corrosive sublimate after fixing, &c. 
50 per cent. Alcohol.—This is the strength of Proof Spirit. 
“ One-third AlcoholP—Made by taking 1 vol. of 90 per cent, 
alcohol, and 2 vols. water. See § 8-3. 502 
APPENDIX. 
882. Table for diluting Alcohol (after Gay-Lussac).—To use 
this table, find in the upper horizontal row of figures the per- 
centage of the alcohol that it is desired to dilute, and in 
the vertical row to the left the percentage of the alcohol it is 
desired to arrive at. Then follow out the vertical and 
horizontal rows headed respectively by these figures, and the 
figure printed at the point of intersection of the two rows 
will show how many volumes of water must be taken to re- 
duce one hundred volumes of the original alcohol to the re- 
quired grade. Thus, if it be required to manufacture some 
70 per cent, alcohol, starting with 90 per cent., we find the 
figure 90 in the upper column, the figure 70 in the vertical 
column, and at the point of intersection we read 31*05, show- 
ing that a fraction more than 31 volumes of water must be 
added to 100 volumes of 90 per cent, alcohol. Or similarly, 
if we wish as before to make 70 per cent, alcohol, but start 
with an alcohol of 85 per cent., we find that 23*14 volumes of 
water must be employed. 
Weaker grade 
required. 
Original Grade. 
90 85 
p. 100. p. 100. 
80 
p. 100. 
75 
p. 100. 
70 
p. 100. 
65 
p. 100. 
60 
p. 100. 
55 
p. 100. 
50 
p. 100. 
p. 100. 
85 6-56 
80 13-79 6-83 
75 21-89 14-48 7-20 
70 31-05 23-14 15-35 
7-64 
65 
41-53 33-03 
24-66 
16-37 
8-15 
60 53-65 44-48 
35-44! 26-47 
17-58 
8-76 
55 
67-87 57-90 
48-07 38-32 
28-63 
19-02 
9-47 
50 
84-71 73-90 
63-041 52-43 
41-73 
31-25 
20-47 
10-35 
45 105-34 93-30 
81-38; 69-541 57'78 
46-09 
34-46 
22-90 
11-41 
40 130-80 117-34 
104-01 90-76; 77-58 
64-48 
51-43 
38-46 
25"55 
35 
163-28 148-01 
132-88 117-82 102-84 
87-93 
73-08 
58-31 
43-59 
30 
206-22 188-57 
171-05 153-61 136-04 
118-94 
101-71 84-54 
67-45 APPENDIX. 
503 
883. Histological Reagents and Apparatus.—See § 216. As 
regards the products of Grubler and Co., so often quoted in 
the foregoing pages, I would add that they should either he 
ordered from them direct, or, if ordered through any agent, 
should be ordered to be sent in the original packages, signed 
arid dated by them. This is in order to ensure the due fresh- 
ness of the products; many of them will not keep well for 
very long. Glass and other apparatus can be obtained as 
well as chemicals from the above-quoted houses. 
AVant of space compels me to suppress the lists of suggested reagents 
given in a former edition under the headings “ The Laboratory Table” and 
“ The Zoologist’s Travelling Case.” Either collection may still be obtained 
from Grubier and Co., the latter in appropriate bottles, fitted into a case 
measuring 1 foot 4 inches X 5J inches X 4| inches, at the price of about 
£2 5s., or a case somewhat larger, yet not too heavy to be carried in the 
hand, at about £3. 
884. Cleaning Slides and Covers.—The readiest way known to me of 
freeing slides from balsam, damar, and cement is to wet with water and 
scrape with an old knife, using afterwards, if necessary, one of the solvents 
mentioned below. 
Hanaman, Journ. Boy. Mic. Soc., i, 1878, p. 295 ; American Naturalist, 
xii, p. 573.—To a cold saturated solution of bichromate of potash add of 
its bulk of strong sulphuric acid (care must be taken on account of the heat 
and vapours evolved). 
Heneage Gibbes, ibid., iii, 1880, p. 392.—Place the cover-glasses in 
strong sulphuric acid for an hour or two, wash well until the drainings give 
no acid reaction; wash first with methylated spirit, and then with absolute 
alcohol, and wipe carefully with an old silk handkerchief. 
Seiler, ibid., p. 508.—New slides and covers are placed for a few hours 
in the following solution : 
Bichromate of potash ..... 3 ounces. 
Sulphuric acid . . . . .3 fluid ounces. 
Water 25 „ 
Wash with water. The slides may be simply drained dry ; the covers 
may he wiped dry with a linen rag. 
Slides and covers that have been used for mounting either with balsam 
or a water medium are treated as follows:—The covers are pushed into a 
mixture of equal parts of alcohol and hydrochloric acid, and after a few days 
are put into the bichromate solution and treated like new ones. The slides 
are scraped free of the mounting medium with a knife and put directly into 
the bichromate solution. 
Fol (Lehrb., p. 132) recommends either a solution containing 3 parts of 
bichromate, 3 of sulphuric acid, and 40 of water; or simply dilute nitric 
acid. 
Garbini (Manuale, p. 31) puts slides for a day into 10 per cent, sulphuric 
acid, then washes, first with water and then with alcohol. 504 
APPENDIX. 
Behkens (Zeit. f, wiss. Mik., 1885, p. 55) treats slides first with con- 
centrated nitric acid, then with water, alcohol, and ether. 
James (Journ. Roy. Mic. Soc., 1886, p. 548) treats used slides with a 
mixture of equal parts of benzin, spirit of turpentine, and alcohol. 
Knauer (Centralbl. f. Baht., x, 1891, p. 8; Zeit. f. wiss. Mik., ix, 2, 
1892, p. 187; Journ. Roy. Mic. Soc., 1891, p. 833) recommends boiling for 
twenty or thirty minutes in 10 per cent, lysol solution, then rinsing with 
cold tap water till clear. 
Nias (Journ., pag. cit.) finds it is sufficient to boil with washing soda, and 
rinse. 
In the employment of the water or alcohol section-fixing 
method, § 186, it is extremely important to work with slides 
absolutely free from grease. After the slides have been 
cleaned by one of the processes given above, they should be 
rinsed with distilled water and preserved in 90 per cent- 
alcohol, from which they should be removed with forceps 
when required for use, not with the fingers, then simply 
drained, or wiped with a very clean cloth. 
885. Gum Mucilage for Labels, &c.—The Journ. of the Chemical 
Soc. says that the adhesive qualities of gum may be very much exalted bv 
the addition of aluminium sulphate (the so-called “patent” alum) to the 
mucilage. “ 2 grins, of crystallised aluminium sulphate, dissolved in 20 
grins, of water, is added to 250 grms. strong gum arabic solution (2 grms. 
in 5 grms. water). Ordinary solutions of gum arabic, however concen- 
trated, fail in their adhesive power in many cases, such as the joining 
together of wood, glass, or porcelain ; prepared, however, according to the 
above receipt, the solution meets all requirements ” (from Public Opinion, 
Feb. 19th, 1886). 
Pol (Lehrh., p. 148) advises that slides be prepared for labelling bv 
spreading over one end a layer of aluminium-chloride gelatin dissolved in 
acetic acid, and allowing it to dry before putting on the label. 
Why do not the glass makers furnish slides with roughened (ground) end- 
surfaces for the reception of labels ? 
For four other receipts for gums and pastes for labels, see Eliel, in 
Engl. Meehan., 1887, p. 535; Amer. Mon. Mic. Journ., 1887, p. 93 ; Zeit. 
f. wiss. Mik., v, 1, 1888, p. 69. 
Vosseler (Zeit. f. wiss. Mile., vii, 4, 1891, p. 459) recommends, for 
attaching protective cardboard ridges to slides, a syrup-thick solution of 
bleached shellac in alcohol. 
886. Green Light.—The suggestion of the employment of green light 
in microscopy is, I believe, due to Englemann (.Pfliiger’s Arch., 1880, 
p. 550). He strongly recommends the use of green light for delicate 
observations, as giving sharper definition, allowing finer details to be seen, 
and tiring the eyes less than white light. Green glass of sufficiently good 
quality is found in commerce. The glass is best put between the mirror APPENDIX. 
505 
and the object, e. g. on the diaphragm. Blue glass (cobalt or ammonio- 
sulphate of copper) is also useful, hut less so than green. Red light is most 
hurtful. “ The explanation of these points, so important in practice, may 
be found in the results obtained by Lamansky in his researches on the 
‘ Limits of Sensibility of the Eye to the Different Colours of the Spectrum ’ 
Arch.f. Ophthalm., xvii, p. 123, 1871).” I would add that for lamp-light 
work, especially fine work with high powers, either green or blue glass is, 
according to my experience, a sine qua non if the best attainable results be 
desired. I always use blue cover-glasses, putting from one to four of them 
on the diaphragm of the condenser. For some unexplained reason I find I 
get better results by means of several superposed thin glasses than by one 
thick one. 
887. Re-staining Old Mounts (Henneguy, from the last edition 
of the Traite des Methodes techniques de Vanat. micros copique, 
Lee et Henneguy) . It is probably not generally known that 
balsam mounts the stain of which has faded, or which it may 
be desired to submit to some other staining process or mount 
in some other medium, may often with great advantage be 
re-stained and re-mounted. All that is necessary is to put the 
slide into a tube of xylol or benzol till the cover falls off 
(about two days), wash well for some hours in clean xylol, 
and pass through alcohol into the new stain. Since this was 
pointed out to me by Dr. Henneguy, I have unmounted and 
re-stained a considerable number of old preparations, some of 
them over fifteen years old, and have been most agreeably 
surprised at the results obtained. I have succeeded in every 
case with series of sections mounted on Mayer’s albumen, 
but I doubt whether the process would be safe with sectious 
mounted on such a fixative as Schallibaum’s collodion or the 
like. INDEX. 
The numbers refer to the Pages* 
A. 
Abbe, mounting medium, 277. 
Absolute alcohol, uses, 4, 5 ; prepara- 
tion, 52. 
Acanthocephali, 480. 
Acepliala, see Mollusea. 
Acetate of alumina, for mounting, 265. 
Acetate of copper, 46, 267. 
Acetate of lead, 61, 392. 
Acetate of potash, 265. 
Acetate of uranium, 46. 
Acetic alcohol, 45. 
Acetic acid for fixing, 44, 45, 467. 
Aceto-carmine, 152, 155. 
Acetone, for dehydration, 4. 
Acid aniliu dyes, 179. 
Acid, free, test for, in tissue, see Congo 
red. 
Acid fuchsin, 219; for nerve tissue, 
403, 414. 
Acid Magenta, see Acid fuchsin. 
Acidophilous cell-elements, 446. 
Acids, see Acetic, Chromic, Formic, 
Hydrochloric, &c. 
Acidulated alcohol, 52. 
Actiniaria, 13—17, 488. 
Adamkiewics, stain for nerve-centres, 
414. 
Adipose tissue, 35, 445. 
Adjective staining, 140, 141, 224. 
Agar-agar, section fixative, 127. 
Agassiz and Whitman,pelagic ova,346. 
Albumen, imbedding mass, 118; in- 
jection mass, 307; section-fixing 
process, 123, 126; removal of, 
from ova, 342 et seq. 
Albuminoids, reactions, 357. 
Alcohol, its action on tissues, 5; fixing, 
51; absolute, 52; “one third,” 
51; acetic, 45 ; acidulated, 52; 
picric, 50; for hardening, 62; 
macerating, 313; preserving tis- 
sues, 4, 5, 266; the usual grades, 
500; table for diluting, 501. 
Alcohol balsam, 280. 
Alcoholic carmines, 157, 158. 
Alcoholic cochineal, 158, 159. 
Alcoholic hfematein stains, 172—174. 
Alcyonaria, 489. 
Alcyonella, 14. 
Alcyonium, 12, 489. 
Aldehyde green, 198. 
Aleerow, silver impregnation, 247. 
Alizarin, artificial, 232. 
Alleger, serial sections, 127. 
Alien, methylen blue, 207. 
Alt, Congo red nerve stain, 418. 
Altmann, fixing methods, 35, 364; 
paraffin stove, 82; bioblasts, 138, 
364, 368; impi’egnation and cor- 
x’osion methods, 323. 
Alum for fixing, 37; ammonia-alum, 
solubility in water, 170. 
Alum-carmine, 150—152 ; ditto with 
acetic acid, 151; with picric acid, 
152. 
Alum-cochineal, 150. 
Alumina acetate, 265. 
Amarcecium, 346. 
Amber varnish, 289. 
Ammonia bichromate, 60, 392 ; neutral 
chromate, 61. 
* In accordance with a wish expressed by readers in whose judgment I have 
confidence, the references are now given to the pages, and not as heretofore to 
the numbered sections. 508 
INDEX. 
The numbers refer to the Pages. 
Ammonia-alum, 170. 
Ammonia-carmine, 155, 415. 
Ammonium molybdate, for impregna- 
tion, 261; for fixing nuclei, 364 ; 
sulphocyanide, 315; vanadate, 310. 
Amphibia, embryology, 342 et seq. 
Amphioxus, 378. 
Amphipoda, embryology, 352. 
Amphiura, 488. 
Ainpliophilous cell-elements, 446. 
Amyl nitrite, 293. 
Amyloid matter, 199, 201. 
Anchinia, 467. 
Ancylus, 469. 
Andeer, pliloroglucin, 326. 
Andre, fixing Ancylus, 469. 
Andres, Actiniae, 13,14, 488; section- 
stretcher, 87. 
Andrews, imbedding apparatus, 77. 
Anilin, for clearing, 70, 109. 
Anilin dyes, generalities, 179 et seq. ; 
classification into acid and basic, 
179; regressive staining process, 
182 et seq. 
Anilin black, 231, 475. 
Anilin blue, 230. 
Anilin blue-black, 231, 416. 
Anilin green, 228. 
Auilin oil for clearing, 70, 109; for 
staining, 190. 
Anilin red, 196. 
Anilin violet, 200. 
Anilin water, 190. 
Aniseed imbedding mass, 118. 
Annelida, 483—4-85 ; embryology, 352. 
Anodonta, 15. 
Antedon, 488. 
Anthea, 488. 
Anthozoa, 488. 
Antipathes, 489. 
Anti pyrin for narcotisation, 495. 
Apathy, celloidin imbedding, 100; 
preserving the blocks, 106 ; orien- 
tation of objects, 103; serial 
sections, 129, 130; cement, 289; 
hsematoxylin stain, 174; methylen- 
blue impregnation, 202,205,207— 
210; mounting medium, 269. 
Apel, Sipunculus and Hcilicryptus, 
481. 
-Aphides, preparation and embryology, 
351. 
Aplysia, 468. 
Aqua Javelli, 322, 330. 
Arabia, 126. 
Araneina, embryology, 351. 
Arcangeli, carmines, 155. 
Architp234. 
Archiplasm, archoplasm, see Cyto- 
logical methods, Plasma stains. 
Arctiscoida, 475. 
Areolar tissue, 444. 
Arnold, critique of Flemming’s mix- 
ture, 32, 34. 
Arnstein, methylen-blue method, 206, 
209, 210; gold method, 374. 
