NOTES 
ON 
HISTOLOGICAL METHODS 
INCLUDING A BRIEF CONSIDERATION OF THE 
METHODS OF 
Pathological and Vegetable Histology, 
AND THE APPLICATION OF THE MICROSCOPE 
TO JURISPRUDENCE. 
For the use of Laboratory Students in the Anatomical Department of the 
CORNELL UNIVERSITY, 
BY 
SIMON H. GAGE. 
Assistant Professor of Physiology and Lecturer on 
Microscopical Technology. 
ITHACA, N. Y., 
Andrus & Church, 
1885-6. Prefatory Note. 
The object of histological methods is to assist the investigator in 
obtaining a complete knowledge of the tissues. Complete knowledge 
of any tissue comprises, in the writer’s opinion, an understanding of: 
1. The gross anatomy. 
2. The form, nature and relations of the structural elements. 
3. The blood vessels. 
4. The lymph-vessels. 
5. The nerve-supply,—the relation of the terminal filaments of the 
nerves to the structural elements. 
6. The histogenesis or development. 
7. The function or physiology of the tissue. 
At the present day, however, not a single tissue is known in all the 
detail indicated above. 
The following pages are designed to supplement the notes printed 
in 1881, and to present improved methods, introduced since that 
time. A list of topics considered in the entire course is also given. 
The permanent preparations required of each student are thought to 
include those tissues, the knowledge of which is essential as a basis of 
histology; and the methods for their study, illustrate the principal 
methods of histological research—methods which, by combination or 
slight modification, will enable the student to obtain a good knowledge 
of all the tissues. 
When not otherwise stated, the methods here given were taken from 
the books and periodicals named at the end; and after the methods of 
study of a tissue are given, references are made to the works in the 
list in which figures and a more or less complete account of its struc- 
ture may be found. Finally, for the benefit of students specially 
interested in Botany, Medicine, and the Relations of Medicine to Law, 
three extra lectures are given on the methods of vegetable and 
pathological histology, and the application of the microscope to 
j urisprudence- 
Special references to the books and periodicals in the alphabetical 
lists at the end are made by giving in brackets : 1st. If a book, the initial letter of the author’s name, or if more than one author has the 
same initial, the initial and one or more of the following letters in 
the name. For periodicals, the initial letters of the title are given. 
2d. If the work consists of more than one volume, the volume is indi- 
cated by a Roman numeral, or with periodicals, the year instead of 
the volume is sometimes indicated by Arabic numerals in parentheses. 
3d. The pages referred to are given in Arabic numbers. Finally, cross 
references to the various sections in these notes are given in paren- 
theses. 
SIMON H. GAGE, 
Anatomical Laboratory of the Cornell University- 
January, 1886. 
Topics. 
1. The Microscope and its Parts.—Simple microscope—compound 
microscope—optical parts of a compound microscope—nomenclature 
of oculars—of objectives—dry, immersion and homogeneous object- 
ives—working distance—mechanical parts. Choice of a simple, and 
of a compound microscope for laboratory use. Care of the mechani- 
cal, and of the optical parts. Care and training of the eyes in micro- 
scopic work. (Notes of ’81. C. Bi. Bau. R. Fi. H. (J.r.m.s.) (Z.w.m.) 
A.s.m. J.n-y.m.s). 
2. Learning to use a microscope.—The simple microscope. Putting 
oculars and objectives in position and removing them—focusing— 
with low and with high objectives. Putting an object under the mi- 
croscope. Revolver or nose-piece. Field of the microscope. Light- 
ing by direct or transmitted, central or axial and oblique light. Dia- 
phragms. Demonstration of the function of the objective,—of the 
ocular—of the mirror. (Notes of’81. Bi. C. Fi. H. R. St.). 
3. Slides and Covers, Interpretation of Appearances.—Slides and 
cover-glasses, cleaning and care (§ 1-4). Mounting an object, (l 5.) 
Dust or cloudiness on the ocular, on the objective. Muscae volitantes 
in the eye of the observer. Relative position of objects having plane 
outlines—having curved outlines—air bubbles and oil globules. Dis- 
tinctness of outline depends on difference in color or refractive power. 
Optical section—highly refractive—doubly contoured. Currents—Pe- 
desis or Brownian movement. Ranvier’s demonstration of pedesis with 
polarized light (R. 175). (Notes of ’81. Bi. C. F.i. R. Popular Scienee 
Monthly, Vol. VII, p. 177. Journal of Science, 1878, p. 167.) 3 
4. Magnification, Micrometry and Drawing.—Magnification of a 
simple microscope—of a compound microscope. Obtained: (A.) Di- 
rectly as for the simple microscope, (B.) With a camera lucida. Forms 
of camera lucidas and their action (Wollaston’s, Abbe’s Grunow’s and 
Schroeder’s). Notes of 81. Bi. C. R. (Z. w. m. I. 1-23.) J. r. m. s. 
(1883) p. 813, (A. m. m. J. Ill, 201.) 
Micrometers and Micrometry—Optical combination and length of 
body—three ways of varying the magnification of a compound micro- 
scope.—(a) by dividing the size of the image by the magnification ; (b) 
by using the camera lucida and stage-micrometer ; (c) by dividing the 
real image as measured, by the ocular micrometer, by the ocular mi- 
crometer ratio. (Notes of ’81.) Accuracy depends on (a) the perfec- 
tion of the instruments, and (b) the skill of the observer. Notes of 
’81. Bi. C. Fi. R.). 
Drawing.—Getting outlines with a camera lucida. Filling in details 
freehand. Getting image of a desired size. Drawing lines of a stage- 
micrometer near the drawing to indicate the magnification. (Bi. C. 
Fi. (Z. w. m. I. pp. 23.) (J. r. m. s. 1884, p. 697). 
5. The Study of living tissues. ($ 7-20). 
6. Isolation and preservation of the structural elements. (§ 21-32). 
7. Hardening tissues. (§ 35-37). 
8. Section cutting and mounting. ($ 34, 38-64). 
9. Serial sections. ($ 60). 
10. Fine injections. (§ 61-67). 
11. Methods of pathological histology. ($ 68). 
12. Methods of vegetable histology. (§ 69). 
13. The microscope in Jurisprudence. (§ 70). 
14. Reagents and their preparation. ($ 71). 
15. Bibliography. (I-VIII). 4 
PERMANENT PREPARATIONS. 
The following permanent preparations are to be made, labelled, cat- 
alogued (£ 25), and submitted for examination and criticism the last 
week of the term. The cross references following each tissue, are to 
the sections in these notes where the methods of preparation are 
given, and references are made to books or periodicals in which may 
be found figures and descriptions of the tissue. Except when other- 
wise stated the tissues are from the Cat. 
1. Blood of Necturus, (£ 21-23). 
2. Blood of Man, (£ 24). 
3. Ciliated epithelial cells, isola- 
ted, (£ 26). 
4. Striated muscular fibers, isola- 
ted, (£ 27). 
5. Muscular fiber-cells, isolated, 
(8.28). 
6. Cardiac muscular fibers, isola- 
ted, (£29). 
7. Myelinic nerve-fibers, isolated, 
(8 39)-. 
8. Amyelinic nerve-fibers, isola- 
ted, (£ 31). 
9. Areolar connective tissue, 
(8 32).. 
10. Endothelium, (£ 33). 
11. Section of cartilage, (£ 34). 
12. Transection of a nerve, (ulnar, 
brachial, or sciatic), (£47). 
13. Transection of myel, (cervical 
or lumbar enlargement), 
(848). 
14- Transection of a striated mus- 
cle, (occipito-scapularis,) 
($49)-. 
15. Transection of an artery, 
($ 50).. 
16. Transection of trachea, 
($.Si). 
17. Section of submaxillary gland, 
($ 52). 
18. Section of stomach of Nectu- 
rus, ($53). 
19. Section of ileum, (§ 54). 
20. Section of liver, ($ 55). 
21. Section of ovary, (§ 56). 
22. Section of human skin, 
(§.57)- . 
23. Section of injected kidney, 
($ 63). 
24. Section of injected small in- 
testine, (§64). 
25. Web of frog’s foot with both 
blood and lymph vessels in- 
jected, (§ 67). 
MICROSCOPIC SLIDES AND COVER-GLASSES. 
(g 1) Slides.—These are strips of clear, flat glass upon which micro- 
scopic specimens are mounted (§ 5). The size most commonly used 
in America is 1x3 inches. In this laboratory, slides 25x45 mm. are 
employed. Slides may be prepared very cheaply from strips of clear 
glass with a diamond or a steel glass-cutter. 
(§ 2) Cleaning Slides.—New slides can usually be well cleaned by 
washing in soft water. Slides that have been used for mounting objects 
can be cleaned by soaking them in the cleaning mixture for glass 
(§ 72), and then washing in water. After the slides are well rinsed, 
take them singly and by opposite edges, and wipe dry with a soft clean 
cloth. 5 
($3) Cover-glasses.—These are circular or quadrangular pieces of 
glass used in covering microscopic objects. They should be very 
thin,—.10 to .18 mm. (1-250 to 1-150 in.) in thickness. Covers may be 
made very cheaply from the laminae or scales of mica, but mica is too 
soft to be used for permanent preparations. 
(§ 4) Cleaning Cover-glasses.—New cover-glasses should be put one 
by one into the cleaning mixture for glass (% 72), and allowed to re- 
main two days or longer. Cover-glasses that have become soiled in 
any way should be placed in the cleaning mixture for a week or more. 
Finally rinse the covers by a gentle stream of water till the color of 
the cleaning mixture is no more visible. In wiping the cover-glasses, 
grasp them singly and by opposite edges between the thumb and in- 
dex of the left hand. Cover the thumb and index of the right hand 
with a soft, clean cloth, grasp the cover and rub its two surfaces. It 
is necessary to have the ball of the thumb and finger on exactly oppo- 
site sides in wiping the covers, or they will be broken. When a cover 
is well wiped, hold it up and look through it toward some dark object. 
It will be seen partly by transmitted and partly by reflected light. If 
it does not look clear, breathe on the faces and wipe them again. 
Handle the cover-glasses and slides by their edges, or handle the 
cover-glasses with fine forceps. Do not touch the surface with the 
fingers. 
As the covers are wiped, place them in pill-boxes, and assort them 
according to thickness. One can judge of the thickness of a cover- 
glass by looking at its edge. It is better, however, to measure the 
thickness of each cover as it is wiped, with Zeiss’ Deckglass-Taster, 
and place those of like thickness in the same box (see $ 25). 
($ 5) Mounting microscopic obje<5ts.—This is preparing them for 
study with the microscope. They are generally put upon slides (§ 1), 
and in some liquid called the mounting medium. To protect them 
from evaporation, dust and mechanical injury they are covered with 
cover-glasses (§ 3). 
(§ 6) Putting on the cover-glass.—Grasp a clean cover-glass of the 
proper size near one edge with the fine forceps, and if necessary, dust 
on both sides with a quill duster. Without relaxing the grasp of the 
forceps, rest one edge of the cover on the slide near the object and 
slowly lower the other till the cover is nearly parallel with the slide, 
then remove the forceps and the cover will fall into position. If this 
method is followed, the cover-glass may be put exactly where it is 
wanted and there will also be avoided the displacement of the object 
and the inclusion of air-bubbles. 6 
LIVING TISSUES.* 
The student is advised to read all that is said upon each special 
subject before commencing work upon that subject. 
(ft 7) Normal conditions.—For the best results in the study of living 
tissues it is necessary to maintain the normal temperature and, 
to surround the object with a normal fluid,—a fluid in which it 
naturally lives. For Protozoa and other aquatic animals, the water 
in which they are found is their normal fluid. For the blood-corpuscles 
of Necturus, frog and man, and for the ciliated epithelium from the 
frog, artificial serum made from egg-albumen, salt and water, answers 
very well (§73). 
AMOEBOID MOVEMENT. 
(ft 8) Amoeba.—This unicellular animal shows admirably the amoe- 
boid movement,—a continual change of form due to the protrusion of 
one or more processes from any part of the circumference. Amoebae 
are especially abundant in soft water regions. Their special habitat 
is the light, superficial ooze at the bottom of still waters. They are 
also usually abundant in the floculent matrials and slimy matter ad- 
herent to submerged stems and leaves, and in wet sphagnum moss. 
[Leidy, 8-13, H. & M., if\. 
Mounting the amoebce.—Grasp some of the floculent matrial and 
dead leaves at the bottom of the aquarium with the fine forceps and 
press them on the center of a clean slide several times. Cover {ft 6) 
the water and other material left on the slide by this process, and ex- 
amine under the microscope, using a 1-5 objective. Every irregular 
translucent mass should be watched for a short time to see if it ex- 
hibits the amoeboid movement. It is not safe for a beginner to call 
any object an amoeba before it has exhibited the amoeboid movement. 
After once becoming familiar with amoeboid movement it is quite easy 
to detect moving amoeboid forms whether they are independent or- 
ganisms like the amoeba or parts of higher animals like the wdiite 
blood-corpuscles. 
(| 9) White blood-corpuscles of Necturus.—To obtain the white 
blood-corpuscles, pith a Necturus. To do this, remove the animal 
from the water; grasp it in a dry towel; wipe the dorsal part of the 
head dry ; ventriduct the head, an<Tpress the thumb nail quite firmly 
* Several free-hand drawings of an amoeba and of each kind of 
white blood-corpuscle should be made so that their various phases 
may be compared. Drawings of an infusorian and a ciliated epithelial 
cell should also be made. against the dorsal side nearly opposite but slightly cephalad of the 
base of the first gill, to find the notch between the atlas and skull; 
pith the Necturus by forcing the tracer through the skin and muscles 
into this space, and thus severing the medulla. Then force the tracer 
into the cranial cavity and destroy the brain to remove the possibility 
of suffering. 