Aronson, gallein stain, 436. 
Arsenic acid for decalcification, 326. 
Arthropoda, general methods, 472; 
clearing and softeningchitin, 472 ; 
depigmentatiou, 473; eyes, 473— 
475; nerve and muscle, 475; em- 
bryology, 348—352. 
Artificial alizarin, 232. 
Artificial fecundation, 331. 
Artificial oedemata, 444. 
Artificial pigment spots, 59, 391, 392. 
Artificial saliva, 315. 
Artificial serums, 264, 265. 
Ascaris, ova, 45, 368. 
16, 346, 467. 
Asphalt varnish, 286. 
Asphyxiation as a killing method, 16. 
Aspinall’s enamel, for cement, 290. 
Astacus, embryology, 352. 
Asteracanthion, 14. 
Asteroidea, 486. 
Astroides, 489. 
Attraction spheres, see Cytological 
methods, Plasma stains. 
Atjbert, cements, 285. 
Augstein, Strongylus, 480. 
Aurantia, 229. 
Aureoline, 329. 
Auricularia, 487. 
Aves, embryology, 338 et seq. 
Axis cylinder, 403 et seq.; stains, 415 
et seq. 
Axolotl, ova, 343. 
Azoulay, nerve-impregnation, 412. INDEX. 
509 
B. 
Baber, picro-carmine, 154. 
Babes, safranin stain, 190; super- 
saturated do., 365. 
Balbiani, “ noyau vitellin,” 368 ; liv- 
ing ova, 332 ; ova of Phalangida, 
351; Protozoa, 495. 
Balfour, embryology of Araneina, 
351. 
Ballowitz, muscle of Cephalopoda, 
383. 
Balsam, 277 et seq. 
Balzer, elastic tissue, 450. 
Barff, boroglyceride, 272. 
Barnes, observation of Trichinae, 480. 
Barrois, embryology of Echinoder- 
mata, 487. 
Basic anilin dyes, 179. 
Basophilous cell-elements, 446. 
Bastian, gold method, 255. 
Bataillon and Koehler, borax 
methylen blue, 365. 
Baumgarten, fuchsin and methylen- 
blue stain, 232; bleu de Lyon, 
237. 
Bayerl, stain for cartilage, 236, 455 ; 
decalcification, 325. 
Beale, digestion fluid, 320; glycerin 
jelly,273; injections, 308 ; varnish, 
289. 
Bechtereff, hardening brain, 392. 
Beck, cements, 285. 
Becker, microtome, 72. 
Bedot, fixing process, 492. 
Behn, hardening skin, 370. 
Behrens, cements, 285; indulin and 
nigrosin, 228; amber varnish, 
289; cleaning slides, 503. 
Bela Haller, macerating mixture, 
318. 
Bellarminow, injection, 310. 
Bellonci, preparation of brain, 413. 
Bell’s cement, 286. 
Benda, iron hsematoxylin, 176; cop- 
per hsematoxylin, 177; double- 
stain, 227; freezing brain, 400. 
Benecke, connective-tissue fibrils, 444. 
Bengal rose, 226. 
Benzin, for imbedding, 79. 
Benzol, for clearing, 70. 
The numbers refer to the Pages. 
Benzo-azurin, 197. 
Benzo-purpurin, 225. 
Benzoyl green, 228. 
Bergamot oil, 68. 
Bergonzini, staining mixture for 
plasma cells, 449. 
Berkley, cerebellum, 398; rapid 
Weigert stain, 411. 
Berlin blue, 302, 309. 
Berlinekblatj, F., regeneration of 
Weigert’s hannatoxylin, 407. 
Bernheim, gold process, 257; bladder 
of frog, 384. 
Bernheimer, hasmatoxylin for retina, 
441. 
Bethe, methylen blue, 212; stain for 
chitin, 475. 
Betz, central nervous system, 392—394. 
Bevan Lewis, see Lewis, Bevan. 
Bianco, S. Lo, chromo-acetic acid, 
45; Molluscoida (Bryozoa), 468; 
Mollusca, 468; Nemertina, 478; 
Gephyrea, 481; Chsetopoda, 484; 
Holothurioidea, 485 ; Medusae, 
491; Siplionopbora, 493 ; Cteno- 
phora, 49-1; Tunicata, 16, 467; 
narcotising mixture, 14. 
Bichloride of mercury, see Corrosive 
sublimate. 
Bichromate of ammonia, 60. 
Bichromate of potash, for fixing, 36, 
60; for hardening, 58 et seq., 
391 et seq.; for maceration, 317. 
Bichromate of silver impregnation, see 
Golgi. 
Bickealyi, digestion fluid, 320. 
Biebricher Scharlach, 226. 
Biederjiann, methylen-blue method, 
207; nerve-endings in muscle, 
379. 
Binet, bleaching osmic objects, 28. 
Biniodide of mercury medium, 275. 
Bioblasts, Altmann’s, 138, 364, 
368. 
Biondi, staining mixture, 219, 223 ; 
methods for blood, 457, 461. 
Bipinnaria, 487. 
Bismarck brown, for regressive stain- 
ing, 196; for progressive, 200. 
Bitume de Judee, 286. 510 
INDEX. 
The numbers refer 
r to the Pages. 
Bizzozero, gentian violet, 192, 193; 
picro-carmine, 154; blood, 458, 
460, 461. 
Bjeloussow, injection, 307. 
Blackley blue, 228. 
Bladder of frog, 384. 
Blanc, Infusoria, 497. 
Blattida, embryology, 350. 
Blaue, glandular epithelium, 370. 
Bleaching, 328 et seq. ; bichromate ob- 
jects, 58; Erlicki objects, 59; 
osmic objects, 27. 
Bleu alcool, 230. 
Bleu carmin aqueux, 231. 
Bleu de Lyon, 230. 
Bleu de nuit, 230. 
Bleu lumiere, 230. 
Blochmann, ova of Amphibia, 342; 
nervous system of Cestodes, 433. 
Blood, 456—461. 
Blue-black, 231, 416. 
Blum, formaldehyde, 53, 62. 
Bobeetzky, embryology of Lepido- 
ptera, 350. 
Boccardi, gold method, 256; motor 
plates, 381. 
Bohm, gold method, 256; Golgi’s im- 
pregnation, 427. 
Bohmer, hajmatoxylin, 170. 
Bohmig, Rhabdoccela, 477. 
Bone, decalcification, 323 et seq.; pre- 
paration, 451—455. 
Borax-carmine, Geenachee’s, 156; 
aqueous, 155; with indigo-car- 
mine, 235, 236. 
Borax-metliylen blue, 365. 
Bordeaux-iron-hsematoxylin, 366. 
Boric-acid carmine, 155. 
Boen, paraffin imbedding, 77 ; section- 
fixing, 127; reconstruction of sec- 
tions, 333; section-stretcher, 87 ; 
embryological method, 344. 
Boroglyceride, 272. 
Bottcher-Hermann staining process, 
182. 
BoUMA, stain for cartilage, 455. 
Boveri, ovum of Ascaris, 368; medul- 
lated nerve, 404. 
Boyer, embryology of Teleostea, 345. 
Brachionus, 482. 
Beady, chloral hydrate medium, 266. 
Bradynema, 480. 
Brain, see Central nervous system and 
Neurological methods. 
Beandt, glycerin jelly, 273; Protozoa, 
496, 499. 
Beass, alcoholic carmine, 158; bleach- 
ing osmic objects, 28; paraffin 
imbedding, 79,94; Protozoa, 497. 
Braun, Alcyonaria, &c., 489 ; Rhabdo- 
ccela, 477; Nematodes, 480. 
Breckenfeld, Hydra, 490. 
Breglia, Weigert’s haematoxylin, 407, 
411. 
Beemee, motor plates, 381; methylen 
blue and eosin, 227. 
Bbide, Mac-, see Macbeide. 
Beistol, regeneration of osmic acid 
solutions, 25. 
Bbock, macerating medium, 317. 
Bbosicke, staining method, 260. 
Brown, stains for flagella, 500. 
BeuCKE, digestion fluid, 320; injec- 
tions, 302. 
Been, mounting medium, 270. 
Beunotti, gelatin imbedding mass, 
98. 
Brunswick black, 286. 
Bryozoa, 14,15, 468. 
Budge, injection, 310. 
Bugula, 468. 
Bumpus, celloidin imbedding, 110. 
Bunge, stains for flagella, 500. 
Bueci, elastic tissue, 451. 
Bueckhabdt, nervous system of Pro- 
topterus, 398. 
Burger, nervous system of Nemertina, 
206. 
Busch, eosin and haematoxylin, 237; 
decalcification, 323, 324. 
Busse, photoxylin imbedding, 100; 
celloidin do., 101, 103, 106. 
Butschli, paraffin imbedding, 81; 
haematoxylin stains, 171, 177. 
C. 
Cajal, Ramon y, silver chromate im- 
pregnation, 425; terminations of 
nerves and tracheae, 380; retina, 
441; sympathetic ganglia, 425. IXDEX. 
511 
The numbers refer to the Pages. 
Calbeela, methyl green, 197, 199; 
ditto and eosin, 226; indulin, 
228; glycerin mixture, 272; ma- 
cerating mixture, 315. 
Caldwell, serial sections, 123. 
Cambridge rocking microtome, 72. 
Canada balsam, for mounting, 279 et 
seq.; imbedding method, 115. 
Canfield, iris, 383. 
Cannel oil (cinnamon oil), 68. 
Caoutchouc, for section-fixing, 128 ; 
cement, 286. 
Cappabelli, dichroism of methyl vio- 
let, 201. 
Caeazzi, bleaching method, 329. 
Carbolic acid, for clearing, 69, 109. 
Carbolic fuchsin, 196. 
Carbolised syrup, 265. 
Carbonic acid narcotisation method, 
17. 
Carcbesium, 495, 
Carcinoma corpuscles, 192. 
Carmalum, 149. 
Carmine, analysis of, 145 ; generalities 
on, 145—149; theory of staining 
with, 145; practice of staining, 
148; for what purposes useful, 
148. 
Carmine blue, 231. 
Carmine stains, aqueous, 149—155 ; 
alcoholic,156—160; choice of, 148; 
carmine combination stains, 235 
et seq. ; and see the names of the 
respective authors, as also the 
separate entries, “ Alum-carmine,” 
“ Borax-carmine,” &c. 
Carminic acid, 146. 
Carminroth, 155. 
Cabnoy, acetic alcohol, 45; chromo- 
aceto-osmic acid, 34; cement, 290; 
cytological methods, 357, 358, 
364; salt solution, 263; tannin 
solution, 267. 
Caepenteb, cements and varnishes, 
284. 
Caebieee, corpuscles of Herbst and 
Grandry (gold method), 375 ; eyes 
of Gastropods, 469. 
Cabteb, injection mass, 300. 
Cartilage, 455, 456. 
Caryophyllia, 489. 
Cassiopeia borboniaa, 491. 
CastellabnaUjDE, Lamellibranchiata, 
468; Medusae, 491; Nemertina, 
479; Ophiuridea, 486; Zoan- 
tharia, 489. 
Castor oil for mounting, 282. 
Cattaneo, corpuscles of Golgi, 381; 
Infusoria, 43, 497. 
Caustic potash and soda, for macera- 
tion, 314; for corrosion, 322. 
Cavazzani, triple stain, 239. 
Cedar oil, for minute dissections, 9-; 
for paraffin imbedding, 80; clear- 
ing, 66; for mounting, 278. 
Cell granules, 368 et seq., 445—450, 
459. 
Cell researches, see Cytological me- 
thods. 
Cellepora, 468. 
Celloidin, 99 et seq. ; the new method, 
110; the dry method, 111; clear- 
ing sections, 108; injection masses,. 
310. 
Cells, paper, for mounting, 285. 
Cements, 284 et seq.; generalities-, 
284 ; comparative tenacity of, 
285. 
Central nervous system, introduction, 
385, 386; fixing, 387, 388; hard- 
ening, 388—398; imbedding and 
cutting, 398—401; nerve-fibre 
stains, 405—414 ; axis-cylinder 
and protoplasm stains, 415—419; 
impregnations, 419; dry processes- 
for preserving brains, 437; me- 
thylen blue for, 443; neuroglia, 
435, 436; myelon of reptiles, 
398; and see Nerves, Retina, &c., 
and the names of Authors. 
Centrosomes, see Cytological methods, 
Plasma stains. 
Cephalopoda, eyes, 470; ova, 346. 
Cercarise, 477. 
Cerebellum, cerebrum, see Neurolo- 
gical methods. 
Cebfontaine, curarising Lumbricus, 
483. 
Ceetes, Protozoa, 496, 497, 498. 
Cestodes, 476. 512 
INDEX. 
The numbers refer to the Pages 
Chsetopoda, 483. 
Chsetopterid®, 484. 
Chain section-cutting, 88. 
Cheatle, dehydration apparatus, 4. 
Cherry gum, 495. 
Chemicals, 143. 
Chenzinsky, methyleu blue and 
eosin, 227. 
Chichkoff, Turbellaria, 478. 
China blue, 231, 403. 
Chinolin blue, 228. 
Chironomus, embryology, 351. 
Chitin, clearing and softening, 472 et 
seq.; staining, 475. 
Chitonidse, embryology, 348; eyes, 
470. 
Chloral hydrate for narcotising, 14; 
preservative solutions, 265; for 
maceration, 314; Gilson’s mount- 
ing medium, 275 ; Geoffroy’s 
ditto, ibid. 
Chlorate of potash for maceration, 318. 
Chloride-of-aluminium carmine, 150. 
Chloride of copper fluid, 46. 
Chloride of gold, see Gold chloride. 
Chloride of magnesium for narcotisa- 
tion, 16. 
Chloride of manganese examination 
medium, 263. 
Chloride of palladium, see Palladium 
chloride. 
Chloride of platinum, 36, 42, 43, 59, 
61. 
Chloride of vanadium, 418, 441. 
Chloride of zinc, for hardening brain, 
396,397 ; for impregnation, 434. 
Chlorine bleaching methods, 328. 
Chloroform, for paraffin imbedding, 
80, 81; for celloidin ditto, 104; 
for bleaching, 330; for narcotisa- 
tion, 13 ; for clearing, 70. 
Cholodkowsky, embryology of Blat- 
tida, 350. 
Chromate of ammonia, 61. 
Chromate of lead stain, 261. 
Chromate of silver impregnation, see 
Golgi. 
Chromates as fixing agents, 37. 
Chromatin, reactions of, 357—360; 
solvents of, 358; stains for, 365. 
Chromic acid, generalities, 29 ; fixing, 
29; hardening, 56; killing, 22; 
maceration, 317; decalcification, 
324, 326; chromic acid with acetic 
acid, 30; with alcohol, 30,57; with 
nitxfic acid, 35; with platinum 
chloride, 36; with osmic acid, 31; 
with acetic and osmic acid, 31 et 
seq. 
Chromic objects, action of light on, 30; 
bleaching, 30. 