(| io) Mounting the corpuscles.—After pithing it is well to moisten 
the place where the cut was made with the normal fluid. Usually a 
mixture of blood and lymph exudes from the wound. Moisten a 
cover-glass with normal fluid and touch it to the exuded fluid. Then 
place the cover, blood side down, upon the center of a slide which 
has been moistened with normal fluid and on which a short piece of 
hair is placed. The hair prevents the too great pressure of the cover 
upon the blood. The normal fluid is for convenience. If one does 
not possess it, he should breathe upon the cover and upon the slide. 
The moisture of the breath prevents the too rapid desiccation. If blood 
cannot be obtained where the animal was pithed, a gill may be wiped 
dry, then moistened with normal fluid and a slight cut made in it with 
scissors. Blood unmixed with lymph runs out and should be mounted 
as directed above, except that more normal fluid should be used to 
dilute it so that the red corpuscles shall not hide all the white ones. 
The best place to get white corpuscles, however, is either from the 
pericardial sac or the abdominal cavity. A dropping-tube is very con- 
venient to get a drop of the liquid in these cavities after a small open- 
ing is made. Seal as in \ u. After the preparation is sealed put it 
under the microscope and examine with a 1-5 or higher objective 
and demonstrate the following: (A) The colored, oval, nucleated 
corpuscles ; (B) Small white corpuscles nearly circular in outline and 
exhibiting slight or no amoeboid movement; (C) Large white cor- 
puscles, some of them nearly as large or even larger than the red ones. 
These exhibit very marked amoeboid movement. The nucleus is in 
most cases very distinct and also undergoes apparently independent 
amoeboid changes [S. VII, 35]. 
(| 11) Sealing for blood.—As it is sometimes necessary or desirable 
to study a fresh preparation for a considerable time—an hour or 
more—it is necessary to prevent evaporation. This may be done by 
painting a ring of castor oil around the cover-glass so that half of the 
ring shall be on the cover-glass and half on the slide. A ring of the 
melted paraffin sealing mixture is to be preferred, however ($ 75). 
($ 12) White blood-corpuscles of a frog.—To obtain the white cor- 
puscles, proceed to pith as described for Necturus (§ 9). The notch 
between atlas and skull in the frog is nearly opposite the caudal mar- 
gin of the inembrana tympani. It is more easily found than in Nec- 8 
turns. Mount and seal the blood from the pithing wound or from the 
pericardial or abdominal cavity, as described for Necturus ($ 9-11), 
and examine it in the same way. Demonstrate the presence of both 
red and white corpuscles and the marked amoeboid movement of the 
white ones; observe also the colored corpuscles [Quain II, 9, 23; (Q. j. 
m. s. (1871) 361); R. 148, 178]. 
(§ 13) White blood-corpuscles of man.—To obtain the white cor- 
puscles of man, flame a needle (heat it red hot in an alcohol or gas 
flame), and after it is cool insert it obliquely a short distance into one 
of the fingers, then remove it and ligate the finger with a string or 
rubber band and also squeeze it. A drop of blood will exude. This 
should be mounted and sealed as described above (§ 9-11). The 
blood of man contains few white corpuscles as compared with the red 
ones,—1 to 350 or 500 [R. 211]. The average size of the white ones 
is greater than that of the red ones (red ones 7 to 8 ju, white ones when 
circular in outline 8 to 10 ju [R. 185, 210; Quain, II, 24, 29]). They 
undergo amoeboid changes as do those of the frog and Necturus. 
Marked amoeboid movements occur in the corpuscles of many persons 
after the blood is mounted for the microscope if the temperature is not 
much below 20 C. The movements in any case increase in energy 
as the blood is raised in temperature to 37 to 38 C [R. 210].* 
General references to blood.—Many of these give numerous special 
references [B-S. 17 ; Heitz, 64 ; (Q. j. 111. s. (1871) 361) ; Quain, II, 9, 23; 
R. 148, 178; St. 67; Str. 263; S2. 7; Milne-Edwards, 1,49,83; Todd, 
I, 404. See also £ 70 of these notes]. 
(| 14) Counting blood corpuscles.—The average number in a cubic 
millimeter of healthy human blood is between five and six million red 
ones and from 40 to 140 thousand white ones. In the cold blooded 
vertebrates the red ones are less in number, and the white ones pro- 
portionately more numerous. For the methods of counting see [Str. 
269 ; R. 203 ; S 2. 6; St. 86 ; Sat. 48]. 
(§ J5) Protoplasmic movement in plants.—In the cells of Chara, 
Vallisneria, Anacharis, etc., may be seen very clearly the movement 
of the protoplasm (Cyclosis). Take a leaf of Vallisneria, for example, 
shave off a thin slice, mount in water and observe with a 1-5 or higher 
objective. If the leaf to be used is kept in water at 25-30° C. for half 
an hour before making the preparation the movement will be more 
satisfactory (H. & M., 52). 
* A convenient warming stage may be prepared from a rectan- 
gular piece of sheet copper about the size of a slide. A perfora- 
tion is made in the middle about the size of the opening in the 
stage of the microscope. To the rectangular piece is soldered a wire 
or narrow strip about 25 cm. long for a conductor. The warming 
stage is placed upon the microscope and the preparation upon it. 
The preparation is warmed by heating the conductor with an alcohol 
flame. 9 
(| 16) Infusoria and ciliated epithelial cells.—Study and comparison 
of a ciliated unicellular organism, and a ciliated cell from a multi- 
cellular organism. 
Infusoria.—Infusoria are usually present in the aquarium contain- 
ing amoebae (§ 8). They are also abundant in water containing hay 
and standing in a warm place. To mount them, place on the slide 
some absorbent cotton, or preferably, some of the separated fibers of 
the absorbent paper used by dentists, and then press some of the wet 
hay or some of the vegetable matrial from the aquarium upon a 
cover-glass and mount upon the fibers placed on the slide; with blot- 
ting paper absorb part of the water around the edge of the cover. The 
fibers will confine the Infusoria so that they cannot get out of the field 
while one is observing them. Examine with 1-5 or higher object- 
ive [C. 497; H. & M. 89]. 
($ 17) Ciliated epithelial cells.—To obtain these, open the mouth of 
a frog and scrape the roof with a scalpel blade. Mount the gelatinous 
substance so obtained on a slide in a drop of normal fluid, or in the 
absence of normal fluid, in a drop of spittle. Put a piece of hair in 
the preparation and cover. Press the cover-glass down quite firmly. 
If the preparation is to be studied for several hours, as in a class dem- 
onstration, seal it as directed for blood ($ 11). Examine with 1-5 or 
higher objective. If no cells are isolated, press directly down on the 
cover-glass to spread out the mass of cells. Make out the form of 
the cells, their ciliated ends (free ends), and the character of the cili- 
ary movement. Compare with the movement of the cilia on the in- 
fusorian [Q. j. m. s. (1871) 308); Todd, I, 606, 628; Quain, II, 48; R. 
240; F 1. 262; S 2, 24; B 1. 193].* 
(§ 18) Anaesthetizing infusoria and ciliated cells.—Prepare a slide 
for this by cementing to it, with sealing wax, a glass tube drawn out 
somewhat at one end. The contracted end should be near the middle 
of the slide. Make a moist chamber with a ring of putty, in which is 
no turpentine, about the size of a cover-glass, and put it on the slide 
.so that it shall enclose the free end of the glass tube. Make a furrow 
in the ring on one side, and place in it a drop of normal fluid or water. 
Place some Infusoria on a cover-glass and put the cover on the ring 
and press it down moderately. Place the preparation under the mi- 
croscope and see that the Infusoria are active, then connect the tube 
opening into the moist chamber, by means of rubber tubing, with a 
wash-bottle. The tubes of the wash-bottle should both be short, and 
the one into which one blows should be connected with a rubber tube 
40-50 cm. long so that one may blow and continue looking into the 
microscope. Put into the wash-bottle a little water and then two or 
* Ciliated cells may be obtained from the trachea of a snake, lizard 
or turtle, or from the gill of a clam or oyster, etc. three cubic centimeters of ether or chloroform. Find an active infu- 
sorian that cannot get out of the field and then blow into the wash- 
bottle. The vapor of the anaesthetic will pass into the moist chamber, 
and will anaesthetize the infusorian. After the movement ceases re- 
move the wash-bottle and blow air into the moist chamber. After a 
few minutes the movements will recommence. 
Ciliated epithelium.—Scrape a frog’s mouth as before ($ 17), and 
put the scrapings on a cover-glass with spittle or normal fluid, 
and put the cover-glass on the moist chamber as for the Infusoria. 
Get in view some active cilia and then blow into the moist chamber 
the chloroform or ether vapor as before. The cilia will gradually 
cease moving; remove the wash-bottle and blow air into the moist 
chamber. The cilia will resume their movement. The experiment 
may be repeated several times. * 
CIRCULATION OF THE BLOOD. 
(| 19) Circulation in the gills of Necfturus.—Select, if possible, a 
small animal with long gill filaments. Inject into the back three or 
four drops of curara solution ($ 76). Replace the animal in the water 
and in an hour or two it will be motionless, f 
Place the Necturus dorsicumbent on the well moistened circulation 
board and cover it with a wet cloth. J Spread some of the gill filaments 
out on the glass cover of the perforated cork, using a well wetted quill 
duster. Place one end of the circulation board on the stage so 
that the gill filaments will be in the field. Use a diaphragm with a 
* If the vapor is too concentrated, the movement ceases almost 
instantly. If it is continued too long, the movement will not recom- 
mence [S 2. 27 ; B-S. 35 ; Str. 5]. 
f A curarized Necturus should be kept in running water, in order 
that the respiration may not be interfered with. After a day or 
two the effect of the curara disappears. As curara paralyzes the motor 
nerves but is not an anaesthetic, no dissection should be performed 
without either destroying the brain ($ 9) or rendering insensible by 
an anaesthetic. 
JAn excellent circulation board for Necturus and frog may be 
prepared by boring a hole about 2 cm. in diameter in a pine board 
8x30 cm. and 15 mm. thick. The hole should be about 5 cm. from 
one end and near one side. A perforated cork or hollow cylinder of 
wood should be fitted to this hole. Over the top of the perforated 
cork should be placed a very thick cover-glass or a piece of thin glass 
slide, and sealed with sealing-wax ; finally the whole board should be 
covered with woolen cloth or cotton flannel. The perforated cork 
should be capable of being moved so that it will stand a centimeter 
above the surface of the board if desired. large aperature, and light with plentiful transmitted light. Employ 
a 3-4 objective and examine some isolated gill filaments. Demon- 
strate : 
(1) Two large vessels, one on each side of the filament, making a 
loop over the free end. 
(2) The blood flows in opposite directions in the two large vessels. 
(3) The current alternately increases and diminishes. This indi- 
cates the heart beats. 
(4) Besides the loop the large vessels make near the end of the fila- 
ment, there is a capillary network connecting the two in all parts of 
the filament. 
(5) Blood corpuscles are in all the vessels and are carried along by 
the current. 
Put a cover-glass over some of the filaments and use a water immer- 
sion objective (1-10 or 1-15) if possible, and demonstrate : 
(1) The capillaries are well defined channels. 
(2) The flow in them is slower than in the larger vessels. 
(3) The blood corpuscles are of two kinds : (a) Large oval colored 
ones which are very numerous, (b) Colorless or white corpuscles 
which are circular in outline or which are very large and appear in 
general form like the colored ones (g 9). 
(4) The corpuscles are elastic and flexible. This may be seen at 
some sharp angle where the corpuscles are often bent at right angles; 
they always resume their shape when free in the current. The nucleus 
is very apparent in most of them. 
(5) Amoeboid movement in the white corpuscles may often be seen 
when the current in a capillary ceases for some time. Under favora- 
ble conditions, the amoeboid movement of the nucleus may also be 
seen in the white corpuscles so stranded (S. VII, 35). 
(g 20) Circulation in the frog’s foot (interdactylar membrane).—Se- 
lect a small frog if possible. The common spotted frog (Rana halecina) 
is a good subject. Curarize by making a small incision through the 
skin in the dorsal region with scissors, and introducing two or three 
drops of curara solution (§ 76) with a dropping tube. Put the frog 
in a moist, cool place, but not into wTater. In half an hour or more 
the frog will be motionless.* 
Put the frog into a woolen sac well saturated with water. The sac 
should be only a little longer and wider than the frog’s body. Leave 
one leg projecting. Pin the mouth of the sac, but do not pin tightly 
* If the frog cannot be curarized it should be pithed, as described 
above ($ 12). enough to compress the projecting leg. Place on the circulation board, 
being sure that the cloth covering the board is well filled with water. 
Spread out the web by tying a thread around the longest dactyl, 
4th, and one around the 3d or 5th and pinning the strings in such a 
position that the web shall be over the glass in the top of the cork. 
Do not stretch the web too tightly for it would be torn or the circula- 
tion impeded. Cover the exposed leg with a wet cloth or a piece of 
blotting paper. Keep the web and the animal moist (§ 19). 