Cliromo-acetic acid, 30; cliromo-aceto- 
osmic, 31—35, 361; chromo-for- 
mic, 31; cliromo-nitric, 35; cliromo- 
osmic, 31, 57[; cliromo-picric, 36, 
57; cliromo-platinic, 36. 
ChrschtschonoWic, gold method, 
255. 
Chun, imbedding Siphonophora, 95. 
Ciaccio, gold process, 256; motor 
plates, 381; corpuscles of Golgi, 
382. 
Ciaglinski, spinal cord, 414. 
Cilia of Infusoria, 499. 
Ciliated epithelium, 471. 
Cinnamon oil, see Cannel oil. 
Ciona, 467. 
Cladocora, 489. 
Clarke’s spirit-proof cement, 286. 
Clasmatocytes, 450. 
Clavellina, 467. 
Claypole, serial sections, 124. 
Cleaning slides and covers, 503. 
Clearing, generalities, 6; practice of, 
64. 
Clearing agents, 64 et seq.: Stieda’s 
experiments on, 65; Neelsen and 
Schiefferdecker’s, 65—68; cel- 
loidin sections, 108; paraffin ditto, 
70. 
Clove oil, 9, 67; for imbedding, 
79. 
Coal-tar chromatin stains, 180 et seq.; 
regressive ditto, 182—197; pro- 
gressive ditto, 197—209; coal-tar 
plasma stains, 215—232. 
Coal-tar colours, see Anilin dyes. 
Cobb’s differentiator, 4. 
Cocain for narcotising, 15. 
Coccidse, 472. INDEX. 
513 
The numbers refer to the Pages. 
Cochineal, theory of staining with, 
147; use of, 159; Heeeick’s 
151; Czokoe’s, 150; Klein’s, 151; 
Mayee’s, 158, 159; Paetsch’s, 
150. 
Cochlea, 442, 443. 
Ccelenterata, 488—493. 
Cohnheim, gold staining, 252 ; silver 
staining, 668. 
Cole, freezing method, 116. 
Collagen, 444 et seq. 
Collin, Criodrilus, 483. 
Collinge, pelagic fish ova, 346. 
Collodion imbedding, 99 et seq.; sec- 
tion-fixing process, 120—122. 
Collodionising paraffin sections, 89. 
Colopbonium, cement, 288 ; imbedding 
mass, 115; mounting medium, 
281. 
Colfcci, retina, 441. 
Comatula larvae, 488. 
Combination stains, 233 et seq. ; and 
see Stains. 
Congelation imbedding methods, 115— 
118. 
Congo red, 225, 402, 414. 
Conjunctiva, 375. 
Connective tissue, 444 et seq. 
Contejean, Ranvier’s haematoxylin, 
173. 
Convoluta, 477. 
Copal, imbedding process, 114; varnish 
for mounting, 282. 
Copepoda, 472. 
Copilia, 472. 
Copper chloride and acetate preserva- 
tive mixture, 267. 
Copper sulphate fluids, fixing, 344, 
492, 493; impregnation process, 
434 ; hsematoxylin stain, 177. 
Coral, 489. 
Corallin, 196. 
Coei, narcotisation, 14,15; Flemming’s 
mixture, 33; preserving osmic 
acid solutions, 25. 
Cornea, 375—377. 
Corpuscles, tactile, 373 et seq.; corpus- 
cles of Golgi, 381, 382; of Herbst 
and Grandry, 374; of Krause, 
375 ; of Meissner, 375. 
Corrosion, 322 et seq.; Altmann’s 
methods, 323. 
Corrosive sublimate, 38 ; fixing liquids, 
38—42; preservative liquids, 266. 
Cotton blue, 231. 
Cottpieb’s blue, 228. 
Covers and slides, cleaning, 503. 
Cox, Golgi’s mercuric impregnation, 
434. 
Creosote, see Kreasote. 
Creseis, 469. 
Criodrilus, 483. 
Cristatella, 14, 468. 
Crocein, 219. 
Crystal violet, 372. 
Crystalline, 315, 377. 
Ctenophora, 493. 
Cuccati, picro-carmine, 154; hsema- 
toxylin, 173 ; retina, 441; soda- 
carmine, 155. 
Cucumaria, 486. 
Cupric fixing mixtures, 492, 493; pre- 
servative ditto, 267; impregna- 
tion process, 434. 
Curare for narcotisation, 16, 354. 
Cueschmann, amyloid matter, 199. 
Cyanide of mercury, for fixing, 481. 
Cyanin, 228. 
Cybulsky, gold method, 258. 
Cynthia, 467. 
Cytological methods, 354 et seq. ; sub- 
jects for study, 354—356 ; obser- 
vation of living cells, 354; stain- 
ing ditto, 356 ; fresh cells,'356 ; 
micro-chemical reactions, 357— 
360; fixing, 360—365 ; cytologi- 
cal stains, 365—368 ; Nebenkern, 
Archoplasmakugel, sphere attrac- 
tive, ibid.; nucleus of Balbiani, 
368; cell granules, 368. 
Czerny, macerating mixture, 316. 
Czokoe, cochineal, 150; turpentine 
cement, 287. 
D. 
Dahlia, 195. 
Dammar, 280. 
Daszkiewicz, Koeybutt-, plasma- 
cells, 446. 514 
INDEX. 
The numbers refer to the Pages, 
David off, ova of Distaplia, 346. 
Davies, injection, 300. 
Deane, mounting medium, 268; gly- 
cerin jelly, 273. 
Decalcification, 323 et seq. 
Decapoda, 472. 
Deckeb, section stretcher, 87. 
Deecke, hardening brain, 396; im- 
bedding and cutting, 400. 
Definition, affected by transparency 
of stains, 188; green light for, 
504. 
Degenerate nerves, 412. 
Dehydration, 3, 4. 
Dejebine, neurological methods, 386, 
400. 
Dekhuysen, fat, 445; blood, 459; 
silver staining, 246. 
Delafield, hsematoxylin, 170. 
Delage, osmium-carmine, 155 ; Rhab- 
docoela, 477. 
Deltapurpurin, 225. 
Dendroccela, 478. 
Dendrophyllia, 489. 
Dendy, Geonemertes, 479. 
Depigmentation, 328 et seq., 472 
et seq. 
De Queevain, fixation of nerve- 
centres, 387. 
Desilicification, 327. 
De Souza, pyridin, 62. 
Dextrin freezing mass, 117. 
Dezso, Tethya, 494. 
Differentiators, 4. 
Diffusion apparatus, 4. 
Diffusion currents, to avoid, 4. 
Digestion, 320 et seq., 358. 
Dimmock, carminic acid, 145, 155. 
Dinitrosoresorcin, 403. 
DioMidoff, hardening brain, 392. 
Diphyes, 493. 
Diptera, embryology, 351. 
Dissections, minute, 6, 9. 
Dissociation, 312 et seq. 
Distaplia, 346. 
Dogiel, olfactive organs, 375; iris, 
383; methylen-blue impregnation 
method, 207, 209, 212 ; corpuscles 
of Herbst and Grandry, 374; of 
Meissner, 375; retina, 441. 
Domeegue Fabbe, see Fabee Do- 
MEBGUE. 
Donaldson, hardening brain, 391. 
Dostoievsky, iris, 383. 
Double imbedding in celloidin and pa- 
raffin, 112. 
Double stains, see Stains, Combination. 
Doyeee, nerve and muscle of Arcti- 
scoida, 475. 
Dbasch, gold staining, 252; tactile 
hairs, 373. 
Deash, serial section method, 122. 
Dboost, Mollusca, 471. 
Deuebin, blood-plates, 461. 
Dunham, clearing celloidin sections, 
109. 
Du Plessis, Nemertians, 479; Pro- 
tozoa, 499. 
Dueham, alcohol section-fixing pro- 
cess, 120. 
Dubig, formaldehyde in Golgi’s im- 
pregnation, 427. 
Duval, silver staining, 246, 248; car- 
mine and anilin blue, 236; col- 
lodion imbedding, 100 et seq.; 
embryology of Aves, 341; harden- 
ing encephalon, 396; purpurin, 
235; paraffin sections, flattening, 
91. 
E. 
Ear, inner, 442, 443. 
Eau de Javelle, 322, 330. 
Eau de Labarraque, 322, 330. 
Ebneb, von, decalcification, 325. 
Echinodermata, 485—488. 
_7fehinorliyncus, 480. 
Echtgelb, 219. 
Echtgriin, 403. 
Edingee, nerve-centres, 59. 
Ehlees, fixing fluid for Annelids, &c., 
31. 
Eheenbaum, imbedding method, 115. 
Ehelich, dahlia, 195; hsematoxylin, 
171; metliylen blue for nerves, 
203; Mastzellen, 445, 447; gen- 
tian violet, 192; blood, 456, 459; 
classification of anilin dyes, 179; 
of cell granules, 446; neutral red, INDEX. 
515 
The numbers refer to the Pages. 
226; acidophilous mixture, 229 ; 
mixture for eosinophilous cells, 
229; triacid mixture, 223. 
Ehrlich-Biondi-Heidenhain stain, 
219—223. 
Ehrmann, structure of epithelium, 
372. 
Eichlee, labyrinth, 443. 
Eijkman, degenerate nerves, 412. 
Eisig, fixing mixture, 61; narcotising, 
14. 
Eisen, serial sections, 128. 
Eismond, quieting Infusoria, 495. 
Elastic tissue, 450, 451. 
Eledone, eyes, 470. 
Eliel, gum for labels, 504. 
Elschnig, celloidin imbedding, 101. 
Embryology, general methods, 331 et 
seq.; Amphibia, 342—344; Am- 
phipoda, 352; Aphides, 351; Ara- 
neina, 351; Arthropoda,348—352; 
Astacus, 352 ; Aves, 338 et seq. ; 
Blattida, 350 ; Cephalopoda, 346 ; 
Diptera, 351;Echinodermata,487; 
Gastropoda, 347; Lepidoptera, 
350; Mammalia, 335; Mollusca, 
346 et seq.; Phalangida, 351; 
Pisces, 344 et seq.; Planaria, 352 ; 
Reptilia, 342; Teleostea, 344— 
346; Tunicata, 346; Vermes, 352 
et seq. 
Embryos, reconstruction from sections, 
333. 
Emeby, injection, 309. 
Encephalon, 385 et seq.; and see Cen- 
tral nervous system, neurological 
methods, &c. 
Endosmosis, to avoid, 4. 
Engelmann, ciliated epithelium, 471 . 
green light in microscopy, 504. 
Entz, G., Infusoria, 498. 
Eosin, 226; eosin stains for blood, 
458, 459. 
Epidermis, 370. 
Epithelium, 370 et seq. ; ciliated, 471; 
glandular, 370. 
Erlicki, hardening fluid, 59. 
Errera, nigrosin, 197. 
Erythrosin, 226. 
Essence, see Oil, and Clearing agents. 
Etebnod, histological rings, 244. 
Ether, for preserving, 5. 
Etherisation, 14, 354. 
Eulenstein, cement, 286. 
Eunice, 484, 
Everard, Demoob and Massabt, 
hsematoxylic eosin, 239. 
Examination media, 262 et seq. 
Exnee, hardening brain tissue, 391; 
process for medullated nerve 
tracts, 412. 
Exosmosis, to avoid, 4. 
Eyclesheimer, orientation of celloidin 
objects, 103 ; cutting same, 110 ; 
clearing, 109; serial sections, 129. 
Eyes of Ai’thropoda, 473 et seq.; of 
Cephalopoda and Heteropoda, 470; 
of Chitonidfe, 470; of Gastro- 
poda, 469; of Pecten, 470; of 
Asteroidea, 486. 
F. 
Fabee-Domeegue, glucose preserva- 
tive medium, 270; Infusoria, 497, 
499. 
Fairchild, dehydration cylinders, 4. 
Fajebstajn, nerve-endings in tongue 
of frog, 375. 
Faeis, glycero-gum, 269. 
Farrant’s medium, 268. 
Fast green, 228; fast blue, 228. 
Fat, 35, 445. 
Fatjssek, critique of Flemming’s mix- 
ture, 32. 
Fecundation, artificial, 331. 
Feist, methylen-blue method, 210— 
212 ; orientating spinal cord, 
401. 
Feeeeei, phloroglucin decalcification, 
326, 327. 
Febbia, elastic tissue, 451. 
Feebiee, examination liquid for blood, 
459. 
Fick, Golgi’s silver chromate method, 
429. 
Fiedler, Spongilla, 494. 
Field and Martin, petroleum-ether 
for imbedding, 80; orientation in 
paraffin, 85; double imbedding, 
113 ; Schallibaum’s collodion, 121. 516 
INDEX. 
The numbers refer to the Pages, 
Fischel, medullated nerve, 404. 
Fischer, soap imbedding mass, 477 ; 
Trematodes, 476; tactile corpus- 
cles, 373 ; motor plates, 379. 
Fischeb, A., cell granules, 369. 
Fish, oil of thyme, 69, 109; celloidin 
section-cutting, 110; decalcifica- 
tion, 323, 324, 326; hardening 
brain, 391,396,397,398; formalde- 
hyde for Golgi’s impregnation, 427. 
Fixing, generalities, 2 et seq., 18 et seq. 
Fixing agents, 24 et seq.; action of, 
18; choice of, 19; methods of 
using, 20; washing out, 2, 20; 
cytological, 360—365. 
Flatatx, Golgi’s sublimate impregna- 
tion, 433. 
Flattening sections, 87, 91. 
Flagella, stains for, 499. 
Flechsig, gold method, 256; redwood 
nerve stain, 411; other nerve 
stains, 434. 
Flemming, chromo-acetic acid, 30; 
chromo-aceto-osmic acid, weak 
solution, 31; strong ditto, 33; cy- 
tological methods, 354, 355, 357, 
360, 363; damar solution, 281; 
double stains, 216, 217; eyes of 
Gastropods, 469 ; fixing liquids, 
30, 31, 33, 50, 361; preservation 
in glycerin mixtures, 5; injection 
of molluscs, 471; staining method, 
182 et seq., 189 et seq., 195; saf- 
ranin solution, 190; gentian 
violet, 192; dahlia, 195; orange 
method, 217; fatty tissue, 445 ; 
bone, 454; goblet cells, 463; con- 
nective-tissue fibrils, 444. 
Flesch, Max, chromo-osmic mixture, 
31 ; dichroism of hsematoxylin 
stains, 165; inner ear, 443; mo- 
dification of Weigert’s hsema- 
toxylin stain, 407; dry preserva- 
tive pi’ocess, 439; dental tissue, 
456; blood, 457. 
Flogel, serial sections, 126. 
Florman, celloidin imbedding, 106. 
Flustra, 468. 
Foa, fixing liquid, 42; hsematoxylin 
and safranin stain, 240; blood,460. 
Foettinger, chloral hydrate narco- 
tisation method, 14; prepared 
paraffin, 94. 