Put the circulation board on the stage so that the web shall be in 
the field. Employ the 3-4 objective and A ocular. Eight as above 
(| 19). Demonstrate the following points : 
(1) The arteries give off branches and hence become smaller. The 
current flows from the larger part of the vessel toward the smaller 
part, and into the branches. 
(2) The veins receive branches and hence become larger. The blood 
in them flows from the smaller part of the vessel toward the larger 
part, and from the branches into the large vessel. 
(3) The branches given off by the arteries finally break up into an 
anastomosing net-work of capillaries. These capillaries unite and the 
vessels so formed are veins. This may be demonstrated, by moving 
the circulation board so that a branch may be followed. 
(4) The stream is most rapid in the larger vessels ; and sometimes 
pulsations corresponding to the heart beats may be seen in the arte- 
ries. Compare $ 19 (3). 
Remove the 3-4 objective and put in its place 1-5 or a higher one, 
a water immersion if possible (§ 19). 
Cover the web with a triangular piece of cover-glass. Demonstrate 
the following points in addition to those already made out: 
(1) Bring a vessel into the field that appears about 2 mm. in diame- 
ter. The central part of the stream is rapid, while that along the 
edges is slower. This part is sometimes called the inert layer. 
(2) Focus sharply on the edge of the stream. In it will be seen 
many white corpuscles. These are mostly smaller than the red ones, 
and they roll along the walls of the vessel as if they were sticky. 
(3) The central part of the stream is mostly composed of red cor- 
puscles. 
(4) Bring some capillaries into the field. The white and red cor- 
puscles are mingled and go along in single or double file. The red ones bend or elongate as conditions may require, showing that they 
are elastic and flexible, like those of Necturus ($ 20, 41).* f t 
References [B 1, 191 ; B-S, 121 ; R, 598 ; St 158 F 1, 246; S 1, 154 
(Ar. m. a., XIII, 483)]. 
PERMANENT PREPARATIONS OF BLOOD. 
(| 21) Amphibian blood.—A permanent preparation of amphibian 
blood, especially for the red corpuscles, may be prepared by spreading 
some fresh blood on a cover-glass upon which has been previously 
spread some normal fluid. Hold the cover-glass over the open mouth 
or in the neck of a bottle containing osmic acid (§ 77) for about one 
minute. The fumes will fix the corpuscles. After the corpuscles are 
fixed, place on the cover a drop of 75 per cent, glycerin containing 
alum carmine and fluorescine (§ 81) ; and then place the cover on the 
center of a clean slide, and allow it to settle down upon the slide by 
its own weight,—do not press it down.$ 
($ 22) Sealing the preparation.—Place four drops of shellac cement 
(§ 84) at equidistant points around the cover-glass so that half the 
cement shall be on the slide and half on the cover. Allow the slide 
to lie flat for a few hours so that the cement may dry and fix the cover. 
Finally wipe away the glycerin around the edge of the cover-glass with 
a moist cloth, then place the slide on the turntable and make a ring 
around the entire cover-glass, putting half the ring on the slide and 
half on the cover-glass. Several thicknesses of the cement should be 
put on, but each one should be allowed to dry before adding another 
[1Seiler, 92]. Any preparation mounted in a medium miscible with 
water may be sealed or cemented as just described. 
* It sometimes happens that the direction of the current is re- 
versed. This is due to some obstruction, or to the contraction of 
some vessels and the expansion of others [R. 606]. 
f The dark irregular bodies seen in the web are pigment cells. 
X If the current is too rapid to admit of the satisfactory de- 
monstration of points (1 to 3) when the high power is used, control 
it by pressing the legs at the knee. This method, so far as is known 
to the writer, was devised by a laboratory student, C. E. Atwood, 
Cornell, ’80. 
$ Preparations may be placed in the center of the cover-glass 
very easily by preparing a card with circles upon it the sizes of ordi- 
nary cover-glasses, and cementing three slides or strips of bristol- 
board so that when the slide on which the preparation is to be made, 
is placed on the card its center will be over the center of the concen- 
tric circles made on the card. (§ 23) Permanent preparation of extended white blood-corpuscles.— 
In the preparation described above (g 21), the white corpuscles are 
mostly in an unextended condition. In order to moke preparations 
in which the corpuscles are fixed in the various phases of amoeboid 
movement, some of the pericardial or abdominal liquid should be ob- 
tained and placed on a cover-glass as directed in (§ 9-10). It should 
then be placed over a moist chamber made of a ring of puttv on a 
slide and containing a drop of normal fluid. This preparation should 
be examined occasionally and when the white corpuscles are satisfac- 
torily extended (after half an hour or more), the cover is grasped by 
the fine forceps and held in or over the neck of a bottle containing 
osmic acid solution (§ 77) for about a minute. Then a drop of alum 
carmine is put upon it and allowed to remain for a minute or two. 
Finally, after removing part of the alum carmine, with filter paper, a 
drop of the colored glycerin is added as above (§ 21), and the cover is 
put on a slide, sealed, etc., as described in ($ 22). In successful prepa- 
rations, both red and white corpuscles are present; and the white 
ones show various amoeboid phases both of cell-body and nucleus 
[(Q. j. m. s. (1871) 371), (S. VII, 35)]. 
(§24) Permanent preparations of mamalian blood.—(1) Moist prep- 
arations may be made as described in (§ 21). The white corpuscles 
are usually circular and their nuclei are in many cases stained by the 
alum carmine. To render more certain the staining of the nuclei of 
the white ones, a drop of pure alum carmine may be used immediately 
after the treatment with psmic acid as described in ($ 23). 
(2) Dry preparations.—The blood to be mounted should be as fresh 
as possible ; and if it is from an animal it should be free from foreign 
material. Human blood may be obtained as directed above (§ 13). 
The blood should be spread on the cover-glass in a layer or film so 
thin that the corpuscles are not in contact. To do this, select a slide 
to spread the blood whose edge is straight and rounded like the base 
of the letter U. Touch the edge of the spreading slide to the blood, 
press it gently against the cover-glass and draw it quickly across the 
cover. If the room is warm and dry the film will dry very quickly ; 
if not, warm the cover over an alcohol lamp. Examine under the 
microscope, and if the preparation is satisfactory, mount as follows : * 
* It is nearly impossible to make a preparation which shall be 
equally good in all parts, hence if some parts of the preparation are 
satisfactory it should be retained. 
f Preparations of spermatozoids, and many other bodies free in a 
liquid may be mounted dry as described for blood. By heating to 
50-60 C. so that the film will not wash off, various stains may be used. 
After the preparation is stained it may be mounted dry as for blood, 
or in balsam, glycerin, etc. Make a shallow cell of shellac or other cement of about the size of 
the cover-glass, in the center of a slide. Let this dry spontaneously, 
or if in a hurry, use heat. When the cement is dry, warm the slide 
and the cover-glass on which is spread the film of blood, and put the 
cover on the cell, film side down. Press the cover-glass against the 
cell with the convexity of the tracer or fine forceps, until there ap- 
pears a shining or polished appearance all around the cell. This in- 
dicates that the cover adheres to it. Seal the cover-glass as directed 
above ($ 22). 
Do not breathe on the slide or cover-glass in any of the operations ; 
for even a slight moisture will ruin the preparation in a short time. 
[C. 713 ; R. 195 ; F 1, 218, 240 ; (Ar. m. a. I, 340) VIII 476 and XIII, 
753) Wormley 740 ; Woodward, The application of photography to 
micrometry, p. 4]. 
$ 25) Storing, labeling and cataloguing microscopical preparations.— 
(1) Storing.—Microscopical specimens should lie flat and be protected 
from dust and light. (2) Labeling.—Each specimen should be labeled, 
and the label should give, (a) the number, (b) the date, (c) the gen- 
eral name, and (d) the source of the preparation ; and (e), if possible, 
the thickness of the cover-glass.* 
(3) A catalogue of each preparation should be made, and should 
contain a summary of the history of the specimen, including the 
methods of preparation. It is only by the possession of such knowl- 
edge that the comparative excellence of methods can be determined, 
and the defective ones discarded or improved, f 
* It is desirable to know the exact thickness of each cover-glass used 
for two reasons : (i) One need then never through ignorance use so 
thick a cover that high objectives cannot be employed in studying the 
preparation ; (2) The perfect adjustment for the cover-glass in adjust- 
able objectives is greatly facilitated by knowing the exact thickness 
of the cover-glass in each case. This is especially true of objectives 
like those of Zeiss, in which the adjusting collar of the objective is 
marked with numbers corresponding to cover-glasses of various thick- 
ness [A. m. s. (1884), 202]. 
f (1) The catalogues are made on thick slips, postal card size, and 
arranged alphabetically in a box as is commonly done in a library 
slip-catalogue. (2) The labels are prepared from linen paper of mod- 
erate thickness. They are gummed with liquid gelatine ($ 86) and 
attached simply by wetting them as in using postage stamps. (3) The 
ink employed is Higgins’ water proof India ink diluted with an equal 
quantity of soft water to which has been added a few drops of ammonia. Formula for cataloguing Micro- 
scopical Preparations :— 
1. General name, and source. 
2. Number and date of the prep- 
aration, and the name of the pre- 
parator. 
3. Special name of the prepara- 
tion ; the common and scientific 
name of the object from which it 
is derived. 
4. Special object of the prepara- 
tion. 
5. Chemical treatment, — the 
method of hardening, dissociat- 
ing, etc. 
6. Mechanical treatment,—im- 
bedded, sectioned, dissected with 
needles, etc. 
7. Staining agent and the time 
required for staining. 
8. Clearing agent, the mounting 
medium and the cement used for 
sealing. 
9. Objectives to use in studying 
the preparation; thickness of 
the cover-glass. 
10. Remarks, including referen- 
ces to good figures and descrip- 
tions. 
Formula for Labeling Microscopi- 
cal Preparations :— 
Number and date of the prepar- 
ation (No. 2 of catalogue). 
General name (No. 1 of cata- 
logue). 
Name of the object from which 
the preparation is derived. 
Thickness of the cover-glass. 
[(A. s. m. (1883) 169), (J, r. m. : 
Beh. 245.] 
A Catalogue Card Written Accord- 
ing to the Formula :— 
i\ Myelinic nerve-fibres. Cat. 
2. No. 31 (Dr’r. II), March 21, 
1880 ; S. H. G.. preparator. 
3. Isolated, myelinic nerve- 
fibres from the sciatic of the cat 
(.Felis dornestica). 
4. This preparation shows well 
the axis cylinder and the nodes of 
Ranvier. 
5. Dissociated 24 hours in 35 per 
cent, alcohol. 
6. Dissected on the slide with 
needles. 
7. Stained over night (12 hours) 
in picrocarmine. 
8. Cleared with turpentine and 
carbolic acid ; mounted in chloro- 
form balsam ; sealed with shellac. 
9. Use three-fourths and higher 
objectives. Cover .15 mm. 
10. See for figures and descriptions 
(Quain’s Anatomy, Vol. II., p. 
141, and Ranvier, Traitd d’Histol- 
ogie, p. 723). 
A Label:— 
No. 96 ; March 12, 1880. 
Myelinic nerve-fibers. 
Cat. 
Cover . 15 mm. 
1. (1883) 924). (Z. w. m. (1884) 280.) 
ISOLATION AND PRESERVATION OF THE STRUCTURAL 
ELEMENTS. 
($ 26) Ciliated epithelial cells.—Soak the trachea of a cat or other 
animal, or the oesophagus or roof of the mouth of a frog in 35 per 
cent, alcohol from one to three days, preferably but one day. Transfer 
to water, and scrape the mucous surface with a scalpel or needle. i7 
Place the substance so obtained on the center of a slide and spread it 
somewhat with needles. Add a drop of fluorescine. After a minute 
remove the fluorescine, but do not wash with water. Finally smear 
a cover-glass with alum carmine glycerin and cover. Press the cover- 
glass directly down quite firmly. Isolated, ciliated cells should be 
found in abundance. Seal the preparation as for blood (g 22) [(Ar. 
m. a. XI, 354) and as in §17]. 
(g 27) Striated muscular fibers.—Select a muscle with parallel 
fibers,—a ribbon shaped muscle like the occipito-scapularis [W. & G. 
217] answers very well. In obtaining the muscle, wet the hair thor- 
oughly before cutting the skin, to avoid hair on the tissue. After the 
muscle is exposed cut one end and grasp it with forceps. Hold the 
muscle tense, isolate and remove it either with scissors or a sharp 
scalpel. Lay the muscle on a strip of cork and put a ribbon pin 
through each end and into the cork. The muscle, and also other tis- 
sues, should be about as tense on the cork as in the body, that is they 
should be in a state of physiological extension [R. 729]. After the 
muscle is pinned to the cork be sure that it does not touch the cork, 
then invert it in 35 per cent, alcohol. In from one to three days trans- 
fer to water, and cut out a piece about 5 to 10 mm long and 2 mm in 
diameter, and transfer it to the center of a slide. Place over it a tri- 
pod magnifier and with a clean, bright needle and the fine forceps or 
two needles, separate the fibers at one end so that the piece of tissue 
shall appear fan-shaped. To do this, grasp the tissue at one end with 
the forceps or hold it down with a needle. With a needle remove all 
the connective tissue as far as possible. The final success of prepara- 
tions of isolated tissues depends largely on the thorougness with which 
this is done. After the connective tissue is removed, fray out the 
fibers at one end by drawing the needle lengthwise something as if 
combing. It is often necessary to use both needles on a fine bundle 
and draw the fibers apart. This is usually best done after a pretty 
thorough longitudinal combing. It is usually not best to try to isolate 
the fibers the entire length of the piece ; for the final result is usually 
more satisfactory if the fibers are more or less combined at one end. 
After the isolating is judged to be sufficient, a drop of alum carmine 
or haematoxylin (g 87, 88) should be put upon the tissue and allowed to 
remain from one to five minutes. This should then be washed off with 
water, and a drop of fluoriscine added. After a minute or two the 
fluorescine is washed off and then the preparation may be mounted in 
glycerin as for the ciliated cells (g 26) or preferably Canada balsam 
(g 93) maybe used (g 27a) [Quain II, 118 ; R. 468; Heitz. 265; (A. 
s. m. (1882) 131) ; (Q. j. m. s. (1885) 371) ; Leydig 2. 124]. 
(g 27a) Mounting in Canada balsam.—(1) The water must be re- 
moved without allowing the object to become desiccated ; this is usu- 
ally accomplished with 95 per cent, or stronger alcohol. (2) The 
alcohol is replaced by the clearing agent—carbolic acid and turpen- i8 
tine, clove oil, etc. ($ 94), which must be miscible with the balsam. 
(3) The clearing agent is replaced by balsam. 
To mount the muscle in balsam, put upon it several drops of 95 
per cent, alcohol with a dropping tube. After a minute allow the alco- 
hol to drain away, and then add a drop of the clearing mixture ($ 94). 
While this is acting, arrange the specimen and separate the fibers still 
more if possible, then remove the clearing agent from around the tissue, 
and place upon it a cover-glass previously spread with balsam. Air bub- 
bles will soon disappear. Balsam preparations need not necessarily be 
sealed. If it is desired to seal them, wait till the balsam has hardened 
around the edge of the cover, then put on two or three rings of shellac 
as in (| 22) [Fi. 210; R. 138]. 
(§28). Muscular-fiber Cells.—Plain, unstriated or smooth muscle. 
Contractile fiber-cells. To obtain these remove a small piece of the 
muscular wall of the stomach or intestine of any animal—or take the 
muscular wall of the urocyst or any other organ where muscular fiber- 
cells are known to exist, and place it in 15 to 20 cc. of 20 percent, ni- 
tric acid (§ 78) for three days. Then pour off the acid and soak an 
hour or more in water, or 35 per cent, alcohol. [Reichert’s method, 
Ki. 89]. Place the tissue in a homoepathic vial about half filled with 
water and shake the bottle. The cells will separate. Allow the bottle 
to stand half an hour for the cells to settle, then remove most of the 
liquid, and add 5 to 10 cc. of alum carmine. Allow the stainer to act 
an hour or more, then remove most of it, fill the bottle with water and 
as soon as the cells have settled, remove most of the water and fill the 
bottle about half full with fluorescine glycerin ($ 82). Mix the 
cells with the glycerin by rolling the bottle. Shaking would fill the 
glycerin with air bubbles. To mount a preparation, place a drop of 
the mixture on the center of a slide and seal as for blood ($ 22). 
Muscular fiber-cells may be isolated by using needles after dissociating 
a day or two in 35 per cent, alcohol, but it is difficult to find unbroken 
fibers. The cells are quite well stained in picrocarmine or liaema- 
toxylin.* [Fi. 318; Q. 530; Ru. 90; Si. 112 : Ki. I, 89; Ar. a. e. 
(1849), 180). 
(§ 29). Cardiac muscular fibers.f—Cut from the surface of a ven- 
tricle a thin section in order to remove the proper serous covering of 
* Picrocarmine usually stains muscular fiber-cells well after they 
have been isolated in nitric acid. Haematoxylin should not be used 
as it is not a permanent stain for objects containing an acid. It an- 
swers well, however, for the cells isolated by means of alcohol. 
t The capillaries and small vessels of a cat’s heart are usually full 
of blood. The corpuscles are rather confusing in the final prepara- 
tion, hence the blood should be washed out. To do this ligate the 
subclavian and brachiocephalic arteries [W. & G. 350], and inject salt 
solution into the aorta toward the heart ($ 74). The semilunar valves 
will close and the liquid will enter the cardiac arteries. When the 
ventricles look pale the injection may cease. the heart. Make a second section, partly tearing it with the fingers, 
in order to get the fibers in their length. Place part of the sections 
so obtained in 20 per cent, nitric acid over night, and part of them in 
35 per cent, alcohol. The branching net work of fibers may be most 
easily demonstrated by using nitric acid, but the nuclei are difficult to 
stain after the acid treatment. They are easily demonstrated in the 
sections dissociated in alcohol, however. 
After about 12 hours remove the sections from nitric acid, and soak 
them in a plentiful supply of water for an hour or more ; a day or 
more in water, or preferably in 35 per cent, alcohol is advantageous. 
When ready to make the preparation, place a small piece on a slide 
and carefully and thoroughly separate the fibers with needles. Use 
the tripod magnifier. The fibers can hardly be too thoroughly isola- 
ted. They cannot be combed out longitudinally as in striated muscle, 
but have to be pulled apart laterally or transversely using two needles. 
After the tissue is well teased or dissected, add a drop of alum car- 
mine, and allow it to remain 5 minutes or longer, then wash it away 
carefully and add fluorescine. Allow the fluorescine to remain 5 minutes 
or more, and then wash it away. Finally, dehydrate by adding alco- 
hol and mount in balsam, as described (§ 27a). The sections dissocia- 
ted in alcohol should be teased with needles, stained and mounted 
as just described. Quain, II., 135; Str. 179; R. 539; Gibbs. 74; 
Prudden, 101 ; Kx. II. 281. (Ar. m. a. XXIII, 500.)] 
(| 30) Myelinic nerve-fibers.—( White, medullated or dark-bordered 
nerve-fibers).—These may be obtained from any of the nerves of the 
limbs. The sciatic nerve is probably the best to use. The nerve 
should be carefully removed from the body and pinned on a strip of 
cork and placed in 35 per cent, alcohol as described for striated mus- 
cle (§ 27). After remaining in the alcohol from one to three days it 
should be treated exactly as for striated muscle. Special pains is nec- 
essary in removing the connective tissue, and as the fibers are smaller 
than those of muscle, the teasing must be carried further. When the 
isolation is deemed sufficient, a drop of alum carmine is added and 
allowed to remain from three to five minutes. This is then washed 
away and a drop of fluorescine allowed to remain half a minute or a 
minute. The tissue is then washed with water and mounted in 
glycerin or preferably in balsam as directed in (§ 27a) [S. 710; Schwann 
166; Sc. 290; Sat. 180; Str. 120; Quain, II, 138]. 
(g 3*1) Amyelinic nerve-fibers.—(Non-medullated, gelatinous or gray 
nerve-fibers, fibers of Remak). In demonstrating amyelinic nerve- 
fibers it is best to select a nerve in which are also present myelinic 
fibers in considerable abundance. A splanchnic nerve [W. & G. 395] 
is perhaps best. A piece of the sympathic [ W. & G. 395] or the ulnar 
or musculo-cutaneous nerve [W. & G. 386, 384] answers very well, also. The nerve should be dissociated in 35 per cent, alcohol, as de- 
scribed in (§ 27, and 30). The amyelinic nerve-fibers are, in the cat, 
usually smaller than the myelinic, and the teasing must be very thor- 
ough. The use of two needles for drawing the fibers apart after comb- 
ing or fraying the fibers longitudinally is recommended (see also $ 29). 
The staining and mounting should be as described in (§ 27, 27a 
and 30).* [R. 746; Quain, II. 145 ; Sc. 290; F. & L. 72 ; Str. 121 ; 
Klein, hi ; Schwann, 169, 170]. 
(§ 32) Areolar tissue.—This is most satisfactorily prepared by form- 
ing an artificial oedema (Ranvier’s method, R. 329) : Place a cat dor- 
sicumbent, and fasten the legs laterad with cords. Thoroughly 
moisten the hair along the cephalic (inner) aspect of the leg, and after 
parting the hair, make an incision through the skin parallel with the 
femur, extending the incision slightly beyond the knee. Tear the 
skin from the muscles for a short distance, and there will appear a 
delicate fibrous substance holding the skin to the muscles. This is 
the areolar or loose connective tissue, composed in part of white 
fibrous and in part of elastic or yellow fibrous tissue and a variable 
number of connective-tissue corpuscles. With a syringe or a pressure 
bottle, ($ 37) inject into the tissue picro-carmine or alum carmine. In 
doing this, insert a small, sharp pointed connula for some distance into 
the tissue and continue the injection until an oedema or swelling of 
considerable size is formed. After 15 or 20 minutes remove the rather 
thick layer on the surface of the oedema with scissors, and then cut 
as thin a piece from the underlying tissue as possible. Scissors should 
be used which are sharp. This small mass will appear almost like 
jelly. Transfer it to a watch-glass of distilled water. Place on the 
center of a cover-glass a drop of fluorescine glycerin (§ 82), and put 
the cover where it may be easily grasped by the fine forceps. Trans- 
fer the tissue to the center of a slide, and with blotting or filter paper 
absorb the liquid around it. spread the tissue as thinly as possible 
upon the slide. Do this under a tripod magnifier. Use a pair of 
bright dissecting needles and grasp opposite edges of the tissue and 
draw them as far apart as possible without tearing. If there is not 
too much liquid on the slide the edges will cling to the slide as they 
are drawn out and thus enable one to spread the tissue very thinly. 
One should work rapidly, and if the tissue is in danger of drying it 
* A very striking and instructive preparation may be made by plac- 
ing the nerve for 12 to 24 hours in a one-twentieth per cent, osmic 
acid solution. The osmic acid blackens the myeline of the myelinic 
fibers. It is also a dissociating fluid, so that the nerves may be easily 
isolated with needles after its action. If the preparation is teased and 
treated exactly as above, the myelinic fibers will appear black or dark 
gray, the axis cylinder being more or less reddened, while the amye- 
linic fibers will be red. should be breathed upon. As soon as the tissue is properly extended, 
the cover-glass should be applied. The preparation should be sealed 
as described in ($ 22). In preparing areolar tissue it is very desirable 
to use a kitten or other young animal, for the white fibrous tissue is 
neither in such large bundles nor so greatly in preponderance over 
the elastic tissue. The connective-tissue corpuscles are also more 
numerous. If the edge of a fat tract is included in the oedema, one 
is very likely to obtain, in addition to the connective tissue proper, a 
network of small blood-vessels and capillaries with fat cells in the 
meshes. The fat cells are often in various stages of development— 
the so-called signet-ring stage being the most common [Schwann, /70]. 
Excellent preparations of areolar tissue may also be made by injecting 
serum ($ 73), salt solution (§ 74), or even water into the tissue in- 
stead of the coloring matter as described above. Pieces of the right 
size are cut out with scissors, and stained 10 to 30 minutes in alum 
carmine, then for five minutes or more in fluorescine, and mounted 
in alum carmine glycerin as above. [R. 329 ; Si, 72 ; Quain II, 61 ; Fi, 
274 ; (J. r. m. s. (1879) 355) ! (Ar- m- a- XI, 176 ; XII, 391; (Ar. a. e. 
(1879) 401); A. s. m. (1882) 109)]. 
(| 33) Endothelium.—{The epithelium of serous membranes). To 
demonstrate endothelium, kill a Necturus by pithing, or by putting it 
in chloroform or alcohol in a small amount of water. Open the body 
cavity by a longitudinal incision 1 cm to the left or right of the vent- 
rimeson. A transparent ligament will be seen extending from the 
ventrimeson to the liver. Remove a piece of this with scissors and 
place it in a watch-glass of water. Force a stream of wTater upon it 
from a wash-bottle to wash away any lymph or lymph-corpuscles from 
the surface. Remove the water, and spread out the membrane on the 
bottom of the watch-glass. Pour upon it a one-half per cent, solution 
of silver nitrate, and allow it to remain a minute or two. Then pour 
off the silver and wash well with water. Finally place the watch-glass 
containing the membrane in a plentiful supply of water in a strong 
light. As soon as the membrane has turned brown, pour off the wa- 
ter, spread out the membrane and examine. If the cell outlines are in- 
dicated by dark lines, the staining is sufficient; if the outlines are not 
distinct continue the exposure till they are. It should be remembered 
that there is a layer of cells on both surfaces of the membrane. When 
the silver staining is sufficient, transfer a piece of membrane of the 
proper size to the center of a slide ; cover it and put a spring compres- 
sor upon it to hold the cover in place ; then put the preparation in a 
jar of 95 per cent, alcohol over night. The cover may then be re- 
moved and the membrane washed with water and stained with a drop of 
alum carmine or haematoxylin for a minute or two, then washed and 
stained a minute or more with a drop of fluorescine. After the fluor- escine is washed away with water, the membrane is dehydrated by 
dropping on it alcohol with the dropping tube (§ 27a). It is then 
cleared and mounted in balsam as directed in ($ 27a). This method 
of mounting membranes in balsam was first suggested, so far as is 
known to the writer, by the late Ur. Woodward [L. (1872) 99, 163]. 
To avoid the wrinkling or folding that is almost sure to occur in the 
ordinary method of transferring the membrane to the slide, a dish of 
considerable size should be used, and the slide partly or wholly im- 
mersed so that the membrane can be floated upon it [(L. I, 158) ; (Z. 
w. m. I. 392) ; (A. m. m. j. VI, 235 ; VII, 13); (J. d. a. p. XII, 54, 
588) ; (J. r. m. s. (1879), 357) ; R. 105, 246; Quain, II; K. 89; B-S- 
43 ; K. & S. 180]. 