Fol, absolute alcohol, 52; albumen 
fixative, 124; bleaching osmic 
objects, 27, 28; chromo-aceto- 
osmic acid, 32; cleaning slides, 
503; decalcification, 326; gelatin 
fixative, 128; glycerin jelly, 295; 
gold-impregnation of marine ani- 
mals, 258; gumming labels, 504; 
imbedding in vacuo, 83; injection 
masses, 299, 303, 305, 306; In- 
fusoria, 499; narcotisation me- 
thod, 17; paraffin oven, 82; per- 
chloride of iron fixing and staining 
process, 44, 260; picro-carmine, 
154; picro-chromic acid, 36; re- 
constructing sections, 333. 
Formaldehyde, properties, 53; for 
fixing, 53; hardening, 62; pre- 
serving, 466; macerating, 314; 
hardening brain, 397, 398; for the 
Golgi impregnation, 427; as a 
mordant, 187. 
Formalin, see Formaldehyde. 
Formalose, see Formaldehyde. 
Formic acid for fixing, 46. 
Formol, see Formaldehyde. 
Foster and Baieoub, embryology of 
Aves, 338, 340. 
Fowl, embryology of, 338, 341. 
Fbancotte, section-stretcher, 87 ; 
vacuum imbedding, 83. 
Fbeebokn, staining nervous centres, 
416 ; connective tissue, 444. 
Freezing microtome, masses for, 115— 
118. 
French cement, 287. 
Frenkel, fixing mixture, 43. 
Frenzel, serial section methods, 126, 
128 ; sublimate solutions, 38. 
Frey, injection mass, 305; iodised 
serum, 264; indifferent liquids, 263. 
Friedlaender, fixing mixture, 493; 
critique of Golgi’s impregnation, 
420. 
Frog, bladder, 384; tongue, 375. 
Fuchsin, basic, 196; Ziehl’s carbolic, 
196. INDEX. 
517 
The numbers refer to the Pages. 
Fuchsin S, see Saurefuchsin, and Acid 
fuchsin. 
G. 
Gage, picro-carmine, 154; chloral 
hsematoxylin, 173; decalcification, 
325; section stretcher, 87; pre- 
servative fluid, 266 ; injection, 
310; smooth muscle, 382; 
section-fixing, 122, 129; clear- 
ing mixture, 69; picric al- 
cohol, 50; preservation of potash 
or soda preparations, 314; macera- 
tions, 314, 317. 
Galeotti, intra-vitam staining, 139; 
neutral red, 226. 
Gallemaerts, section-fixing process, 
121. 
Galli, stain for neuroceratin funnels, 
403. 
Gannal, mounting medium, 265. 
Garbini, safranin staining, 191; Al- 
cyonaria, 489; cleaning slides, 
503. 
Gaskell, paraffin sections, water 
method, 91. 
Gastropoda, embryology, 347; eyes, 
469; preparation, 468 et seq.; 
killing, 16, 468. 
Gable, section-fixing process, 119; 
hardening brain, 392; fixing liquid, 
40; choice of paraffin, 94. 
Gay-Lbssac, table for diluting al- 
cohol, 502. 
Geberg, gold method, 257 ; corpuscles 
of Grandry, 374. 
Gedoelst, digestion, 321; medullated 
nerve, 404. 
Gehbchten, van, acetic alcohol, 45; 
cytologieal methods, 364; nerve 
centres, 399, 403. 
Gelatin, imbedding masses, 97, 98, 
117; injection masses, 293 et seq.; 
cement, 285; section fixative, 
127. 
Gelpke, Weigert’s hsematoxylin, 406. 
Gentian blue, 230. 
Gentian violet, 192—194. 
Geopfroy, mounting medium, 275. 
G-eonemertes, 479. 
Gephyrea, 481. 
Gerlach, gold method, 255, 256; 
gelatin imbedding, 98; injection, 
300; embryology of Aves, 339; 
nerve-endings in muscle, 379. 
Geza Entz, Infusoria, 498. 
Giacomini, gelatin process for serial 
sections, 134; dry process for pre- 
serving brains, 437. 
Gibbes, Heneage, borax-carmine, 155; 
cleaning slides, 503. 
Gierke, maceration of nerve tissue, 
316; staining nervous centres, 
416; action of chloral on ditto, 
58; uranium carmine, 155; im- 
pregnation, 242. 
Giesbrecht, clearing method, 64; 
serial section method, 122; section- 
stretcher, 87; paraffin imbedding, 
81, 82. 
Gieson, van, clearing celloidin sec- 
tions, 109; formaldehyde, 397. 
Gilson, acetate of uranium fixing 
liquid, 46; fixing with osmic acid, 
26; bleaching bichromate objects, 
58,330; celloidin imbedding, 111; 
chloral hydrate jelly, 275 ; double 
imbedding, 113; mercuro-nitric 
fixing mixture, 40; preservative 
fluid, 266. 
Glandular structures, 461 et seq. 
Glucose preservative media, 270. 
Glue, marine, 287. 
Glycerin, 270 et seq.; glycerin mounts, 
to close, 285 ; injections, 308 et 
seq.; glycerin jelly, 97, 98, 273 
et seq.; preservative mixture, 
272. 
Glycero-gum, 269. 
Glychaemalum, 169. 
Goadby, preservative fluids, 267. 
Goblet cells, 463. 
Goette, hardening liquid, 344. 
Gold chloride, for impregnation, gene- 
ralities, 250 et seq. ; composition 
of the commercial salt, 251; and 
see the names of Authors. 
Gold orange, 219. 
Gold size, 287. 518 
INDEX. 
The numbers refer to the Pages. 
Goegi, chromate of silver impregnation, 
introduction, 419; slow method, 
421—423 ; rapid method, 423 ; 
mixed method, 424; critique of 
the same, 424 et seq.; variations, 
425—431; fixation of nerve-cen- 
tres by injection, 387; mercuric 
impregnation of nervous centres, 
431; corpuscles of Golgi, 381, 
382; nerve-endings in muscle, 
381. 
Goodald, spinal cord, 399. 
Oorgonia, 489. 
Goronowitsch, embryology of Tele- 
ostea, 345. 
Graf, fixing Hirudinea, 485. 
Graff, yon, Turbellaria, 477. 
Gram, stain for bacteria, 193. 
Grandry, corpuscles of, 374. 
Granule cells, 445 et seq. 
Granules, protoplasmic, 368, 445 et 
seq., 459, 460. 
Grape-sugar imbedding mass, 114. 
Graser, staining process, 201. 
Gravis, section, fixative, 127. 
Gray, serial sections, 127. 
Green light in microscopy, 504. 
Grenacher, alum-carmine, 150; alco- 
holic carmine, 157; bleaching 
mixture, 330; borax-carmine, 
aqueous, 155; alcoholic, 156; 
castor oil for mounting, 278; 
hsematoxylin, 170; purpurin, 235; 
eyes of Mollusca, &c., 470. 
Greppin, Golgi’s silver chromate 
method, 430. 
Grieb, alum-carmine, 150. 
Griesbach, benzo-purpurin,225; Ben- 
gal rose, 226; Biebriclier Schar- 
lacli, 226; blood-stains, 456, 457; 
Congo red, 225; elastic tissue, 
450; iodine green, 228; metanil 
yellow, 219; metbyl green, 200; 
gold orange, 219. 
Grouven, Ehrlicli-Biondi mixture, 
222. 
Grubber, histological reagents, 144; 
cherry gum, 495. 
Grunpulver, 197. 
Griinstichblau, 230. 
Guernsey blue, 228. 
Guignet, soluble Prussian blue, 301. 
Gull and, flattening and mounting 
paraffin sections, 91, 120; blood, 
460. 
Gum, arabic, pure, 126 ; preservative 
media, 268 et seq.,- imbedding 
masses, 113, 114, 116, 117; injec- 
tion mass, 307 ; mucilage for labels, 
504; fixative for sections, 126. 
Gum damar, 280. 
Gum sandarac, 283. 
Gutta-perclia, cement, 287; section- 
fixing process, 128. 
H. 
Hacke, hsematoxylin, 173. 
Hsemacalcium, 172. 
Hsemalum, 167, 168. 
Hsemateate of ammonia, 164. 
Hsematein, generalities, 161—167 ; 
properties, 163 ; where obtainable, 
163; formulae for hsematein stains, 
167 et seq. ; alumina-hsemate'in 
stains, 167—173; other hsematein 
stains, 173—178; and see Hsema- 
toxylin. 
Hsematein-ammoniak, 163. 
Hsemateinum crystallisatum, 163. 
Hannatoxylic eosin, 238. 
Hsematoxylin, generalities, 161—167; 
theory of staining with, 161; 
ripening of solutions, 161—103; 
dichroism of the stains, 164, 
165; affiuitiesfor plasma granules 
and for mucin, 165; the practice 
of staining, 164—166; how to 
“blue” stains, 164,165; formulae 
for stains:—alum solutions, 167 — 
173; stains depending on the 
formation of other lakes, 173— 
178, 367; combination stains, 237 
et seq.; and see the separate en- 
tries, such as “ Iron hsematoxylin,” 
“ Hsemacalcium,” &c.; and see 
Hsematein. 
Hannoglobin, 458. 
Haensel, liquid of, 36. 
Hairs, tactile and others, 372. INDEX. 
519 
The numbers refer to the Pages. 
Halicryptus, 481. 
Halle and Born, orienting celloidin 
objects, 103. 
Haller, Bela, macerating fluid, 318. 
Halliburton, chemistry of the cell, 
359. 
Hamann, carmine, 155; Echinoderms, 
486; Acanthocephali, 480. 
Hamilton, freezing process, 116; 
hardening brain, 395; freezing 
ditto, 399. 
Hanaman, cleaning slides, 503. 
Hansen, haematein stain, 169; elastic 
tissue, 451. 
Hantsch, glycerin medium, 273. 
Hardening, generalities, 21; practice 
of, 22 ; nerve-centres, 388, 398. 
Hardening agents, 56 et seq.; choice 
of, 23. 
Hardy, Kotifers, 482. 
Harmek, silver-staining marine ani- 
mals, 249. 
Hartig, white injection mass, 305. 
Harting, preservative fluid, 266; 
cement, 287. 
Haswell’s dehydration apparatus, 4. 
Haug, borax-carmine, 155; luema- 
toxylin, 327; decalcification, 323, 
325, 326; modification of Wei- 
gert’s nerve stain, 412. 
Hayem, blood, 456, 457. 
Heat, killing by, 11; fixing by, 365. 
Heidenhain, M., cytological methods, 
355, 364, 366; lanthanin, 364; 
hsematoxylin (iron), 175, 366; 
discussion as to the Ehrlich - 
Biondi stain, 220, 222; paraffin 
imbedding, 80; cause of “ shrink- 
age ” in paraffin sections, 80, 87; 
sublimate solution, 40 ; water sec- 
tion-fixing process, 120; thionin, 
194; Bordeaux-iron-haematoxy lin, 
366; vanadium haematoxylin, 368. 
Heidenhain, II., haematoxylin, 173; 
glands of small intestine, 465. 
Header, collodionising sections, 90. 
Helix, eyes, 469; ova, 347 ; narcotisa- 
tion, 15, 16. 
Heller and Gumpertz, osmic acid 
impregnation, 260. 
Henchman, embryology of Limax, 
347. 
Henking, collodionising sections, 90; 
embryology of Diptera, 351; of 
Phalaugida, 351; of Insecta in 
general, 348; examination liquid 
for ova, &c., 349. 
Henle, staining nervous tissue, 415. 
Henneguy, alum-carmine, 151; em- 
bryology of Aves, 340; of Axolotl, 
343 ; of Gastropoda, 347 ; of Te- 
leostea, 344; Protozoa, 495, 496 ; 
washing out carmine stains, 148; 
permanganate staining method, 
186; mounting methyl green 
stains, 199; mounting paraffin 
sections, 91, 125, 127; L. John- 
son’s platinic mixture, 59, 60; 
staining with thionin, 195; re- 
staining old mounts, 505. 
Henocque, gold process, 255. 
Hebbst, corpuscles of, 374. 
Herla, stain for attraction spheres, 
368. 
Hermann, silver staining, 246; papillae 
foliatae of rabbit, 375; platino- 
aceto-osmic mixture, 43, 362; 
osmium and pyroligneous acid 
impregnation, 258; cytological 
stains, 367; formaldehyde, 53, 54, 
62, 63. 
Hermann-Bottcher staining process, 
182, note. 
Herrick, cochineal alum-carmine, 
151. 
Hertwig, silver staining, 246, 249; 
macerating methods, 318; Actiniae, 
488; Medusae, 491; embryology 
of Rana, 343; of Triton, 343; 
tobacco-smoke method, 12. 
Herxheimer, elastic tissue, 450; epi- 
thelium, plasma fibrils of, 372. 
Heschl, amyloid matter, 199. 
Hessert, stains for flagella, 500. 
Heteropoda, eyes, 470. 
Heurck, van, mounting medium, 277. 
Heydenreich, amber varnish, 289. 
Heymons, embryology of Biattida, 
351. 
Heys, balsam, 279. 520 
INDEX. 
The numbers refer to the Pages. 
Hickson, hannatoxylin and eosin, 237; 
maceration, 314; eye of Musca, 
473. 
Hirota, blastoderm of fowl, 341. 
Hirudinea, 15, 208, 484. 
His, fixing, 35 ; cornea, 376. 
Hochstetter, injection, 310. 
Hoeer, narcotisation method, 15. 
Hoffmann, vacuum imbedding, 83. 
Hofmann, ovum of fowl, 340. 
“ Hofmann’s Griin,” 228. 
Hog GAN, silver staining, 246; histo- 
logical rings, 244; perchloride of 
iron stain, 260. 
Holl, toluol for imbedding, 79; note 
concerning liquid of Ivleinenberg, 
361. 
Holothurioidea, 485. 
Homarus, eyes, 474. 
Hopewell Smith, decalcification, 
325; teeth, 453. 
Hopkins, maceration, 318. 
Horn, 372. 
Hoyer, picro-carmine, 154; silver 
staining, 246; gold ditto, 255; 
gum-arabic mounting medium, 
268; gelatin injection masses— 
carmine, 298; Berlin blue, 303; 
lead chromate, 304; silver nitrate, 
305; green, 305; shellac mass, 
310; oil-colour masses, 310; chlo- 
ral for preserving masses, 294; 
water section-fixing process, 120; 
reactions of mucin, 461. 
Hoyer, jun., formaldehyde, 54. 
Huber, Golgi’s silver chromate method, 
429; medullated nerve, 404. 
Hudson, Rotifers, 482. 
Hyatt, imbedding method, 114. 
Hydatina, 482. 
Hydra, 15, 490. 
Hydrate of chloral for narcotising, 
14; and see Chloral. 
Hydrochlorate of cocain, 15. 
Hydrochloric acid for decalcification, 
323, 325. 
Hydrofluoric acid for desilicifieation, 
327. 
Hydrogen peroxide for bleaching, 25, 
28, 30, 329. 
Hydroidea, 490. 
Hydroxylamin, 15, 
Hypochlorite of potassium, see Eau de 
Javelle. 
Hypochlorite of sodium, see Eau de 
Labarraque. 
I. 
Ide, double imbedding, 113; epi- 
thelium, 371. 
Igacuschi, gold process for liver, 464. 
Iijima, embryology of Planaria, 352. 