($ 34) Cartilage.—An excellent source for hyaline cartilage is an 
end of one of the long bones of Necturus. The larynx of the cat also 
furnishes good material. Sections may be made free-hand of the 
fresh material as follows : Remove most of the soft parts covering 
the cartilage, wrap it in newspaper or other strong paper, and grasp 
it by the thumb and index of the left hand so that it projects slightly 
above the nail of the thumb and index. Wet the cartilage well with 
salt solution ($ 74) and wet the razor or section knife with the same. 
Grasp the razor firmly, rest the blade on the nail of the thumb or in- 
dex ; move the razor forward across the tissue with a drawing cut, 
making each section with a single sweep of the knife. Make several 
sections, and transfer them to a dish of picric acid solution ($ 83). 
They should be left in this half a day or more. They may then be 
washed with water and mounted in glycerin or glycerin jelly ($ 80). 
If it is desired, the sections may be stained wTith alum carmine after 
soaking in water till the picric acid is removed. Stained sections may 
be mounted in balsam, but glycerin or glycerin jelly is preferable. 
As it is sometimes impossible or undesirable to make sections of fresh 
cartilage, the cartilage in small pieces may be kept in the picric acid 
for a few days and then in 75 to 80 per cent, alcohel until one is ready 
to make the sections. In that case the sections are transferred to 
water or 35 per cent, alcohol as they are cut, and the razor and tissue 
are wet with the 35 per cent, alcohol, with salt solution or with water. 
The sections must not be allowed to dry in any case. It is difficult 
for the inexperienced to make free-hand sections of sufficient thinness 
and evenness, hence the cartilage may be cut in the microtome as 
follows : Fill the well of the microtome nearly full of melted im- 
bedding mass (§ 103) ; dry one end of the cartilage with filter paper, 
and insert it into the melted mass so that the other end of the cartil- 
age is about on a level with the top of the microtome. To prevent 
drying, place a small mass of absorbent cotton, wet with salt solution, 
over the exposed end of the cartilage. Cool the imbedding mass as soon as possible by the use of snow, ice or cold water. Wet the razor 
with salt solution and make the sections as rapidly as possible, using 
a drawing cut as in free-hand sections. (See also $ 40; 58).* [R. 274 ; 
Quain, II, 77 ; Heitz. 198; Sat. 82 ; Str. 83; K. 45 ; Leydig 2, 73]. 
HARDENING TISSUES. 
(£ 35) Hardening agents.—A great many agents are used for hard- 
ening and preserving tissues for histological purposes. Those most 
used are alcohol, chromic acid and various salts of chromium, as di- 
chromate of potassium and ammonium. Picric acid is also often 
employed. None of them are equally good for all tissues, and in most 
cases two or more of them are employed on each tissue. A perfect 
hardening and preserving agent for histological purposes would leave 
the structural elements with exactly the appearance they possess dur- 
ing life, color perhaps excepted. 
Fifty to seventy-five (50-75) times as much liquid should be used as 
tissue, and if the tissue is of a massive character, like liver, only two 
to three cubic centimeters should be taken. Label everything. Put 
the tissues into the hardening agent as soon as possible after the death 
of the animal. 
(§ 36) Hardening in picric acid solution.—($ 97).—Nerve (12), stri- 
ated muscle (14), trachea (16), stomach of Necturus (18), ileum (19), 
and ovary may be hardened in picric acid solution as follows: Fasten 
a piece of sciatic nerve and of the occipito-scapularis muscle, obtained 
as directed in (§ 27, 30), to a strip of cork and in physiological 
extension by wooden pegs—pins would be corroded by the picric 
acid. Invert the tissue in a vessel of the solution. Suspend the trachea, 
stomach, ileum and ovary in the solution by a string tied to some of 
the adherent connective tissue. In from one to three (1 to 3) days 
pour off the picric acid solution and add 95 per cent, alcohol. This 
should be changed once or twice a day until the alcohol is but slightly 
stained with the picric acid. 
(§ 37). Hardening in Muller’s fluid (§ 96).—Myel, artery, submax- 
illary gland, liver and human skin may be hardened in Muller’s fluid 
as follows : The artery should be pinned out on cork as described in 
* So far as I know this method of cutting cartilage in a microtome, 
without complete imbedding (§ 39) was first suggested by B. L. Oviatt, 
Cornell, ’87. The cells of cartilage are very liable to shrink and ap- 
pear quite unlike those of fresh cartilage if the ordinary methods of 
preservation are employed. The picric acid solution has been found 
very satisfactory for all kinds of cartilage. ($ 27) and inverted in the liquid. Myel, submaxillary gland, liver 
and human skin may be laid on a bed of absorbent cotton and the 
liquid poured upon them. This is practically suspending them in the 
liquid. The Muller’s fluid should be changed at least twice, viz.: on 
the 2d and 4th days after the tissues were put into it. After the tis- 
sues have remained in the fluid for 12 to 14 days, pour it off, wash the 
cotton thoroughly or use new cotton, and place the tissues in 50 per 
cent, alcohol for one or two days, then into 75 per cent, for the same 
time, and finally into 95 per cent, alcohol till ready to make sections 
[F. & L. 245; Si and S2.; Ru.; Fi.; B-s.; Stowell; R.; Heitz.; Sat.; 
Lee; W.; Fol.; Car.; Str.; Tr.; Beh.; (J. r. m. s.); (Z. w. m.)]. * 
IMBEDDING AND SECTION CUTTING. 
(§ 38) Infiltrating with paraffin.—At the present day most objects to 
be imbedded in paraffin are previously infiltrated or thoroughly per- 
meated with it so that all the cavities of the tissues are finally filled 
with paraffin. To do this : (A) All the water must be removed from 
the tissue by means of 95 per cent, or stronger alcohol. (B) The al- 
cohol is replaced by chloroform by placing the tissue in a wide mouth 
bottle with 10 to 15 times as much chloroform as tissue. After 12 to 
24 hours the tissue is usually thoroughly permeated with chloroform. 
A longer stay in the chloroform does no harm. (C) The tissue is 
placed in a mixture of paraffin 4 parts chloroform 1 part, which is kept 
just melted, the temperature never being allowed to exceed 55 or 60 C. 
The tissue should remain in this from 12 to 24 hours and then (D) It 
is placed in pure paraffin (§ 103) kept just melted, and allowed to re- 
main from 12 to 24 hours. 
($ 39) Imbedding.—After a tissue is infiltrated it should be imbed- 
ded in pure paraffin as follows : A small paper box, made of a piece 
of strong paper 4x7 cm, is nearly filled with melted imbedding mass, 
and the tissue is placed in it and quickly arranged, so that the object 
will be cut at the proper angle by cutting at right angles to the length 
of the box. As soon as a film is formed by the cooling of the imbed- 
* An excellent method of hardening tissues is to inj ect the harden- 
ing agent into the arteries, going to the tissue or into the ducts of 
glands. This may be done for all tissues and for all hardening agents 
by boring two holes in the top of a preserving jar, and making a 
kind of wash-bottle of it. Pressure may be obtained by using a con- 
stant pressure apparatus, or an atomizer bulb. The connection with 
the vessel or duct may be readily made by a rubber tube and glass 
cannula. This form of injecting apparatus is especially adapted for 
corrosive liquids like picric, and osmic acid, and silver nitrate, etc. 25 
ding mass the box may be placed in cold water or in any cold place so 
that the paraffin will cool quickly and avoid air bubbles. It is well 
to indicate on the box the name of the tissue and the end where the 
cutting should commence. The tissue so imbedded and labeled may 
be put away until one is ready to make sections of it. 
(£ 40) Sectioning.—When one is ready to make sections of a tissue 
it is put into the holder of a microtome. If the microtome is the well 
form, the screw is lowered until the imbedded tissue, from which the 
paper box has been removed, will reach the top of the microtome. 
The end of the imbedding mass is cut away until the tissue is visible, 
then it is placed in the well of the microtome and melted paraffin 
poured in to fill the well. The paraffin should be hot, 65 or 70 C, so 
that it will melt the surface of the imbedding mass containing the tis- 
sue and become one with it. After the paraffin is cool, trim it away 
from the tissue so that only a thin layer supports the tissue on every 
side. The appearance of the imbedded tissue when it is ready to be 
cut is something like a bluntly sharpened lead pencil, the sharpened 
end being square or rectangular in outline. In making the sec- 
tions, if the edge of the knife is made parallel with one edge of the 
imbedding mass, consecutive sections will adhere to each other and 
form a kind of ribbon (see also £ 60).* 
If the imbedding mass is loose in the microtome, it may be made 
firm by crowding a thin wedge of wood between the wall of the micro- 
tome and the imbedding mass. This must not be allowed to proj ect 
above the top of the microtome, however. 
In making the sections, carry the knife directly across the tissue. 
In dry sectioning, as this is called, the more direct the cut the more 
successful the section ; and usually a very rapid movement gives the 
best results. Perfect sections should consist of but a single layer of 
structural elements. The sections may be preserved for an indefinite 
time without deterioration ; and they may be stained and mounted at 
any time (£41-46). For wet sectioning see (£ 34 and 58). 
(£ 41). Fastening the sections to the slide.—In order that no part of 
a section shall get lost or disarranged during the staining and mount- 
ing processes, the sections should be fastened to the slide. (See also 
£ 60). To do this a thin coating of collodion or celloidin (£ 101, 104) 
is put upon the center of the slide with a brush and allowed to dry. 
The sections are arranged on the slide and pressed down gently if 
they are not flat, and then several drops of ether and alcohol (£ 105) 
are put upon them with a dropping tube. The ether and alcohol 
soften the collodion, and the sections are cemented to the slide 
* One may imbed directly in the well of the microtome and not use 
the paper box, but it is not so satisfactory. by the collodion. After the ether and alcohol have evaporated 
(in 3 to 5 minutes), the slide on which the sections are fastened, 
is placed in a jar of xylol (§102) to dissolve out the paraffin. This 
will require about half an hour. The sections may remain in xylol 
for several days, perhaps indefinitely, without being injured. Before 
the sections are placed in xylol they may be exposed to the air with- 
out injury (§ 40), but after the paraffin is removed from them they 
would become desiccated if exposed to the air. * f 
(| 43). Staining the sections.—After the paraffin is removed from 
the sections by the xylol, the slide bearing them is placed for a min- 
ute or longer in 95 per cent, alcohol to wash away the xylol. The 
sections are then washed with water by a gentle stream from a wash- 
bottle. The stainer, if it acts within 1 to 20 minutes, is placed di- 
rectly upon the sections and the slide laid flat. If a long time is re- 
quired to complete the staining, place the slide in a jar containing the 
stainer. After the sections are stained sufficiently the superfluous 
dye is removed by a gentle stream of water.% 
(\ 44). Dehydration.—After the stainer is washed away, the sections 
are placed in ajar of 95 per cent, alcohol to remove the water. The sec- 
tions may remain in the alcohol a considerable time without injury. 
They should remain in the jar five minutes or more, or the dehydra- 
tion may be hastened by moving the slide or by pouring alcohol from 
a bottle over the sections. 
(| 45). Clearing.—The dehydrated sections are prepared for the final 
mounting in balsam, by putting upon them a few drops of clearing 
mixture (§ 94). The clearing is usually completed in two or three 
minutes or even less. 
When sections are cleared they can hardly be seen if held over some 
dark object. If, on the other hand, they are held up between the eye 
and the light, they appear very translucent. 
* This method of fastening sections to the collodionized slide by- 
using ether and alcohol, was first suggested in the laboratory, so far 
as I know, by H. E. Summers, Cornell ’86. See also $ 58 for fasten- 
ing celloidiu sections to the slide. 
f The method originally suggested for fastening sections to the 
slide is as follows : Collodion ($ 101) is mixed with three to four times 
its volume of clove oil, and some of this mixture is brushed on the 
slide just before using it, or the collodion may be put upon the slide 
as described above ($ 41) and just before putting the sections on the 
collodionized surface it is lightly brushed with clove oil. In either case 
the slide, after the sections are upon it, is heated to 35—40 C. for a fewT 
minutes to drive off the clove oil. After the clove oil is expelled the 
sections are treated precisely as described in $ 41. (See references to 
§60). 
t Although the sections may appear greasy when washed with wa- 
ter, the stainer will act if the paraffin was removed as described in 
(§ 4i). (| 46). Mounting the sections.—As soon as the sections are cleared, 
the clearing agent shonld be removed with blotting or filter paper, 
then a cover-glass is spread with balsam ($ 93), and placed over the 
sections as in \ 27a. For references see ($ 60). 
ORDER OF PROCEDURE IN MAKING HISTOLOGICAL PREPA- 
RATIONS BY THE PARAFFIN METHOD. 
1. Dehydrating the tissue with 
95 per cent, alcohol (§ 38). 
2. Soaking the tissue in chloro- 
form (1 38). 
3. Infiltrating with paraffin and 
chloroform (§ 38). 
4. Infiltrating with pure paraffin 
(1 38). 
5. Imbedding (g 39). 
6. Sectioning ($ 40). 
7. Cementing the sections to the 
slide with collodion (§ 41). 
8. Removing the paraffin from 
the sections with xylol ($ 42). 
9. Removing the xylol from the 
sections with alcohol ($ 43). 
10. Removing the alcohol from 
the sections with water (§ 43). 
11. Staining the sections with a 
nuclear dye ($ 43). 
12. Washing away the superflu- 
ous dye with water (l 43). 