Imbedding defined, 74; generalities 
on, 74 et seq.; double imbedding 
in celloidin and paraffin, 112; and 
see also Celloidin, Paraffin, &c. 
Impregnation, distinguished from 
staining, 241; negative and posi- 
tive ditto, 241; primary and 
secondary ditto, 242. 
Impregnation methods, 241 et seq.; 
and see Gold chloride, Nitrate of 
silver, Metliylen blue, &c. 
Inchiostro di Leonardi, 200. 
India-rubber cement, 287. 
Indian ink injection, 310. 
Indifferent liquids, 262 et seq. 
Iudigen, 228. 
Indigo substitute, 228. 
Indigo-carmine, 235; Thiersch’s 
staining method, 235; Seiler’s, 
235; Merkel’s, 236; with saf- 
ranin, 229. 
Indirect staining method, 137 et seq. 
Indulin, 228. 
Indulin-aurantia-eosin mixture, 229. 
Infusoria, 495 et seq., and see Protozoa. 
Injection of Mollusca, 471. 
Injections, 293 et seq.; injection masses, 
—albuminous, 307,- aqueous, 309 
etseq.; collodion, 310 •, fatty, 310 ; 
gelatinous, 293 et seq.; glycerin, 
308 et seq. ; gum, 307; resinous, 
310; starch, 310; and see the names 
of Authors. 
Injections, natural, 311. 
Inner ear, 442, 443. 
Insecta, see Arthropoda. 
Insecta, embryology, 348 et seq. INDEX. 
521 
The numbers refer to the Pages. 
Intra-epidermic nerve-fibres, 373. 
Intra-vitam staining, 138, 203. 
Inversion of stains, 142, 224. 
Iodic acid for fixation, 458. 
Iodide of palladium nerve stain, 413. 
Iodine for fixing, 46, 47; for harden- 
ing, 62 ; Lhgol’s solution, 46; 
iodine as a mordant, 141, 143. 
Iodine green, 228. 
Iodine lisematoxylin, 173. 
Iodised serum, 263, 264; ditto for 
macerations, 313. 
Iris, 383. 
Iron, perchloride, for fixing, 44; for 
staining, 260; pyrogallate of, 260; 
iron-hsematoxylin stains, 175— 
177; with Bordeaux, 366; iron- 
carmine, 152; detection of iron in 
cells, 359. 
Israel, orcein, 234. 
J. 
Jackson, cloudiness in clearing media, 
65. 
Jacobs, freezing mass, 117. 
Jacqyet, injection of leeches, 485. 
Jadassohn, structure of epithelium, 
272; thionin for plasma-cells, 
450. 
Jager, glycerin medium, 273. 
Jakimovitch, silver staining, 248; 
medullated nerves, 404. 
James, cleaning slides, 504; damar 
solutions, 281. 
Janssens, carmine blue, 231. 
Javelle, eau de, see Eau de Javelle. 
Jelgeksma, staining nervous centres, 
416. 
Jelinek, extraction of picric acid, 47 ; 
Stabilit imbedding blocks, 108. 
Jellies, glycerin, 273—275. 
Jensen, immobilisation of Infusoria, 
496. 
Johnson, Lindsay, keeping osmic 
solutions, 24; sunning solutions 
of metallic salts, 24, 243 ; platiuic 
hardening mixture, 59; gold im- 
pregnation, 257; retina, 439, 440; 
cementing celloidin blocks, 107- 
Joliet, imbedding method, 113. 
Joseph, injection mass, 307. 
Julien, cement, 289; stains for flagella, 
500. 
Juno, microtomes, 71. 
K. 
Kadyi, imbedding mass, 95. 
Ivaes, nerve stain, 411. 
Kaiser, glycerin jelly, 97, 274 ; naph- 
thylamin brown for spinal cord, 
417; Bismarck-brown stain, 196; 
nerve stain, 410. 
K allies, Golgi’s silver chromate 
method, 426, 431. 
Kanthack, histological reagents and 
apparatus, 144. 
Kastschenko, reconstruction from sec- 
tions, 333. 
Keiseb, sublimate solution, 40; fixa- 
tion of Acanthocephali, 481. 
Kemp, blood-plates, 460. 
Kent, S., Infusoria, 46, 497. 
Kent, A. F. S., cement, 290. 
Kernschwarz, 233. 
Killing tissues and organisms, 11— 
17. 
Kingsley, imbedding method, 77; 
section-fixing, 123. 
Kionka, blastoderm of fowl, 341. 
Kishinouye, embryology of Araneina, 
351. 
Kitton, asphalt, 286; cement, 290. 
Klebs, glycerin jelly, 97. 
Klein, fixing mixture, 30; cornea, 375; 
cochineal, 151. 
Ivleinenberg, colophonium for mount- 
ing, 281; embryology of Lum- 
iricus, 352; picro-sulphuric acid, 
48; hamiatoxylin, 173. 
Klemensiewics, picro-carmine, 154. 
Knauer, cleaning slides, 504. 
Knotting, for cement, 287. 
Koch, von, copal imbedding method, 
114. 
Kofoid, embryology of Agriolimax, 
348. 
Koganei, iris, 383. 522 
INDEX. 
The numbers refer to the Pages. 
Kollikeb, ovum of rabbit, 335, 337 ; 
bone, 454. 
Kollmann, fixing mixture, 345. 
Kolossow, gold method, 256; osmic 
acid mixture, 27 ; osmic acid stain, 
260; regeneration of osmic acid 
solutions, 25. 
Koppen, elastic tissue, 451. 
Kopsch, formaldehyde for Golgi’s im- 
pregnation, 427. 
Koeotnepe, killing Siplionophora, 13. 
Koeschelt, Infusoria, 497. 
Kobybtjtt-Daszkiewicz, plasma-cells, 
446. 
Kossel, chemistry of the cell, 359. 
Kossinski, double stains, 229, 230. 
Kotlaeewsky, central nervous system, 
.32, 61, 392. 
Kowalewsky, embryology of Teleos- 
tea, 345. 
Keause, corpuscles of, 375; molybdate 
of ammonium, 261; motor plates, 
381; silver staining, 248; retina, 
441, 442; tliiophen green, 228; 
Ehrlicli-Biondi mixture, 219. 
Kreasote, for bleaching, 329; for 
clearing, 70, 109. 
Keomayeb, plasma fibrils of epithe- 
lium, 371. 
Ivboneckee. artificial serum, 265. 
Kbonig, cement, 288. 
Keysinsky, pliotoxylin imbedding, 
100. 
Kuhne, digestion fluids, 321; macerat- 
ing mixture, 318; motor plates, 
381. 
Kuhne, H., aniseed freezing mass, 
118. 
Kuhnt, retina, 441. 
Kukenthal, Annelids, 14, 483, 484. 
Kultschitzky, fixing mixture, 36; 
double imbedding, 112; preserva- 
tive fluids, 5; tactile corpuscles, 
374; stain for nervous centres 
with hsematoxylin, 410; neuro- 
glia, 436; elastic tissue, 451; 
“ patentsaures liubin,” 219. 
Kupffee, embryological methods, 342; 
axis-cylinder, 403; liver, 464. 
Kuskow, digestion fluid, 320. 
L. 
Lababbaqite, eau de, see Eau de 
Labarraque. 
Labelling slides, 504. 
Labyrinth of ear, 442, 443. 
Lacerta, ova of, 342. 
Lachi, formaldehyde for brain, 397. 
Lactic acid for decalcification, 324,326. 
Lamansky, sensibility of the eye, 505. 
Lamellibranehiata, 468, 471. 
Landois, impregnation methods, 261; 
macerating mixture, 316. 
Lanholt, retina, 442. 
Landsbeeg, Infusoria, 497. 
Lang, fixing liquids, 40; methods for 
Platyhelmia, 40. 
Langebhans, Amphioxus, 378; gum 
and glycerin medium, 269 ; tactile 
corpuscles, 374; fixation of ma- 
rine animals, 21. 
Langhans, dichroism of hsematoxylin 
stains, 165. 
Lankestee, eyes of Limulus, 473. 
Lanthanin, 364. 
Lauth’s violet, 194. 
Laydowsky, blood, 458; sandarac me- 
dium, 283; chloral hydrate me- 
dium, 265; macei-ating fluid, 314; 
inner ear, 442 ; formaldehyde, 55. 
Lawbence, glycerin jelly, 273. 
Lead, acetate, 61, 392; chromate, 261. 
Lebeb, impregnation methods, 261; 
retina, 441. 
Lecithin, reactions, 357. 
Leclebcq, stains for blood, 459. 
Lee, A. B., preservation in paraffin or 
cedar oil, 5; cedar oil for paraffin 
imbedding, 80; mounting eelloidin 
sections, 128; intra-vitam stain- 
ing, 139,140; sublimate solutions, 
38, 364; formaldehyde, 53, 54; 
eelloidin imbedding, 101,105,106; 
dry celloidiu process, 111; Mayer’s 
albumen, 125, 128 ; toluidin blue, 
197; malachite green, 228; ni- 
grosin, 228; bleu de L\on, 230; 
Wasserblau, 231; Kernschwnrz, 
233; orcein, 235; carmine and 
indigo-carmine, 235; impregnation INDEX. 
523 
The numbers refer to the Pages. 
with gold, 250; osmic acid and 
pyrogallol stain, 258; glycerin 
mixture, 272 ; resinous mounting 
media, 277—280; colophonium 
mounting medium, 282; cedar oil 
for mounting, 278; paper cells for 
mounting, 285; Nemerteans, pre- 
paration, 478; fixing Hirudinea, 
485. 
Leech, 485. 
Legal, alum-carmine, 152. 
Legbos, silver staining, 248. 
Lemon juice for fixing nuclei, 364; for 
killing, 12. 
Lendenfeld, von, staining collar- 
cells, 494. 
Lenhossek, von, tongue of rabbit, 
375; nerves of annelids, 484. 
Lennox, retina, 441. 
Lens, crystalline, 315, 377. 
Leonabdi, incliiostro di, 200. 
Lepidoptera, embryology, 350. 
Lepkowsky, gold method for teeth and 
bone, 454. 
Letellieb, injection, 310. 
Leuckhabt, imbedding boxes, 76. 
Levulose for mounting, 270. 
Lewis, Bevan, freezing process for 
brain, 388; hardening ditto, 394 ; 
staining, 416. 
Lichtgrun, 227. 
Liebebmann, analysis of carmine, 145; 
carminic acid, 146. 
Light, action of, on alcohol containing 
chromic objects, 30; on metallic 
salts, 242; green, in microscopy, 
504. 
Ligula, 476. 
Lilienfeld, blood-plates, 461; che- 
mistry of the cell, 359. 
Lilienfeld and Monti, test for phos- 
phorus in tissues, 359. 
Limax, 469; embryology, 347. 
Limulus, 473. 
Lindsay Johnson, see Johnson, 
Lindsay. 
Linseed-oil injection, 310. 
Liquid of Muller, of Erlicki, of Kult- 
schizky, of Merkel, &c., see the 
names of the respective Authors. 
Liquidambar, 283. 
List, haematoxylin and eosiu, 237; 
methyl green and eosin, 226; 
other combination stains, 464; 
macerations, 313; Antliozoa, 488; 
Coccidse, 472; goblet cells, 464. 
Lithium-carmine, 155. 
Litmus paper, to make, 297. 
Liver, 464. 
Lo Bianco, S., see Bianco, S. Lo. 
Loct, embryology of Agelena, 351. 
Loeffleb, stains for flagella, 499. 
Loligo, eyes, 470. 
Longhi, eserin for Infusoria, 499. 
Longwobth, corpuscles of Krause, 375. 
Lonnbebg, cytology of the snail, 356; 
Tricenophorus, 476 
Looss, softening chitin, 322; Nema- 
todes, 479. 
Lovett, cement, 290. 
Lowe, crystalline, 377. 
Lowenthal, nerve-centres, 59; picro- 
curmine, 154. 
Lowit, gold method, 252; blood, 456, 
457, 459. 
Lowt, macerating skin, 371. 
Loxosoma, 468. 
LugoIi, solution of, 46. 
Lumbricus, 483; embryology, 352. 
Lustgabten, elastic tissue, 195, 450. 
Luys, noir Colin, 416. 
Lymphatics, see Impregnation methods 
and Injections. 
Lysol, properties, 319; for maceration, 
319. 
M. 
Maas, carmine and malachite green, 
237. 
Macallum, Sphyranura. 477; double 
stain, 236; detection of iron in 
cells. 359. 
MacBbide, development of Ampkiura, 
488. 
Maceration, 312 et seq.; epithelium, 
370; Mollusca, 471. 
Macrotoma, 475. 
Magdala red, 195. 
Magenta, 196; Magenta S, 219. 
Magini, stain for nervous centres, 434.. INDEX. 
The numbers refer to the Pages. 
Magnesium chloride, for narcotisation, 
16; sulphate, ibid. 
Maheenthal, yon, osmium impreg- 
nation, 260. 
Malachite green, 228. 
Malassez, ammonia-carmine, 155. 
Maleatti, chemistry of the cell, 359. 
M alloby, phosplio-molybdic acid hae- 
matoxylin, 178, 418. 
Mammalia, embryology, 335 et seq. 
Manchester brown, 200. 
Manfeedi, gold method, 256. 
Manganese chloride, 263. 
Mann, osmic acid mixture, 27; picro- 
sublimate mixtures, 41; toluidin 
blue, 197; Wasserblau and eosin, 
231; fixative for sections, 126; 
fixing nerve-centres, 387. 
Maechi, yon, Limax, 469; corpuscles 
of Golgi, 381; degenerate nerves, 
412. 
Mabcus, formaldehyde for nerve- 
centres, 397. 
Marine animals, killing, 17; fixing, 21; 
silver impregnation, 248; gold 
ditto, 258. 
Marine glue, 287. 
Mabk, collodionising sections, 89; 
embryology of Limax, 317; sec- 
tion-fixing, 123. 
Mabschalko, von, plasma-cells, 450. 
Mabsh, bleaching, 328; gelatin ce- 
ment, 285; decalcification mix- 
ture, 326. 
Maeshall, Mays’ gold process, 381. 
Mabtin, benzoazurin, 197, 232. 
Mabtinotti, safranin staining, 191; 
damar solutions, 281; aniliu black, 
416; picro-nigrosin, 417; elastic 
tissue, 450; intra-vitam staining, 
138. 
Mason, nervous system of reptiles, 
&c., 398. 
Mastzellen, 445—450. 
Matschinsky, bone, 453. 
Maueice and Schulgin, double stain, 
237; embryology of Amaroecium, 
346. 
Mauvein, 196. 
Max Flesch, see Flesch. 