13. Staining with a general 
dye ($ 43). 
14. Washing away the superflu- 
ous dye with water (§ 43). 
15. Dehydrating the section with 
alcohol (| 44). 
16. Clearing the sections with the 
clearing mixture (§45). 
17. Mounting the sections in 
Canada balsam (§ 46). 
18. Sealing the cover-glass(§ 27a). 
19. Labeling the slide (§ 25). 
20. Cataloguing the preparation 
(3 25). 
(§ 47) Transedtion of a nerve.—The sciatic is to be preferred. Ar- 
range the nerve in the imbedding box and then in the microtome so 
that the sections shall be exact transections. Harden, etc., as ip $ 36, 
38-42* 
Stain with carmine, or picro carmine, 15 to 20 minutes, or with alum 
carmine 5 minutes, fluoriscine 20 seconds. References: Same as in 
| 30, and R. 740, 756; Sc. 804; K. 103; Quain, II, 150; S2. 73]. 
* Any organ or tissue may be cut across, or parallel with its own 
long axis without regard to the body axis. Sections at right angles to 
the long axis are called transverse sections, or more shortly transec- 
tions. Sections parallel with the long axis are longitudinal sections, 
or more shortly longisections. See also $ 60. ($ 48) Myel {Spinal cord).—The cervical or lumbar enlargement is 
to be chosen. Imbed so that an exact transection can be made. If 
the nerve-roots and the ganglia are left on the myel, and held at right 
angles to it by the use of pins during the imbedding, the sections, 
when at the proper level, will include both nerve-roots and ganglia. 
For hardening, cutting, etc., see § 37-42. 
Staip 15 to 20 minutes with carmine or picrocarmine, or 5 minutes 
with alum carmine and 15 to 30 secs, with fluorescine. References: 
Quain II, 269; S2 185; K. 127; Fi. 348; Sat. 298; F. & L. 212; Sc. 
335; Str. 623; St. 242; W. 48, 59; Lee, 361]. 
(§ 49) Transedtion of a striated muscle.—The occipito-scapularis or 
other ribbon shaped muscle should be chosen. Arrange the muscle 
in the imbedding box and in the microtome so that exact transections 
can be made. For hardening, cutting, etc., see \ 36, 38-42. 
Stain 5 minutes with haematoxyliu (§ 88), and 15 to 20 secs, with 
fluorescine. References: R. 496; K. 69; St. 136; Fi. 319; and as in 
l 27.] 
(§ 5°) Transedtion of an artery.—One of the arteries of the limbs,— 
femoral, brachial, etc., should be chosen for the muscular type of ar- 
teries. The aorta gives excellent preparations if the elastic type of 
arteries is desired. Arrange the artery for exact transactions. The 
hardening, sectioning, etc., areas in \ 37-46. 
Stain the sections with haematoxylin 3 to 5 minutes, or alum car- 
mine may be used. References: R. 552; Str. 194; Sat. 151; Ki. II, 
294; Quain II, 183; (J. d. a. p. (1865) 536)]. 
(| 51) Sedtion of trachea.—Longisections or transections may be 
made. If longisections are to be made, the trachea should be cut 
lengthwise and pinned out flat during the hardening and imbedding 
processes, otherwise the sections are liable to be oblique. Harden, 
imbed and section as directed in \ 36, 38-46. 
Stain 5 minutes with haematoxylin and then 15 to 30 seconds in 
fluorescine. [References: Ki. II, 165; Quain, II, 511 ; K. 217 ; Str. 
435 ! Todd, V, 260; Allen, 689; Heitz. 709.] 
(§ 52) Section of submaxillary gland.—Harden the submaxillary 
gland, as directed in \ 37. Excellent preparations may also be made 
by hardening in picric acid and alcohol, or simply in 95 per cent al- 
cohol. The hardening should commence as soon as possible after 
death. Imbed, section, etc., as in \ 39 to 49. 
Stain 3 to 5 minutes with haematoxylin, and then 15 to 30 seconds 
with fluorescine. Picrocarmine stains fairly well[also. [References : 
Quain, II, 578; S2. 136; F. & L. no; Str. 294. (Ar. m. a. XIII, 
281, 359)]- 29 
(§ 53) Section of stomach of Ne<5turus.—The most satisfactory 
preparations are obtained by injecting 95 per cent, alcohol, or picric 
acid and alcohol (§ 36) into the dorsal aorta before the removal of the 
stomach from the animal. The stomach should be cut lengthwise and 
placed immediately in 40 to 50 times its volume of the hardening 
agent. As longitudinal rugae of the stomach are usually present it is 
better to make transections.' Imbed, section, etc., as in §38-46. 
Stain with haematoxylin, or alum carmine 2 to 3 minutes and fluor- 
escine 15 to 30 seconds. Or stain 5 to 10 minutes with picrocarmine. 
[References : Quain, II, 593 ; Sat. 388 ; Str. 370 ; Fi. 430 ; St. 178 ; K. 
196; (Ar. m. a. (1885) 174).] 
(§ 54) Section of the ileum.—The sections should be near the 
caecum where the villi are comparatively few. Either transections or 
longisections may be made. If longisections are to be made the 
ileum should be cut lengthwise and pinned out flat during the hard- 
ening and imbedding processes. The dorsal or attached aspect of the 
terminal part of the ileum usually contains so many Peyer’s follicles 
that the relations of the villi and crypts of Lieberkiihn are obscured, 
hence sections of the ventral or free aspect are to be preferred, or if 
transections are made they should include the ventral part. Some of 
the sections should be thick, .04 mm., others should be very thin. 
Stain with haematoxylin or alum carmine, 2 to 3 minutes. [.Refer- 
ences : Quain, II, 599 ; S2. 146 ; Heitz. 597 ; Str. 380 ; K. 201 ; F. & L. 
132]. 
(§ 55) Section of liver.—Harden, imbed, etc., as directed in § 37-46. 
Stain -with haematoxylin 3 to 5 minutes, or with alum carmine the 
same time, and then 15 to 30 seconds with fluorescine. [References : 
Quain, II, 626; Heitz. 675; B., 159; Str. 407]. 
(§ 56) Setftion of ovary.—The greatest care should be taken not to 
touch the surface of the ovary in the various processes, as there is 
danger of removing the surface epithelium. Imbed and place in the 
microtome so that transections shall be made. Some of the sections 
should be as thin as possible in order to see clearly the primordial 
ova; others should be thicker, so that the larger ova may be seen. 
Harden, etc., as in § 36, 39-46. 
Stain with carmine or picrocarmine 15 to 20 minutes, or with alum 
carmine 3 to 5 minutes and fluorescine 15 to 20 seconds. References : 
Quain II, 716; Str. 510; K. 257; Bal. I, 43, II, 614; (Q. j. m. s. (1878) 
384, and (1876) 190) K2; (Ar. m. a. I. 162, and XIX 464); Si. 238; Ru. 
142; F. & L. 232]. 
(§ 57) Section of human skin.—It is desirable to have sections from 
two regions,—from the scalp and from the palmar aspect of the hand 
or the plantar aspect of the foot. The skin may be hardened as in $ 37. The sections are best made 
by the celloidin method (§ 58). If the paraffin method is used, the 
greatest care is necessary not to overheat the tissue or the epidermis 
will become too hard to cut. 
Sections from the hand or foot should be vertical to the surface. In 
making sections of the scalp, the tissue should be arranged, if possi- 
ble, so that the sections, while vertical to the surface, shall include 
the entire length of the root of the hair. 
Sections may be stained with haematoxylin or with alum carmine 
1 to 3 minutes, and then with fluorescine. Paraffine sections may be 
colored with picro carmine. References: R. 876; QuainII,236; Heitz, 
553; K. 274; F. & L. 165; (Ar. m. a. XIII, 205, XVI, 343)]- 
CELLOIDIN METHOD. 
(? 58) Celloidin sections.—The heat required for infiltrating and 
imbedding with paraffin acts injuriously on some tissues, and some 
others cannot be so well prepared by the use of paraffin as by the use 
of celloidin or collodion (a solution of pyroxylin). If it is undesirable 
to remove the fat from a tissue, both the paraffin and celloidin meth- 
ods must be avoided and the sections must be made with the freezing 
microtome ($ 59). 
Imbedding in celloidin.—All of the water must be removed from 
the tissue with 95 per cent, or stronger alcohol; then it should remain 
from 12 to 24 hours in equal parts of ether and 95 per cent, alcohol. 
It should then be transferred to a thin solution of celloidin ($ 101, 104) 
and allowed to remain 12 to 24 hours. It is then placed in thick cel- 
loidin (§ 104) for a day, and is finally imbedded as follows : A small 
amount of thick celloidin is placed in a short test tube, and a cork 
slightly smaller than the test tube is forced down upon it, after wdiich 
sufficient celloidin is added to cover the cork for 2-3 mm. After an 
hour or more the tissue is placed in the test-tube and properly ar- 
ranged, and finally covered with thick celloidin. The tube is left 
uncorked until a film forms over the exposed celloidin. The tube is 
then nearly filled with pure chloroform and corked. The chloroform 
should be changed two or three times, and in the course of five or six 
days, or even less, the celloidin will become hard and transparent. 
The tissue is then ready to be cut. It is usually easy to remove it from 
the tube after pouring off the chloroform. 
Cutting the sections.—If a well microtome is used the imbedded 
tissue must be wedged in with a cork. A sliding microtome is prefer- 
able. The cork on which the tissue is supported is fastened into the 
holder of the microtome, the knife is made very oblique so that a 
drawing cut will result. The tissue and knife must be thoroughly and constantly wet with alcohol of 70 to 80 per cent, and the sections 
as they are cut should be transferred from the knife to 70 to 80 per 
cent, alcohol with a quill duster. 
Staining and mounting the sections.—The slides to be used for 
celloidin sections are lightly brushed with collodion or celloidin as 
directed in 41), the sections are transferred to the collodionized 
slide and dehydrated by pouring upon them 95 per cent, alcohol with 
a dropping tube, and then after removing most of the alcohol several 
drops of equal parts of ether and alcohol are added exactly as for 
paraffin sections ($ 41). The slide is kept in the air until most of the 
ether and alcohol have evaporated,—until the section begins to look 
dull—then the slide bearing the sections may be placed in a j ar of 80 
per cent, alcohol or several drops may be poured upon the sections 
with a dropping tube. The sections may now be-washed with water, 
stained, dehydrated, cleared and mounted, exactly as described for 
paraffin sections (§ 43-46). * f J \ References: W. 108, Lee., 193; 
A. s. m. (1884) 209). 
FROZEN SECTIONS 
(§ 59) Sections with a freezing microtome.—For the study of 
many objects it is undesirable to subject them to the processes neces- 
sary for paraffin or celloidin sections. This is especially true of tissues 
in which the fat is to be studied. In such cases the sections may be 
made with a freezing microtome. The freezing microtome also ena- 
bles one to obtain sections of fresh or hardened tissues in a minimum 
of time, or when it is desired to treat the sections with silver nitrate 
or gold chloride. 
Cutting the sections.—A piece of tissue of the proper size, either in 
the fresh state or after hardening, may be placed in the freezing mi- 
crotome and frozen either with ether vapor, etc., or ice and salt, or ice 
and 95 per cent, alcohol [S. r. II, 134]. Preferably, however, the tis- 
sue is first infiltrated with gum arabic for a day or more and then fro- 
zen in the same, or gelatin may be used for the infiltration and also 
* Collodion made from the best gun cotton, that used in photogra- 
phy, is nearly or quite as celloidin. 
f The clearing mixture composed of carbolic acid and turpentine 
(| 94) answers well for celloidin sections. Chloroform may be used in- 
stead [W. 114, Lee, 199]. 
J Tissues imbedded in celloidin may be kept a long time, perhaps 
indefinitely, in chloroform or 70 to 80 per cent, alcohol. The sections 
may be kept in a vial of 80 per cent, alcohol until one is ready to 
stain and mount them. 
\ The method here given of fastening the sections to the slide was 
suggested by H. E. Summers (§ 41) : Unless the sections are to be 
kept in serial order it is not absolutely necessary to fasten them to the 
slide. If they are to be in series the mass should be trimmed asymme- 
trically, and the sections as they are cut must be tranferred to num- 
bered watch-glasses. The asymmetrical outline will enable one to get 
them right side up. See £ 60. for surrounding the object during the freezing and cutting. If gelatin 
is used, the tissue and gelatin must be kept warm during the infiltra- 
tion. Alcohol must be removed from the tissue if it is present before 
infiltrating and freezing in either gum arabic or gelatin. 
In cutting the sections the knife should be cooled, and carried di- 
rectly across the tissue with a rapid movement as for paraffin sections 
(2 4oj. 
Mounting, staining, etc.—As the sections are cut they may be trans- 
ferred to slides, and a drop of glycerin added to each if they are not 
to be colored. Or if gelatin is used for imbedding, etc., a drop of the 
desired coloring matter is put upon the sections, and later washed 
away with a gentle stream of water, and then a drop of glycerin added 
to each. Seal the preparation as in $ 22. 
If gum arabic is used for the infiltrating, etc., and the sections are 
to be stained, they must be placed for half an hour or so in water to 
dissolve the gum arabic. They may then be transferred to slides, 
stained, mounted in glycerin and sealed as directed in § 27. [Refer- 
ences : Ru. 164; R. 83; W. 76; Sat. 17; Si. 123; (Q. j. m. s. (1884) 
163), (S. r. II. 134)]. 
SERIAL SECTIONS. 
(§ 60) Serial sections.—“ For the investigation and understanding 
of einbryological and histological facts, only next in importance to 
the proper hardening of the specimen is the method of serial section- 
ing and mounting : for the parts may then be compared in the sequence 
they occupied in the animal or organ, and the most delicate or loosely 
connected part is preserved in situ." For serial sections the object, 
whatever it may be, is properly hardened, and then dehydrated, infil- 
trated and imbedded as described in \ 38-39; but in order that the 
most complete and certain knowledge may be obtained from the sec- 
tions, it is necessary to know with absolute certainty all the aspects 
of each section ; and if the object is an embryo or small animal it is 
desirable to know the position of the various sections with reference 
to the ends of the body or definite landmarks. Hence (1) An outline 
drawing of the embryo or animal should be made before imbedding it. 
This drawing should be exactly natural size or some definite propor- 
tion ; as twice natural size, etc. (2) The object should be imbedded 
so that the sections shall have a definite relation to the axon (body 
axis) and to the three principal planes of the body [W. & G. 33; (A. 
m. s. (1884) 203); Wilder, New York Medical Journal, Aug. 2, 1884, 
P- 113]- 
As the tissue is taken from the body it is arranged in a definite man- 
ner and pinned to a cork. This arrangement is stated on the label 
which should accompany the tissue until it is finally mounted. The 
aspects of a small animal or embryo may of course be determined by 
simple inspection any time before imbedding. The sections should 
be made in one of the three principal planes of the body : (A) Transections.—The object is cut in a plane at right angles to 
the axon (long axis of the body) and the object is so imbedded that 
the caudal end of the animal or organ is up and the dorsal aspect 
toward some fixed part of the microtome. Then the sections are 
placed on the slide with the caudal aspect up and the dorsal aspect 
toward the top of the slide (away from the label). The right and left 
of the sections will then correspond to the right and left of the ob- 
server. 
(B) Dextro-sinistral longisections.—The sections are made parallel 
with the axon (body axis), and the object is imbedded with the dorsal 
side up and the cephalic extremity in a known position. The sec- 
tions are placed on the slide so that their cephalic end shall point 
toward the top of the slide. These sections, if of an animal or em- 
bryo, are bi-laterally symmetrical like the preceding, and right and 
left correspond to the right and left of the observer. 
(C) Dorso-ventral longisections.—The sections are cut parallel with 
the axon. The object is imbedded so that the cephalic extremity is 
known and the left side is up. The sections are arranged on the slide 
so that the cephalic end shall point toward the left side of the slide, 
and the dorsal aspect toward the top of the slide. 
(D) When ready to make the sections, the distance the microtome 
screw is out is noted, and when a slide is filled with sections it is again 
noted. The distance that the screw is raised in cutting the sections 
on a slide is equal to the total thickness of all the sections on that 
slide ; or in other words, the length of the object cut in making the 
sections. The amount should be put on the temporary label of the 
slide and the same should be done for each succeeding slide. In this 
way one may know, by comparison with the previously made diagram 
the exact locality from which each slide of sections was obtained. 
(E) If an enlarged model is to be made, the sections should be cut 
of uniform thickness. Born recommends that this thickness be not 
greater than .04 mm. [Ar. m. a. (1883) p. 584); (M. j. (1876) 578); 
(A. n. (1884) 446); W. 216]. 
Cutting the sections.—After the tissue is properly imbedded and 
placed in the holder of the microtome the imbedding mass is cut away 
so that only a thin layer surrounds the tissue. This should be trimmed 
so that it is square or rectangular in outline. The knife is set or held 
so that its edge is parallel with one side and then it is carried directly 
and quickly across the tissue. If the imbedding mass is of the right 
hardness for the temperature, ribbons of indefinite length may be 
made with a sliding microtome. Short ribbons may also be made, 
after a little practice, with a well microtome. 
Placing the sections on the slide.—The sections may be placed on 
the collodionized slide (g 47) singly as they are made, or the ribbon of 34 
sections may be broken into segments of the proper length. In 
either case the first section should be placed in the upper left hand 
corner of the slide, and the others should follow as in columns of 
figures. The sections are fastened to the slide, stained, mounted, etc. 
as directed in \ 42 to 46. 
Slides for serial sections.—Unless the object is very small, slides 
50x75 mm. and rectangular cover-glasses 48x58 mm. are recom- 
mended. The covers can be more safely spread with the balsam if they 
are laid flat. 
Label for the slides in a series.—This should give the following in- 
formation. (a) The number of the series, (b) The number of the 
slide in the series, (c) The date on the label belonging to the animal 
or organ, (d) The name of the object, (e) The number in the series 
of the first and last section on the slide, (f) The thickness of all the 
sections on the slide or preferably the location in the length of the 
object, (g) The thickness of the individual sections, if a model is to 
be made from the series, (h) The thickness of the cover-glass: 
Series No. 5 Feb. 22, 1886, 
Slide No. 1' 
Transections of Necturus. 
Sections 1 to 50. 
Microtome screw raised from o to 2 mm. 
Sections each .04 mm. thick. 
Cover-glass .16 mm. thick. 
Serial sections may be made by the celloidin method also. The ar- 
rangement of the object in the imbedding mass should be as described 
above. The mass should be trimmed asymmetrically so that one may 
get the sections a desired side up on the slide and with the desired 
aspect toward the top of the slide.* 
References.—The literature of section cutting and related matters is 
very extensive. Original papers or summaries may be found in : 
W. ; Lee. ; Sat. ; Fi. ; Ru. ; Si. ; Beh. Fol. ; St. ; Car.; (J. r. m. s.) ; 
(A. n.) ; (Z. w. m.) ; (Z. a.) ; (M. z. s. n.) ; (A. m. s.) ; (J. a. p.) ; 
(Q. j. m. s.) ; (Ar. m. a.). The Medical Student, 1883.] 
* The perfect sharpening of section knives requires great skill and 
experience. A large, fine oil stone that has been flattened on a sur- 
face plate, with emery, and a strop of fine leather nailed or pasted to 
asmooth board are desirable. [(Deecke, A. m. s., 1882, p. 275) ; Fol. 
129; W. 85, and A. n., 1885,p. 831.] 35 
FINE INJECTIONS. 
(| 61) The purpose of a fine injection of the vascular system is to 
determine the finest vessels in a tissue by the aid of a microscope with 
the same certainty that coarsely injected vessels may be determined 
with the unaided eye. 
(A) Specimen.—Choose a young but nearly mature, lean animal. 
Kill with chloroform, and leave the animal in the anaesthetic at least 
half an hour. 
(B) Inject before the rigor mortis appears. 
(C) Inject only the part desired, and tie all anastomosing vessels, 
and all vessels to other parts. 
(D) Inject into the artery of the part, leaving the vein open until 
nearly pure injecting mass escapes, then tie the vein and continue the 
injection till the part feels hard and is the color of the injecting mass. 
(E) After the injection is finished, if a gelatin mass was used (§ 106),. 
cool the part injected by means of snow, ice or cold water. 
Injection of the small intestines of a cat.—Place the cat dorsicum- 
bent on the tray and tie the legs laterad with cords. Open the abdomen 
by a longitudinal and a transverse incision through the skin and ab- 
dominal wall. Isolate the vessels of the intestine by tying (i) a large 
string, like cotton twine, around the large intestine near the caecum. 
(2) A similar string should be tied around the duodenum including 
the pancreas. (3) Tie a string around the superior mesentric vein 
(W. & G. 355) near the pylorus, and open the vein centrad of the 
string. 
Insert a cannula into the superior mesentric artery near its origin 
[W. & G. 359]. Pill the cannula with warm water or warm salt solu- 
tion by means of a dropping tube. Warm the syringe, if a syringe is 
used, by drawing warm water into it and expelling the water; then 
fill the syringe with injecting mass (§ 106), which should be at a tem- 
perature of 33 to 38° C. Connect the syringe with the cannula, and 
slowly, but continuously force down the piston. As soon as nearly 
pure injecting mass escapes from the incision in the vein, tie it, and 
continue the injection till the intestines feel firm and are of the color 
of the injecting mass. Then tie the superior mesenteric artery peri- 
pherad of the cannula and put snow, ice or cold water on the intestines 
to cool the injecting mass. When the mass is firm in the large vessels 
it will not escape from the small ones. It usually takes half an hour 
or more for the mass to set firmly. 
After the mass has set, open the free surface of the ileum, and with 
scissors cut off some of the villi. Mount them in glycerin and exam- 
ine with the microscope. If the injection is successful the villi will 
be quite long and the injected blood-vessels will be comparatively 
straight. If the villi are short and broad and the vessels are wavy, satisfactory preparations cannot be obtained. If the villi are not sat- 
isfactory in the ileum examine them in various parts of the intestine; 
for often some parts are satisfactory even if the whole intestine is not. 
Injection of the kidney of a cat.—The same animal may be used as 
for the intestines. Insert the cannula into the renal artary, tie a 
string loosely around the renal vein and open it centrad of the string. 
Fill the cannula with water or salt solution with a dropping tube, and 
then connect the syringe containing the injection mass. Force the 
piston down gradually, and soon the mass will escape from the vein. 
Tie the vein and continue the injection till the kidney feels quite 
firm and is of the color of the mass. Cool it as directed for the intes- 
tine.* 
($ 62) Hardetiing injected tissues.—Injected tissues should be hard- 
ened one or two days in 50per cent., two in 75 per cent., and then one 
or more in 95 per cent, alcohol. Or they may be placed two days in 
picric acid and alcohol, and then in 95 per cent, alcohol (§36). In- 
jected bones and teeth may be decalcified if desired by the picric 
acid, or preferably the 2.5 per cent, solution in 95 per cent, alcohol, 
($97). It is especially necessary to harden injected organs in a cool 
place. 
($ 63) Sections of injected kidney.—These should be made precisely 
as described for cartilage (§ 34), except that the razor and tissue should 
be wet with 95 per cent, alcohol. As the sections are cut they should 
be placed in a dish of 95 per cent, alcohol. The sections should in- 
clude both cortical and medullary substance. 
Mounting the sections.—Place the sections on a slide, clear and 
mount in balsam ($ 27a). f 
(| 64) Sections of injedted intestine.—These are best made free-hand 
by grasping the injected and hardened intestine in the left hand, and 
with the razor cutting from the periphery toward the center. The 
edge of the razor should not go beyond the base of the villi. A section 
of the whole intestine may be made in this way by rotating it as it is 
cut. The sections need not be made very thin, and they cannot be 
made witth one sweep of the knife. The sections should be mounted 
in balsam. It is desirable to stain them (see note to $ 63). 
(g 65) Injection of the vessels of a frog’s foot.—Kill the frog by 
placing it in a jar of water containing chloroform or ether for half an 
hour. Expose the heart, cut off the apex of the ventricle, and insert 
* One can make very satisfactory fine injections with the injecting 
jar described in the note to £ 37. If gelatin mass ($ 106) is used the 
tube extending from the jar to the vessel should be wrapped with 
woolen cloth to prevent the too rapid cooling of the mass. 
f A very transparent, contrasting dye to bring out the tissue in in- 
jected sections is desirable. Picric acid is very good when red and 
fluorescine when blue is used for the injection. a cannula through the ventricle into the bulbus aorticus. Tie a soft 
string around the bulbus so that it will press upon the cannula. Fill 
the cannula with water or salt solution with a dropping tube, and in- 
ject red mass (§ 108). If the injection is successful the skin of the 
legs, and the web will become very red, and if the web is held up to 
the light, the injected vessels may be seen. 
( 66) Injection of the lymphatics of the frogs web.—To inject the 
lymphatics of the web, a small hole is made through the skin distad 
of the knee and a cannula filled with blue mass ($ 109) is inserted. 
The syringe is connected with the cannula and the leg is compressed 
with the thumb and index just bejond the point of insertion of the 
cannula. After forcing the piston down slightly the leg and foot are 
pressed by the thumb and index to force the mass by the valves into 
the lymphatic capillaries. By alternately forcing down the piston and 
compressing the foot, one can usually fill the lymphatics in one or 
more of the interdactylar membranes. If the injection is successful 
the web will be blue, although the blood vessels are injected with red. 
By examining the web with a magnifier or the fths objective one can 
judge of the success of the injection. Both feet should be injected. 
When the injection is finished, a string should be tied tightly around 
the leg beyond the incision for the lymphatic injection and the frog 
should be cooled with snow, ice or cold water. 
(§ 67) Preparing and mounting the web.—When a successful injec- 
tion has been made and the frog cooled, the legs should be cut off 
near the knee. The toes should be pinned apart on cork so as to 
moderately stretch the web. Then the foot should be hardened in 50, 
75 and 95 per cent, alcohol one day each. The web may then be cut 
out and mounted in balsam [ R. 653]. 
References for fine injections.—Br.; Fi.; Si.; St.; W.; Lee; Robin; 
Ru.; R.; Sat.; J. r. m. s.]. 
METHODS OF PATHOLOGICAL HISTOLOGY. 
A thorough knowledge of normal histology is necessary (a) to ena- 
ble one to determine whether there are any pathological modifications 
in the structure of a tissue; and (b) the exact nature and extent of 
those modifications when present. 
($ 68) The methods of pathological histology are based on the same 
principles as those of normal histology: (A) The tissue should be 
put into the hardening agent as soon as possible after the death of the 
animal or after its removal from the body in a surgical operation. 