Maybe, Paul, acidulated alcohol, 52 ; 
alcoholic carmine, 157, 158; albu- 
men tixative for sections, 123; 
bleaching, 328; borax-carmine, 
156; carmine staining, theory of, 
115—147; Carmalum, 149; chlo- 
ride of aluminium carmine, 
150; Paracarmine, 156; carmine 
and indigo-carmine, 236; cochi- 
neal staining, theory of, 147; 
cochineal, old formula, 158; new 
formula, 159 ; desilicitication, 327; 
general principles of preparation, 
9 ; liasmatoxyliu (hannatem) stain- 
ing, theory of, 161—167; Haema- 
lum, 167, 168; Htemacalcium, 
172; Glyehmmalum, 169 ; pre- 
paration of the ammonia-salt of 
haematein, 164; bluing haema- 
tein stains, 164, 165; mounting 
haematein stains, 278 ; critique of 
Ivleinenberg’s liaematoxylin, 173; 
injection, 309; picro-carmine, 
154; picro-sulphuric acid, 48; 
piero-hydrochloric acid, 50; picro- 
nitric acid, 50; section-stretcher, 
87; section-fixing methods, 123— 
125; shellac fixative, 123; theory 
of staining, 137; staining mucus, 
462; Mucicarmine, 463; Muchae- 
matein, 463; water-bath for 
paraffin, 82. 
May'eb, S., “ violet B.” for connective 
tissue, 231, 444; methylen-blue 
method, 207, 209, 212. 
Mays, nerve-endings (gold process), 
381. 
Maysel, Bismarck brown, 200. 
Medullated nerve, 403, 404; and see 
Neurological methods. 
Medusae, 490—492. 
Meissneh, corpuscles of, 375. 
Mebcieb, haematoxylin nerve stain, 
412; Upson’s nerve-impregna- 
tions, 436, 437; Golgi’s impreg- 
nation methods, 419, note. 
Mercury bichloride, see Corrosive sub- 
limate; biniodide, mounting me- 
dium, 275; cyanide, for fixing, 
481; nitrate and picrate, 364. INDEX. 
525 
The numbers refer to the Pages. 
Mere, making up chromo-aceto-osmic 
acid, 34. 
Merkel, cliromo-platinic liquid, 36; 
impregnation method, 261; car- 
mine and indigo stain, 236; cel- 
loidin imbedding, 100; staining 
nervous tissue, 415. 
Metagelatin, 306. 
Metallic salts, action of light on solu- 
tions, 242. 
Metallic stains, 241 et seq. 
Metanil yellow, 219. 
Metcalf, embryology of Chiton, 
348. 
Methanilin green, 197. 
Methanilin violet, 200. 
Methyl alcohol for narcotising, 14; 
for safranin staining, 191. 
Methyl blue, 230. 
Methyl green, 197—200; combination 
stains with, 226. 
Methyl mixture for maceration, 319. 
Methyl violet, 200. 
Methylal, for dehydration, 5, 211. 
Methylated spirit, 501. 
Methylen blue, for impregnation, 202 
—214, 443; generalities, 202; 
staining in toto during life, 203 ; 
Staining nerve-tissue during life, 
204; influence of oxygen and of 
ammonia, 205; staining by in- 
jection or by immersion, 206; 
the solutions employed, 206; pre- 
servation of the stain, 209; im- 
pregnation of epithelia, lymph- 
spaces, &c., 212, 213; of central 
nervous system, 443; polychro- 
matic, 202; with eosin, 227; 
borax-metbylen blue, 365 ; stains 
for hardened nervous tissue, see 
Rehm, Sahli, and Adamkiewics ; 
for plasma-cells, 448. 
Meyer, salicylic solutions, 267 ; silver 
staining, 249. 
Meyer, Semi, methylen blue for cen- 
tral nervous system, 443. 
Mibelli, elastic tissue, 451. 
Michrochemistry of the cell, 357—360. 
Microtomes, 71—74. 
Migttla, glycerised serum, 265. 
Miller, injection, 305; caoutchouc 
cement, 286. 
Milnesinm, 475. 
Minot, hsematoxylin stains, 178; clear- 
ing celloidin sections, 109; mace- 
rating skin, 371; carmine, 155 ; 
microtome, 73. 
Mitrophanow, gold process, 371; 
maceration, 371; sense organs of 
Amphibia, 375 ; Wasserblau, 231; 
prickle-cells, 371. 
Mitsukuri, embryology of Reptilia, 
342. 
Mobies, macerating media, 317. 
Moerner, tracheal cartilage, 456. 
Moleschott, potash or soda solution, 
314; Moleschott and Piso 
Borme’s macerating fluid, 314. 
Molgula, 467. 
Mollusea, 468—471; embryology, 346 
et seq. 
Molluscoida, 11, 18, 19, 468; and see 
Bryozoa. 
Molybdate of ammonium, 364. 
Monobromide of naphtlialin, 277. 
Monti, sulphate of copper impregna- 
tion method, 434; test for phos- 
phorus in tissues, 359. 
Moore, stain for blood, 458. 
Moore, Y. A., aniseed imbedding, 118. 
Mordants for staining, 140—143. 
Morgan, ova of Arthropods, 348,472 ; 
removing albumen from ova, 343, 
348 ; test-cells of Ascidians, 346. 
Morphia for narcotising, 16. 
Moseley, shell, 470; Chitonidae, 470. 
Mosso, blood, 457. 
Motor plates, 378 et seq. 
Mounds of Doyere, 475. 
Mounting media, see Examination 
media. 
Muchsematein, 463. 
Mucicarmine, 463. 
Mucilage for labels, 504. 
Mucin, 461 et seq. 
Mucus, removing, from Mollusea, 469. 
Mucus glands, 461 \et seq. 
Muller, blood, 456; hardening liquid, 
58; silver staining, 247; injection 
mass, 309. 526 
INDEX. 
The numbers refer to the Pages. 
Munder, G., histological reagents, 144. 
Munson, chloral hydrate fluid, 266. 
Musca eyes, 473. 
Muscle, dissociation of, 378; nerve- 
endings in, 379 et seq.; smooth, 
382 et seq.; sections, 378. 
Myelin, 403; myelin stains, 405—414. 
Myelon, see Central nervous system. 
N. 
Nails, 372. 
Nansen, maceration of nerve-tissue, 
316. 
Naphtha, for clearing, 70; for im- 
bedding, 79. 
Naplithalin, monobromide, 277. 
Naphthylamin brown, 417. 
Narcotisation, 12—17. 
Nathusius, von, hairs and nails, 373. 
Nealey, bone, 453. 
Nebenkern, see Cytological methods, 
Plasma stains. 
Neelsen and Schieeferdecker, 
clearing agents, 65. 
Negro, motor plates, 381. 
Nematodes, 479. 
Nemertina, 478. 
Nereis, embryology, 353. 
Nerves, degenerate, 412. 
Nerves, see Neurological methods. 
Nerve-endings in muscle, 378 et seq. ; 
in skin and others, 373 et seq.; 
methylen-blue method, 204 et seq.; 
and see Neurological methods. 
Nervous system, central, see Central 
nervous system and Neurological 
methods. 
Nessler, test for ammonia, 298, note. 
Nesteroeesky, gold process, 256. 
Neuroceratin, 403, 404. 
Neuroglia, 435, 436. 
Neurological methods, 385—443 ; in- 
troduction, 385, 386; section 
methods, 387—401; cytological 
methods of neurology, 401-—404; 
nerve-fibre stains, Weigert and 
others, 405—414 ; axis-cylinder 
and protoplasm stains, 415—419; 
axis-cylinder and protoplasm im- 
pregnations (Golgi and others), 
419—434; and see Impregnation 
methods, Retina, Inner Ear, 
Nerves, Central nervous system, 
&c., and the names of Authors. 
Neutralisation of carmine masses, 296. 
Neutral red, 226. 
Neutral tar colours, 179. 
Neutrophilous cell-elements, 446. 
New green, 228. 
Neyt, see van Beneden and Neyt. 
Nias, cleaning slides, 504. 
Nietzki, carminic acid, 146 ; hserna- 
tein, 161. 
Nicoile and Cantacuzene, impreg- 
nation method, 261. 
Nicotin for narcotisation, 13. 
Niessing, fixing mixtures, 363. 
Nigranilin, 231. 
Nigrosin, 197, 228; with safranin, 230. 
Nikieorow, borax-carmine, 155; stain 
for nervous centres, 414; clearing 
celloidin sections, 108; acidoplii- 
lous mixture, 229. 
Nissen, gentian violet staining pro- 
cess, 193. 
Nissl, stains for nerve-cells, 401, 402 ; 
for nerves, 414. 
Nitrate of silver, for impregnation, 
242 et seq. ; for marine animals, 
248; and see the names of Authors. 
Nitrate of uranium, fixing mixture 
with osmium, 27. 
Nitric acid for fixing, 35 ; for harden- 
ing, 57, 392 ; for maceration, 318; 
for decalcification, 324, 325; for 
bleaching, 329. 
Nitric and chromic acid mixtures, 35. 
Nitrite of amyl as a vaso-dilatator for 
injections, 293. 
Noir Colin, 231. 
Noll, corrosion, 322; sponge spicules, 
494; salicylic acid medium, 268. 
Nordmann, plasma-cells, 447. 
Normal salt solution, 263. 
Norris and Shakespeare, carmine 
and indigo stain, 236. 
Nuclein, reactions of, 357—360, and 
see Chromatin. 
Nucleus, see Cytological methods. 
Nucleo-albumins, 358. INDEX. 
527 
The numbers refer to the Pages. 
O. 
Obersteiner, central nervous system, 
386; hardening, 391; staining, 415. 
Obregia, Golgi’s silver chromate me- 
thod, 430; serial section method, 
133. 
Oceania, 491. 
Octopus, eyes, 470. 
Odenius, tactile hairs, 319. 
Ogata, hardening brain, 392. 
Ohljiacher, reactions of safranin with 
iodine and picric acid (pseudo- 
carcinoma corpuscles), 192; sec- 
tion-fixing process, 125; formalde- 
hyde staining process, 187. 
Oil, of aniseed, imbedding mass, 118; 
of bergamot, 68; cedar wood, 80; 
cloves, 9, 67; origanum, 68, 109; 
sandal-wood, 69; turpentine, 69; 
and see Clearing agents. 
Ommatostrephes, eyes, 470. 
One-third alcohol, 51, 313. 
Onuphis, 484. 
Opal blue, 230. 
Ophiuridea, 486. 
Opisthobranchiata, 468. 
Opisthotrema, 476. 
Oppel, liver, 427, 464. 
Oppitz, silver staining, 248. 
Orange G, 216, 217. 
Orcein, 234, 445, 451. 
Orchella, 234. 
Orchestia, embryology, 352. 
Orcin, 234. 
Orientation in paraffin; incelloidin, 103. 
Origanum oil, 68, 109. 
Orseille, 234. 
Orth, methyl violet, 200; lithium- 
carmine, 155. 
Osborn, embryology of Triton, 343; 
brain of Urodela, 400. 
Osmic acid, for fixing, 24—29; for 
hardening, 57, 391; for macerat- 
ing, 317; regeneration of solution, 
25; for impregnation, 258; 
blackening of preparations with, 
27; how to keep solutions, 24; 
Cori’s solution, 24 ; Koiossow’s 
solution, 25, 27; osmic acid and 
alcohol, 27; aud chromic acid 
mixture, 31 j and chromic and 
acetic acid mixture, 31—35 ; pla- 
tinic mixture, 43; osmic and 
oxalic stain, 260; osmic and pyro- 
gallic ditto, 258. 
Osmicated cells, 28. 
Osmium-blackened fat, 35, 445. 
Osmium-carmine, 155. 
Osmosis, to avoid, 3, 4. 
Ostracoda, 472. 
Otocyst of Mysis, 475. 
Ova, see Embryology. 
Ovens for paraffin, 82. 
Overton, bleaching osmic objects, 28; 
fixing with iodine vapours, 47. 
Oviatt, injection method, 294. 
Ovum, see Cytological methods, and 
Embryological methods. 
Owen, preservative liquid, 267. 
Oxalic acid for maceration, 319. 
Oxygenated water, 25, 28, 30, 329. 
P. 
Pacini, preservative liquids, 266. 
Pal, hsematoxylin stain, 409; staining 
nervous centres, 433. 
Pabadino, iodide of palladium nerve 
stain, 413. 
Palladium chloride, for fixing, 43 ; de- 
calcifying, 323; hardening, 61; 
staining, 261. 
Palladium iodide nerve stain, 413. 
Palythoa, 489. 
Pancreatin digestion fluids, 320. 
Paneth, making Weigert’s hsema- 
toxylin, 407; goblet-cells, 463. 
Pansch, injection mass, 310. 
Paper, imbedding trays, to make, 75, 
76; paper cells, 285. 
Papillae foliatae, 375. 
Paracarmine, 156. 
Paraffin, imbedding processes, 79 et 
seq.; review of, 92; paraffin 
masses, 93, 94; solvents of, 79, 
91; ovens, 82; paraffin for luting 
mounts, 289. 
Paralinin, 359. 
Paris green, 198. 528 
INDEX. 
The numbers refer to the Pages. 
Paris violet, 200. 
Parker, turpentine cement, 287. 
Parker, G. H., dehydration methods, 
5; methylen-blue method, 211, 
474; eyes of scorpions, bleaching, 
329; eyes of Homarus, 474; of 
Astacns, 474. 
Parker and Floyd, formaldehyde for 
brain, 397. 
Parma blue, 230. 
Partsch, cochineal stain, 150. 
“ Patentsaures ltubin,” 219. 
Patten, embryology of Blattida, 350; 
orientation in paraffin, 84; eyes 
of Molluscs and Arthropods, 470; 
maceration of Mollusca, 471. 
Paulsen, goblet cells, 463. 
Pecten, eyes of, 470. 
Pedicellina, 468. 
Pelagic ova, 345. 
Pennatulidse, 489. 
Pentacrinus, 488. 
Pepsin digestion fluids, 320. 
Perchloride of iron, for fixing, 44; for 
impregnation, 260. 
Peremeschko, cytological methods, 
354. 
Perenyi, fixing fluids, 35, 342. 
Pergens, picro-carmine, 154. 
Periplaneta, ova of, 350, 472. 
Perl, soluble carmine, 155. 
Permanganate of potash for washing 
out carmine stains, 148; for 
macerating, 317; for mordanting, 
186; for reducing silver impreg- 
nations, 248. 
Perophora, 467. 
Peroxide of hydrogen, 25, 28, 30, 329. 
Peroxide of sodium, 329. 
Perrier, narcotising Lumbricus, 483. 
Perruthenic acid, 464. 
Petroleum-ether, 80. 
Peitzner, safranin solution, 190; 
damar solution, 281; Infusoria, 
497. 
Phalangida, 475 ; ova of, 351. 
Phallusia, 467. 
Phascotosoma, 481. 
Plienicienne, la, 200. 
Phenylen brown, 200. 
Phloroglucin, 326. 
Phloxin, 226. 
Phoronis, 481. 
Phospho-inolybdic-acid hsematoxylin, 
178, 418. 
Phosphoric acid for decalcification, 
326. 
Phosphorus in tissues, test for, 359. 
Photoxylin imbedding method, 100. 
Phyllosoma, 472. 
Physalia, 493. 
Physiological salt solution, 263. 
Physophora, 493. 
Pianese, double-stain for connective 
tissue, 444; methylen blue and 
eosin, 227. 
Picric acid, fixing fluids, 47 et seq. 
hardening ditto, 61; staining with, 
216; decalcification with, 324, 
326. 
Picric alcohol, 50, 317. 
Picro-alum-carmine, 152. 
Picro-carminate of ammonia, 153. 
Picro-carminate of soda, 154. 
Picro-carmine, 153, 154. 
Picro-cliromic acid, 36. 
Picro-liydrochloric acid, 50. 
Picro-nigrosin, 417. 
Picro-nitric acid, 50. 
Picro-osmic acid, 50. 
Picro-platinic mixture, 50. 
Picro-sublimate mixtures, 41. 
Picro-sulpliuric acid, 48. 
Pictet, examination liquid, 263. 
Piersol, embryology of Mammalia, 
338. 
Pigment spots, artificial, 59. 
Pintner, Taeniae, 476. 
Pisenti, alum-carmine, 150. 
Plakina, larvae, 494. 
Planaria, 40; embryology, 352. 
Plasma-cells, 445—450. 
Plasma fibrils, 371. 
Plasma stains, 215—232, 365. 
Plastic reconstruction of sections, 333. 
Plastin, 359. 
Platinic mixture, Merkel’s, 36, 61; 
Lindsay Johnson’s, 59. 
Platino-aceto-osmic mixture, 43. 
Platino-osmo-bichromate mixture, 59. INDEX. 
529 
The numbers refer to the Pages. 
Platino-sublimate mixture, 332. 
Platinum chloride, for fixing, 42, 43. 
Plaxnee, Kernschwarz, 233; medul- 
lated nerve, 403. 
Platyhelmia, 476—479. 
Plessis, du, see du Plessis. 
Pluteus, 487. 
Podwyssozki, fixing mixture, 34; saf- 
ranin staining, 191; carcinoma 
corpuscles, 192. 
Poisoning methods, 16. 
Polaillon, impregnation method, 260. 
Poli, serial sections, 128. 
Polxtzeb, inner ear, 443. 
Polychaeta, 484. 
Polychromatic methylen blue, 282. 
Polzam, imbedding mass, 95. 
Porifera, 493. 
Porpita, 493. 
Potash acetate, mounting medium. 
265. 
Potash solution for maceration, 314. 
Potassium bichromate 58 et seq. 
Potassium hypochlorite, 322, 330. 
Potassium permanganate, see Perman- 
ganate of potash. 
Pouchet, bleaching methods, 329. 
Pbenant, safranin staining, 191; 
cochlea, 442. 
Preservative media, 4, 5, 262 et seq. 
Prickle-cells, 371. 
Primrose, 226. 
Pbingle, the water section-fixing pro- 
cess, 120; vacuum imbedding, 83. 
Pbitchaed, hardening fluid, 57; re- 
ducing solution, 256; inner ear, 
442. 
Progressive staining, 136, 197 et seq. 
Prosobranchiata, 468. 
Protula, 484. 
Protozoa, introductory, 495; quieting, 
495; staining intra vitam, 496; 
cilia, 499; flagella, 499; other 
methods, 497—499. 
Pbudden, haematoxylin, 170. 
Prussian blue, soluble, 301; impreg- 
nation with, 261; injection 
masses, see Injections. 
Prussic acid, for killing, 16. 
Pteropoda, 469. 
Pubcell, eyes of Phalangida, 475. 
Purpurin, 235. 
Pyrenin, 359. 
Pyridin for hardening and clearing, 
62. 
Pyrogallate of iron stain, 260. 
Py rosin, 226. 
Q. 
Quebvain, de, fixation of nerve- 
centres, 387. 
Quieting Infusoria, &c., 495. 
Quince-mucilage fixative, 127. 
Quinolein blue, 228. 
It. 
Rabbit, embryology, 335 et seq. 
Rabl, fixing methods, 31, 41, 42, 332 ; 
embryos of Salamandra, 343; 
serial sections, 121; alum-co- 
chineal, 151; picro-sublimate 
mixture, 41; platino-sublimate 
mixture, 332; medullated nerve, 
404; staining method, 239; cyto- 
logical methods, 363; collodion- 
ising sections, 90. 
Rabl-RuCKHAED, embryology of 
Telostea, 345. 
Ramon y Cajal, see Cajal. 
Sana, embryology, 343. 
Ranviee, alcohol, absolute, prepara- 
tion of, 52; one-tliird ditto, 51, 
313; areolar tissue, 444; bladder 
of frog, 384; bone, 451; carmine, 
neutral, 155; clasmatocytes, 450; 
cornea, 376; corpuscles of Golgi, 
381; decalcificatiou, 325; epithe- 
lium, 370; gland-cells, 464; gold 
chloride impregnation, 253, 254 
(and see Motor plates, Nerve- 
fibres, &c.) ; hsematoxylin, 173 ; 
injection masses, carmine, 296; 
Prussian blue, 300, 301; silver 
nitrate,306; glycerin, 309; aqueous, 
309; iodised serum, 263 ; macera- 
tion, 313, 319 ; medullated nerve, 
404; motor plates, 380; mucus 
cells, 464; nerve-fibres, intra-epi- 530 
INDEX. 
The numbers refer to the Pages. 
dermic,373; neutral carmine,155; 
nitrate of silver impregnation, 
243, 245; perruthenic acid, 464; 
picro-carmine, 153,154; purpurin, 
235; quinole'in blue, 228; retina, 
439, 442 ; tactile hairs, 373. 
Ranvier and Vignal, osmium mix- 
ture, 27. 
Rawitz, hsematein staining, 166 ; “ in- 
vert” stain,224; artificial alizarin, 
232; Golgi’s impregnation me- 
methods, 419, note. 
Reagents, how to procure, 143 ; selected 
collections of, 503. 
Recklinghausen, von, impregnation 
methods, 242, 246, 247. 
Reconstruction of objects from sections, 
333. 
Redding, gold process, 258. 
Redenbaugh, narcotisation, 16. 
Regaud, silver impregnation, 247. 
Regressive staining, 137, 182 et seq. 
Rehm, nerve-tissue stains, 402, 417. 
Reich, silver nitrate, 246. 
Reichenbach, embryology of Astacus, 
352. 
Reinbach,triacid mixture, 223; cover- 
glass preparations, 460. 
Reinhold-Giltay microtome, 73. 
Reinke, Flemming’s orange stain, 218; 
lysol for maceration, 319; staining 
horny tissues, 373. 
Remak’s copper hardening fluid, 344. 
Renaut, haematoxylin, 173; hsema- 
toxylic eosin, 238 ; silver impreg- 
nation, 247; cornea, 376. 
lteptilia, embryology, 312; myelon, 398. 
Resegotti, coal-tar stains, 185, 189; 
staining by substitution, 185 ; on 
the kinds of safranin, and on saf- 
ranin staining, 189. 
Resins, mounting media, 277 et seq. ; 
imbedding masses, 114, 115. 
Retina, 439—442. 
Retterer, natural injections, 311; ova 
of rabbit, 337; smooth muscle, 382. 
Retzius, methylen-blue method, 209. 
Rezzonico, medullated nerve, 404. 
Rhabdoccela, 477. 
Rhizophysa, 493. 
Rhopalea, 467. 
Ribbon section-cutting, 88. 
Richard, Bryozoa, 15. 
Rindfleisch, maceration, 317. 
Ringing wet mounts, 285. 
Ripart and Petit, preservative fluid, 
46, 267. 
Ritter, embryology of Chironomus, 
351. 
Robert, fixing Aplysia, 469. 
Robertson, imbedding mass, 114. 
Robin, injection masses, 294,295, 305; 
natural injections, 311. 
Robinski, silver nitrate, 246. 
Rocking microtome, 72. 
Rollett, Carminroth, 155; freezing 
process, 118; cornea, 376; sec- 
tions of muscle, 378. 
Roosevelt, pyrogallate of iron, 260. 
Rose B. a l’eau, 226. 
Rose de naphthaline, 195. 
Rose, preparation of bone, 452. 
Rosein, 196. 
Rosin, stain for nerve-cells, 402 ; for 
axis-cylinders and protoplasm,417. 
Rossi, stain for nervous centres, 412; 
blood, 457. 
Rossler, Phalangida, 475. 
Rotatoria, 482. 
Rouge, fluorescent, 196. 
Rouget, silver nitrate, 246, 248; 
methylen blue, 207. 
Roijsselet, permanent preservation of 
Rotifers, 482. 
Roux, embryological methods, 344. 
Rubin, 196. 
Ruffini, corpuscles of Golgi, 382. 
Ruprecht, bone, 453. 
Ruthenium, impregnation method, 261. 
Rutherford, picro-carmine, 154. 
Ryder, double imbedding, 112. 
S. 
Sacerdotti, Golgi’s impregnation, 
426. 
Sachs, motor plates, 381. 
Saefftigen, Echinorhyncus, 480. 
Saftrosin, 226. INDEX. 
531 
The numbers refer to the Pages. 
Safranin, 189 et seq.; with indigo-car- 
mine or nigrosin, 229, 230; with 
Wasserblau, 231; with Lichtgrtin 
or Saureviolett, 227; for elastic 
tissue, 450; for cartilage, 455. 
Sagartia, 488. 
Sahli, balsam, 280; nerve-centres, 
hardening, 390; stain for, 414. 
Sala, Golgi’s silver chromate impreg- 
nation, 430. 
Salamandra, embryology, 343; cyto- 
logy of, 354, 355 ; breeding larvae, 
355. 
Salicylic vinegar, 267, 268. 
Saliva, artificial, 315. 
Salivary glands, 464. 
Salt solution, 263; for maceration, 
313, 314. 
Salts, metallic, action of light on, 242. 
Samassa, Golgi’s silver chromate me- 
thod, 429; sections of Ctenophora, 
493. 
Sandal-wood oil, 69. 
Sandarac, gum, 283. 
Sanfelice, liaematoxylin, 173. 
Sankey, staining nervous centres, 416. 
Santoritts, paraffin stove, 82. 
Sapphirina, 472. 
Sarasin, embryology of Reptilia, 342. 
Sarcolemma, 378. 
Sabgent, bleaching, 328. 
Sattlee, silver staining, 248. 
Saurefuchsin, 219. 
Sauregelb, 219. 
Saurerubin, 219. 
Saureviolett, 227. 
Saville Kent, see Kent. 
Sawtschenko, carcinoma corpuscles, 
192. 
Sazepin, antennae, 473. 
Schafer, muscle-cells, 378. 
Schaffer, cartilage, 455 ; retina, 441; 
reconstructing sections, 333; bone, 
451. 
Schallibaum, serial sections, 120. 
Schenk, fixing fluid, 46; carbolic 
fuchsin, 196. 
Schewiakoff, Protozoa, 499. 
Schiefferdeckeb, cartilage, 456; cel- 
loidin imbedding, 100, 101, 104; 
injection masses, 310; levulose for 
mounting, 270; xnethyl mixture, 
319; pancreatin digestion fluid, 
320; serial sections, 120, 129; 
maceration of retina, 442 ; me- 
dullated nerve, 404; Mastzellen, 
447; the water section-fixing pro- 
cess, 120. 
Schmatts, uranium - carmine, 155 ; 
staining nervous tissue, 416. 
Schmidt, embryology of Limax, 348. 
Schneider, aceto-carmine, 15£. 
SCHOLTZ, intra-vitam staining, 138. 
Schulgin, double stain, 237 ; embry- 
ology of Amaroecium, 346; pa- 
raffin mass, 94. 
Schtjltze, P. E., palladium chloride, 
613; embryology of Spongiae, 
494 ; Pennatulidae, 489; section- 
stretcher, 87; hardening skin, 
370. 
Schultze, Max, acetate of potash 
mounting medium, 265; sulphuric 
acid macerating mixture, 319; 
retina, 442; iodised serum, 263. 
Schurmayer, Infusoria, 495. 
Schwalbe, smooth muscle, 382; coch- 
lea, 442; impregnation, 242. 
Schwarz, chemistry of the cell, 359. 
Schwaeze, Cercaria, 477. 
Schweiggee-Seidel, carmine, 155. 
Sclavo, stains for flagella, 500. 
Scorpions, eyes of, 329. 
Scott and Osborn, embryology of 
Triton, 343. 
Sealing-wax varnish, 290. 
Seaman, cement, 290; glycerin jelly, 
274. 
Sections, cutting, 85 ; chain or ribbon, 
88; collodionisation, 89; serial 
mounting, 119 et seq.; section- 
stretching and section-stretchers, 
86—88; unrolling, 87,91; shrink- 
age in, 87 ; reconstruction, 333. 
Seeligee, Antedon, 488. 
Segall, medullated nerve, 404. 
Sehrwald, Golgi’s silver chromate 
method, 428, 429, 431. 
Seiler, cleaning slides, 503 ; alcohol 
balsam, 280; double stain, 235. 532 
INDEX. 
The numbers refer to the Pages. 
Selenka, imbedding method, 77. 
Sepia, eyes, 470. 
Serial section mounting, 119 et seq. 
Serum, artificial, 264, 265 ; iodised, 
263, 264, 313. 
Shell, 471. 
Shellac, cement, 290; imbedding mass, 
114; fixative for sections, 122; 
varnish, 289. 
Shimee, mounting medium, 269. 
Shrinkage in elements of sections, 87. 
“ Siebdosen,” 4. 
Siebenmann, labyrinth, 443. 
Sieve-dishes, 4. 
Sihlee, nerve-endings, 381. 
Silver chromate impregnation, 419 
et seq., and see Golgi. 
Silver nitrate, see Nitrate of silver. 
Siphonophora, 492. 
Sipunculus, 481. 
Skin, hardening, 370; nerves of, 373. 
Slides, cleaning, 503 ; labelling, 504. 
Smibnow, tactile corpuscles, 374; 
Golgi impregnation, 426. 
Smith, Hopewell, decalcification and 
sections of teeth, 325, 453. 
Soap, imbedding masses, 94 et seq., 
477. 
Sobetta, ovum of mouse, 338. 
Soda solution for maceration, 314. 
Soda hypochlorite, see Eau de Labar- 
raque. 
Soda-picro-carmine, 154; soda-car- 
mine, 155. 
Sodium peroxide, 329. 
Solferino, 196. 
Solgeb, sarcolemma, 378; salivary 
glands, 464. 
Solid green, 228. 
Sollas, gelatin imbedding (freezing 
method), 117. 
Soluble blue, 231. 
Solution of Eeltcki, of Mulleb, of 
Mebkel, &c., see the names of the 
respective Authors; normal salt, 
263; macerating salt, 313. 
Solutions of metallic salts, how to 
keep, 242. 
Sommeb, Macrotoma, 475. 
Souliee, macerating mixtures, 315. 
Souza, de, pyridin, 62. 
Spee, Gbaf, prepared paraffin, 94. 
Spek, van deb, and Unna, plasma- 
cells, 449. 
Spheres, attraction, see Cytological 
methods, Plasma stains. 
Sphgranttra, 477. 
Spicules of sponges, 494. 
Spinal cord, see Neurological methods. 
Spirit blue, Spirit-soluble blue, 230. 
Spirographis, 484. 
Spongise, 493. 
Spongilla, 494. 
Squibe, histological reagents, 144; 
oil of origanum, or white thyme, 
69; methyl green, 199; commer- 
cial salts of gold, 251; glycerin 
jelly, 275; damar medium, 281; 
granule cells, 446 ; Sauref uchsin 
and orange, 239 ; mounting 
methyl-green stains, 199; decal- 
cification, 325, 326; picro-car- 
mine, 154; hsematoxylin, 171. 
Stabilit imbedding-blocks, 108. 
Stahl, immobilisation of Infusoria,496. 
Staining, generalities, 135 — 144 ; 
choice of stain, 143; on slide, 7 ; 
in toto, 7, 10; intra vitam, 138, 
203, 496; reagents, how to ob- 
tain, 143; staining by substitu- 
tion, 184; substantive and adjec- 
tive, 140; progressive and regres- 
sive, 136; theory of, 137; direc- 
tions for the regressive method, 
182; staining nerve-tissue, see 
Nerves, Central nervous system, 
Neurological methods, &c. 
Stains, combination, 233 et seq., 669. 
Stains, general, selective (nuclear, 
plasmatic, and histologically spe- 
cific), defined, 135; aqueous and 
alcoholic stains, 9, 10; effect of 
transparency and opacity of stains, 
188; generalities, 135 et seq. ; in- 
vert stains, 142, 224; cytological, 
365; and see Anilin black, Anilin 
blue, Carmine, Cochineal, Eosin, 
Gold, Hmmatoxylin, Indulin, Iron, 
Orchella, Purpurin, Safranin, 
Silver, &c. INDEX. 
533 
The numbers refer to the Pages. 
Starch injection mass, 310. 
Steinach, sieve-dishes, 4. 
Stentor, 495. 
Stephenson, mounting medium, 275. 
Sternaspidaj, 484. 
Stieda, cements, 290; clearing media, 
65. 
Stilling and Pfitznee, stomach of 
Triton, 384. 
Stibling, sulphocyanides of ammo- 
nium and potasssium, 315 ; anilin 
blue-black, 416. 
Stomach of Triton, 384. 
Storax, 283. 
Stoves and water-batlis, 82. 
Stbahl, embryology of Reptilia, 342. 
Stbassen, ztte, Bradynema, 480. 
S tbasseb, plastic reconstruction, 333 ; 
section-stretcher, 87; serial sec- 
tions, 122. 
Stbickeb, imbedding mass, 114. 
Stboebe, stain for nerve-centres, 414. 
Steong, fixing nerve-centres, 388; 
formaldehyde for the Golgi im- 
pregnation, 427. 
Strongylus, 480. 
Strychnin for killing, 16. 
Styrax, 283. 
Sublimate, corrosive, see Corrosive 
sublimate. 
Substantive and adjective staining, 
140. 
Substitution of stains, 184. 
Suchannek, the water section-fixing 
process, 119; bergamot oil, 68; 
anilin oil for clearing, 70 ; Venice 
turpentine, 282; sieve-dishes, 4. 
Sulphate of copper fixing fluids, 344, 
492, 493; impregnation, 434. 
Sulphate of magnesia for narcotisa- 
tion, 16. 
Sulphindigotate of soda or potash, 
235. 
Sulphocyanides, 315. 
Sulphuric acid for maceration, 319. 
Sulphurous acid for bleaching, 330. j 
Scmmebs, serial sections, 122, 129. 
“ Sunning,” solutions of metallic salts, 
243. 
Stjssdobf, mucus cells, 462. 
Sycandra, 494. 
Sympodium, 489. 
Synapta, 486. 
Syrup, imbedding masses, 116; exa- 
mination media, 265. 
T. 
Tactile corpuscles, 373 et seq. 
Tactile hairs, 373. 
Tcenia, 476; embryology of, 352. 
Tapani, inner ear, 443. 
Tagttchi, injection, 310. 
Tax, Golgi’s mercury stain, 433. 
Tannin solution, 267; for Infusoria, 
499. 
Tar colours, see Anilin dyes. 
Taetufeei, impregnation, corneal 
cells, 377. 
Tegumentary organs of Vertebrates, 
370 et seq. 
Teichmann, white injection, 305; oil 
mass, 310. 
Teleostea, embryology of, 344 et seq. 
Tendon, 381,382. 
Test-cells of Ascidians, 346. 
Tethya, 494. 
Thenea, 249. 
Thermostats, 82. 
Thieksch, carmine injection, 300; 
Prussian blue ditto, 302; lead 
chromate ditto, 304; green ditto, 
305; oxalic acid indigo-carmine, 
235. 
Thimbles, paper, to make, 76. 
Thin, retina, 442. 
Thioniu, for plasma-cells, 450; for 
mucin, 461; as a chromatin stain, 
194. 
Thiophen green, 228. 
Tiioma, microtome, 71; decalcifica- 
tion, 325. 
Thompson’s high refractive medium, 
277. 
Thbelfall, section-fixing method, 
128. 
Thyme, oil of, 69. 
Tiara, 491. 
Tintinnodea, 499. 
Tibelli, medullated nerve, 404. 534 
INDEX. 
Tizzoni, alum-carmine, 150 ; harden- 
ing skin, 370. 
Tobacco-smoke killing method, 12. 
Toison, blood, 459. 
Tolu balsam cement, 290. 
Toluidin blue, 197. 
Toluol, for clearing, 70; for preserv- 
ing, 5; for imbedding, 79. 
Tornier, haematoxylin staining, 174. 
Torneux and Hermann, silver stain- 
ing, 246; epithelium, 370. 
Trays, paper, to make, 75. 
Trematodes, 476. 
Trenkmann, stains for flagella, 500. 
Triacid mixture, Ehrlich’s, 223. 
Tricenophorus, 476. 
Trichinae, 480. 
Trinchese, nerve-endings in muscle, 
379. 
Triton, stomach, 384. 
Tropaeolin, 219. 
Trypsin, digestion fluid, 321. 
Trzebinski, spinal cord, 392. 
Tschisch, nerve-centres, 59. 
Tubby, celloidin imbedding, 103. 
Tullberg, narcotisation with mag- 
nesium chloride, 16. 
Tunieata, 467; embryology of, 346. 
Turbellaria, 477. 
Turpentine for clearing, 69; for 
mounting, 282; cement, 287 ; for 
imbedding, 79. 
U. 
Ulianin, embryology of Amphipoda, 
352. 
Underwood, gold process, 257. 
Unna, bleaching chromic objects, 30 ; 
ripening of haematoxylin solutions, 
161, 163; instanteously ripe hae- 
matoxylin stain, 163; half-ripe 
stock solution, 163; plasma-cells 
and Mastzellen, 448, 449; elastic 
tissue, 451; mucin, 463; collagen, 
444; plasma fibrils of epithelium, 
372; stain for smooth muscle, 383. 
Upson, gold and iron method, 436; 
gold and vanadium method, 437; 
other stains, 416. 
The numbers refer to the Pages 
Uranium acetate, 27, 46. 
Uranium nitrate, 27. 
Uranium-carmine, Gierke and 
Schmaus, 155. 
Ussow, embryology of Cephalopoda, 
346. 
V. 
Vacuum imbedding, 82. 
VAN Beneden, acetic acid fixing 
method, 44; acetic alcohol, 45; 
Ascidians, 467; corrosive subli- 
mate liquid, 38; cytological me- 
thods, 364; embryology of Mam- 
malia, 335, 337 ; of Ta>nia, 352. 
van Beneden, and Nett, cytological 
methods, 45, 228, 364, 368. 
VAN DER Spek, Mastzellen, 449. 
VAN DER Stricht, bergamot oil, 68. 
van Ermengem, stains for flagella, 
500. 
van Gehtjchten, acetic alcohol, 45; 
cutting brain sections, 399; 
Nissl’s stain, 402; medullated 
nerve, 403 ; Golgi’s impregnation, 
428. 
VAN Gieson, clearing with anilin oil, 
109 ; formaldehyde for brain, 397. 
van Hetjrck, mounting medium, 277. 
VAN Walsem, hot or cool microtome- 
knife, 88; flattening sections, 91; 
paraffin mass, 94; serial sections, 
128; Weigert nerve-stain, 412. 
Vanadate of ammonium, injection 
mass, 310. 
Vanadium haematoxylin, 368. 
Varnishes, 284 et seq. 
Velella, 493. 
Venice turpentine, for mounting, 282; 
cement, 287. 
Veretillum, 12. 
Veridine, 198. 
Vermes, 476; embryology, 352 et seq. 
Vert d’alcali, 198. 
Vert d’Eusebe, 198. 
Vert en cristaux, 197. 
Vert lumiere, 197. 
Verworn, narcotisation, 14. 
Vesuvin, 200. INDEX. 
535 
The numbers refer to the Pages. 
VlALLANES, gold method, 254; cel- 
loidin imbedding, 104; brain of 
Arthropods, 475. 
Vialleton, embryology of Aves, 341. 
Victoria blue, 195. 
Victoria green, 228. 
Vignal and Ranvier, osmium mix- 
ture, 27; picro-carmine, 153. 
Ville, carmine injection, 296. 
Violet B, 231, 444. 
Violet of Lauth, 194. 
Virchow, action of light on chromic 
objects, 30. 
Vivante, bone, 453, 454. 
Vogt and Yung, Cucumaria, 486; j 
Rotifers, 482; Sipunculus, 481; i 
cleansing intestine of Lumbricus, j 
483; Cestodes, 476. 
vom Rath, picro-sublimate mixture, 
41; picro-sublimate osmic, 42; j 
picro-osmic, 50; picro-platinic, 
50. 
von Ebner, decalcification mixture, 
325. 
von Graef, Turbellaria, 477. 
von Koch, copal imbedding process, ! 
114. 
von Lendeneeld, staining collar- 
cells, 494. 
von Lenhossek, nerves of Annelids, 
484; tongue of rabbit, 375. 
von Mahrenthal, osmium impreg- 
nation, 260. 
von Marchi, nerve stain, 412. 
von Marschalko, plasma cells, 450. 
VON Nathusius, hairs and nails, 373. 
VON Wistinghausen, neutralising 
hajmatoxylin-stained tissues, 166; 
hsematoxylin stain, 173; embry- 
ology of Nereis, 353. 
Vosmaek, epithelium of sponges, 249. 
Vosseler, Mayer’s albumen fixative, 
125; Venice turpentine, 282; wax 
feet, 312; cardboard ridges for 
slides, 504. 
W. 
Waddington, arabin, 126; Infusoria, 
499. 
Wagnerella, 328. 
Waldeyer, inner ear, 442; plasma- 
cells, 445; decalcification, 323. 
Warburg, Ehrlich-Biondi mixture, 
222. 
Ward, narcotisation methods, 17; 
Sipunculus, 481. 
Washburn, embryology of Limax, 
348. 
Washing out, 2, 20, 21. 
Wasserblau, 231, 445. 
Watase, embryology of Cephalopoda, 
347. 
Watch-glass imbedding method, 77. 
Water, fresh or warm, for killing, 17. 
Water section-fixing process, 119. 
Water-baths, 82. 
Water-blue, 231, 445. 
Watney, dichroism of hsematoxylin 
stains, 165. 
Wax feet, 312. 
Wax imbedding masses, 94. 
Webb, dextrin freezing mass, 117. 
Weber, preservation of Holothurids, 
486; of Asteroidea, 486; of Ecliino- 
idea, 486; of Siplionophora, 493; 
Rotifers, 482. 
Webster, naphtha for paraffin im- 
bedding, 79. 
Wedl, orchella stain, 234. 
Weigert, Bismarck brown, 200; 
picro-carmine, 154; hsematoxylin 
nerve methods, 405—412 (the 1885 
method, 405; the 1891 method* 
407; variations, 409—412) ; clear- 
ing celloidin sections, 109 ; mount- 
ing serial sections, 131; varnish 
for mounting, without cover, 283 ; 
hardening nerve-centres, 390,397 ; 
specific neuroglia stain, 435 ; fibrin 
stain, 460. 
Weil, section method, 115; bone, 452. 
Weysse, embryology of Sus, 338. 
Wheeler, embryology of Blattida,350. 
White, sections of bone or dental 
tissue, 453. 
Whitelead cement, 290. 
White of egg, section-fixing process, 
123; imbedding methods, 118; 
injection mass, 307. 536 
INDEX. 
The numbers refer to the Pages. 
White zinc cement, 290. 
Whitman, fixing mixture, 61; ova of 
Amphibia, 342; pelagic ova, 345 ; 
Hirudinea, 484. 
Wickebsheimeb, preservative fluid, 
267. 
Will, Aphides, 351. 
Wilson, Alcyonaria, 489 ; embryology 
of Lumbricus, 352. 
Wintebsteineb, serial section method, 
133. 
Wissowsky, blood, 458. 
Witt, shellac, 290. 
Wlassax, serial section mounting, 
120. 
Wolff, motor plates, 381; bladder of 
frog, 384. 
Woltees, hsematoxylin nerve stains, 
411, 418; cartilage, 456; skin 
nerves, 373; vanadium nerve 
stain, 418. 
Woodwaed, borax-carmine, 155. 
Woodwoeth, orienting paraffin ob- 
jects, 85. 
Weight, Sphyranura, 477. 
X. 
Xylol, for clearing paraffin sections, 
70; ditto celloidin sections, 109; 
for paraffin imbedding, 80; for 
preserving tissues, 5. 
z. 
Zachabiades, bone, 455. 
Zachaeias, acetic alcohol, 45; acetic 
carmine, 152; iron-carmine, 152. 
Zachaeias, E., chemistry of the cell, 
359. 
Zellnee, natural injections, 311. 
Zenkeb, fixing liquid, 42. 
Zenthoeeee, elastic tissue, 451. 
Zebnkcke, preparation of Ligula, 476. 
Zieglee, white cement, 290. 
Ziehen, gold and mercury impregna- 
tion method, 434. 
Ziehl’s carbolic fuchsin, 196. 
Zimmebmann, microtomes, 73. 
Zimmeemann, sieve-dishes, 4; hone, 
453. 
Zimmeemann, A., chemistry of tlie 
cell, 359. 
Zinc, chloride, for hardening brain, 
396, 397; impregnation method, 
434. 
Zinc-white cement, 290. 
Zoantharia, 489. 
Zoja, methylen blue, 204; bioblasts of 
Altmann, 368 ; Hydra, 490 ; Pro- 
tozoa, 499. 
Zoobothrium, 468. 
Zoological methods, 466 et seq. 
Zschokke, deltapurpurin, benzopur- 
purin, 225 ; cartilage, 456. 
ZUB Steassen, Bradynema, 480. 
Zwaabdemakeb, safranin stain, 190. 
ERRATA. 
Page 7, line 20, for “ side ” read “ slide.” 
Page 68, line 3, for “ cannel ” read “ cinnamon.” 
Page 459, line 2 from bottom, for Lowitz” read “ Lowit’s.” 
FEINTED BY ADLABD AND SON, 
BAETHOLOMEW CLOSE, E.C., AND 20, HANOVEE SQFABE, W.