This is even more necessary in pathological than in normal histology, 
for there is in many cases a greater tendency to disorganization. 
(B) The tissue should not be allowed to dry. Vials of the hardening 
agent should be at hand, and only pieces of moderate size should be hardened; and plenty of liquid should be used. (C) If possible, 
scraped or teased preparations of the perfectly fresh tissue should be 
studied in salt solution or normal fluid to see the appearances in the 
tissue elements before they are hardened and possibly modified by the 
hardening agents. Staining by drawing fluorescine under the cover- 
glass by a piece of blotting paper is frequently desirable ; and the use 
of osmic acid to fix the tissue elements is sometimes of advantage 
(§ 23). In all cases where possible, some healthy tissue should be left 
with the morbid so that the sections shall include both, and then the 
transition from the normal to the pathological tissue can be more 
easily appreciated. 
Muller's fliiid is suitable for hardening most pathological speci- 
mens (§ 37). The imbedding and sectioning should be as described 
in ($ 38-46), or in many cases it is better to use the celloidin method, 
as no heat is necessary (§ 58); and if it is desirable or necessary to 
study the fat of a tissue, the sections should be made with the freezing 
microtome {\ 59). Sections of perfectly fresh tissue may also be made 
with the freezing microtome, but the crystals of ice which form in 
freezing tend to tear the tissue and give it a pathological appearance. 
References: The microscopical journals; Ziegler; Gibbs; C. & R.; 
Fol.; Richardson; Fi.; B.etG.; B24 Buck; Green; Heitz.; Peaslee; 
Seiler; Tyson; Virchow; IVoodhead; Wythe; (A. p. a.) ; Mittheilun- 
gen aus dem Kaiserlichen Gesundheitsamte; Reports of the U. S. 
bureau of animal industry]. 
METHODS OF VEGETABLE HISTOLOGY. 
(| 69) The methods of Vegetable Histology, are naturally some- 
what similar to those of animal histology as protoplasm, and that 
which has been formed by protoplasm, ‘ 'formed material, ’ ’ are investi- 
gated in the two cases. The formed material of plants differs greatly 
from that in animals, and in too many cases far greater attention is 
paid to it in the study of vegetable histology than to the protoplasm. 
This is comparable with a too exclusive study of “dry bones” in 
animals. 
In vegetable histology as in animal histology, the tissues should be 
studied : 
(A) Fresh, and alive if possible, (see $ 15). Normal conditions are as 
necessary as for the study of living, animal tissues (§ 7). Protoplasm 
in plants may be anaesthetized by ether, chloroform, etc., as in ani- 
mals. [.Bernard, I, 251]. Free-hand sections and teased preparations 
of fresh tissues often show the structure well. Osmic acid fixes uni- 
cellular organisms and plant cells in a natural condition. It may be 
applied as directed in $ 21, or a drop may be drawn under the cover- 
glass after the object is mounted in water or some normal fluid. 
(B) Vegetable tissues should be hardened and cut into sections. 
Picric acid ($ 97), especially Kleinberg’s mixture, is often used. Al- 
cohol of 50 and 75 per cent., each one day, and then 95 per cent, is 
often used also. The hardened tissues may be sectioned freehand 
without further treatment, or they may be imbedded, sectioned, etc., 
by the paraffin method (§ 38-46), or the celloidin method, which 
seems to be preferable in some ways as heat is unnecessary. The 
use of celloidin precludes the use of most aniline dyes, however, 
and they are very largely used in staining vegetable tissues. When- ever it is necessary or desirable to preserve the chlorophyll, oil or 
resin in a plant, the freezing method (g 59) is to be preferred. 
(C). Staining vegetable tissues with aluin carmine, haematoxylin 
and fluorescine has the same general advantages as in animal tissues, 
and the stains may be applied in the same manner. Permanent pre- 
parations may be made by mounting in balsam, glycerin or glycerin 
jelly,—or if the color of the chlorophyll is to be retained a saturated 
solution of potassium acetate, is recommended by Bower & Vines, p 
J5- 
Excellent directions for work in vegetable histology, and for per- 
manent preparations may be found in : Beh.; Bower & Vines ; C ; 
Car.; Goodale; H. & M.; Strasburger; Tr.; (J. r. m. s.) (Z. w. m.) 
(Q. j. m. s.) (Ar. m. a.) (A. m. m. j.) M. The works of Behrens, 
Bower and Vines, Goodale, Strasburger, and Trelease are devoted ex- 
clusively to vegetable histology and methods for the same. 
Permanent preparations of expanded plasmodium, may be made 
by placing pieces of rotten wood on which masses of plasmodium are 
found on moistened slides, so that the plasmodium touches the slide. 
Over the whole should be placed a bell jar, or other cover to pre- 
vent drying. In an hour or more the plasmodium will, in many 
cases, spread out on the moistened slide. The slide should be placed 
in the picric acid solution (§97) for half an hour or longer. The 
picric acid kills and fixes the extended plasmodium. It may then be 
rinsed in water and mounted, by covering with a cover-glass well 
spread with glycerin jelly or it may be dehydrated, cleared and 
mounted in balsam ($ 27a). Preparations of the common yellow 
plasmodium prepared and mounted in Canada balsam, as here des- 
cribed, are at present as perfect in color and in every way as when 
first made five years ago. \_Proc. Amer. Assoc. Adv. Sc., 1880, p. 377) ; 
(A. m. m. j. (1880) 130)]. 
THE MICROSCOPE IN JURISPRUDENCE. 
($ 70) While an entire human body may be distinguished as such 
with certainty, histological knowledge is not, in my opinion, sufficient- 
ly advanced at the present day to enable one to say that any micro- 
scopic structure is absohitely characteristic of and peculiar to a human 
being. 
While it is true that no one can say that a given microscopic struct- 
ure is part of a human being, and not of any other animal, he might 
say with certainty, that it could not be from some animals in which 
the given structure is known to be very different. The histological or 
microscopical expert, in my opinion then, cannot give positive evidence 
with regard to the exact source of any microscopic structure. The 
most he can do is to say what it may be, and what it cannot be. Even 
this, unsatisfactory as it may seem, requires a profound knowledge of 
human and comparative histology, and of the changes that may be 
produced in the various structures by drying and dampness, by chem- 
ical and mechanical means. There is also often required a thorough 
knowledge of optics, and great manipulative skill. He, who, in the 
name of Science, allows himself through ignorance or design to be- 
come an advocate and not an expounder of the whole truth, so far as 
it is known, has been well characterized by Woodward as possibly 
more dangerous to society, than the criminals he is called upon to 40 
convict; and the lawyer who through ignorance or design allows 
truth and justice to be so betrayed, is no better. 
The histological or microscopical expert is called upon most fre- 
quently to give evidence concerning the following subjects : (i) Blood 
or blood stains. (2) Semen or seminal stains. (3) Foetal liquids, 
excreta or their stains. (4) Contents of the stomach, especially food. 
(5) Various tissues of the body. (6) Hair, wool, and various textile 
fibers. (7) Foods; adulteration, or the presence of trichinae, or larval 
tape-worms in the same. (8) Adulteration of medicines and other 
commercial produces. (9) Poisons, animal, vegetable and mineral. 
(10) Handwriting. 
In the University Library, or the Anatomical Laboratory may be 
found the following references to these subjects :* 
(1) Blood and blood stains.—(a) Normal structure of blood; L. 
Elsberg, Annals of N. Y. Acad, of Sciences, Vol. I, Nos. 9 and 10. 
Also in Heitz, p. 64 ; Fi. 229; Ki. II, 320; Quain II, 23; R. 178; Str. 
263; Todd I, 404 ; Milne-Edzuards I, 49, 83 ; Sat. 34. 
(b) Examination of blood stains.—Richardson, 284; also Monthly 
Microscopical Journal of London, 1874 (Reprint). Chemical News, 
1875 (No. 132, p. 291); Taylor's Med. Jurisp.; Wharton and Still?s 
medical jurisprudence, p. 678; Hofmann, 273, 776. The application 
of photography to the micrometry of blood in criminal cases. Re- 
printed from the Phila. Med. Times, 1876. Fi. 239; Wormley, 708; 
(A. m. m. j. III. 6, I. 184); Stowell's micr. diagnosis, p. 30; St. 82. 
(c) Measurement of blood-corpuscles and precautions necessary in 
micrometry in general.—The value of the results vary in proportion 
to the accuracy of the micrometer and the microscope, and the skill 
of the observer: Bi. 156; C. 95, 97, 184, 713; R. 31; Wormley 701; 
Richardson, 59. Monthly Micr. Journal of London, 1874 and 1875 
(Reprints). Woodward, the application of photography to micromety; 
and in American Journal of the Medical Sciences, 1875, p. 151. (These 
papers, and the photographs made by Dr. Woodward of blood and 
blood stains, should be in the hands of every expert and every lawyer); 
(Rogers in A. q. m. j. 97); (Q. j. m. s. (1861 p. 87); American Jour- 
nal of Science, 1872; Milne-Edwards I. 83; Heitz. 66; International 
Medical Congress 1876; (Q. j. m. s. (1861) 81); Taylor, 307; Wharton 
& Stillk, 679; Proc. Zool. Soc. of London, 1862, 96, 1875, 474; (A. m. 
m. j. IV 175, V 17, VI 107, 138, 127); (M. Ill, 157); Buck I, 584. 
(d) Mistaking spores ofplants for blood-corpuscles.—Bi. 156; Tay- 
lor; Richardson, Monthly Micr. Journal of London, 1874 (Reprint). 
(e) Menstrual blood.—Dalton, Physiology, 559 ; Richardson, 300 ; 
Littre, Diet, de med.; Taylor; Wharton & Stilll, 677; Buck, I, 584. 
(f) Blood crystals.—Wharton & Stille, 681; Chemical News (1873) 
p. 291; (Fi. 237; F2.; Qua n II. 27; Richardson, 294; Taylor. 
(g) Microspedroscopic tests for blood.—B1, 273; Chemical News, 
1873, P- 291; Foster’s physiology; Hermann’s Physiologie; (J. a. p. 
IV, 119); Richardson, 298; (Q, j. m. s. (1869) 358); (P. r. s. 1866-7, 
433; 1863-4, 355); Buck, I, 578. 
(2) Semen and Seminal Stains.—(a) Normal semen : Quain, II, 
697; Str. 459. (b) Seminal Stains: B. et G., 547, 552,863; Rich- 
* These references do not include Bouvier’s Taw Dictionary, or the 
Merritt King Law Library, recently obtained by the University. ardson, 300; Taylor; Wharton & Stille, 452; Hofmann, 673; Ham- 
lin, in A. m. s., 1883, p. 21, gives the best directions for the treat- 
ment of seminal stains. 
(3) Foetal Liquids, and excreta and their stains.—Robin et Tar- 
dieu, memoir sur l’examen microscopique des taches formees par le 
meconium et l’enduit foetal. Paris, London and New York, 1857 ; B. 
et G- 575, 581,836/ Taylor; Wharton & Stille, 680. 
(4) Contents of the stomach, especially food.—B. et G. 715 ; Stow- 
ell, diagnosis, 35 ; Hofmann, 551 ; Taylor; Wharton & Stille. 
(5) Various tissues of the body.—The works of Quain, Strieker, 
Ranvier, and the journals containing histological structure. 
(6) Hair, Wool and various textile fibers.—Quain, II ; Str. ; Todd, 
II, 478 ; B. et G., 805 ; Bulletin of the National Assoc, of Wool Grow- 
ers, 1875, p. 470 ; Wurtz, dictionnaire de chemie, II, p. 154, III, 253 ; 
Hofmann, 291; Fi. 270; Ru. 127; C. 703; B et G. 89; Stowell, diag- 
nosis, 115 ; (A. m. s. 1884, 59-). 
(7) Foods, and their Adulteration.—Stowell, micr. diagnosis, p. 33 ; 
(J. r. m. s., 1879, P- 938 and 1881, p. 962) ; (M. II, 107); Blyth, foods, 
composition, etc. ; (A. m. s., 1883, p. 97) ; Report, S. M. Babcock, 
Chemist N. Y. State Agr. Experiment Station, 1885 ; B. et G., 515. 
For trichinae, etc. See Taylor; B. et G., 716, 718; Phin : How to de- 
tect Trichinae and how to avoid them. Rochester, 1881. 
(8) Adulterations of medic;nes and other commercial products.— 
Hoffmann, Examination of medicinal chemicals, N.Y., 1877; Naquet’s 
legal chemistry. For the adulteration of textile fabrics, see references 
to (6). 
(9.) Poisons—Taylor; Wharton and Stille; Hofmann; Wormley; 
B. et G., 718 ; (A. m. m. j., I, 118). 
(10) Handwri ing.—,A. m. s., 1879, p. 46, 1884, p. 47); (A. j. m. s., 
IV, 194) ; (A. m. m. j., I, 48, 124) ; (A. n., I, 167). 
REAGENTS AND THEIR PREPARATION. 
Employ only pure materials. Keep all volatile substances (alcohol, 
ether, xylol, etc.) well covered. Use glass stoppers for the cleaning 
mixture for glass, nitric and hydrochloric acid. Glass or cork stop- 
pers may be used for the other substances. 
($ 71) Water.—The water to be used in microscopial work should 
be (a) distilled; (b) fresh snow or rain water boiled in an agate or 
porcelain dish and filtered ; (c) soft spring water, boiled in an agate 
or porcelain dish and filtered. Wherever buildings are heated by 
steam, distilled water may be easily obtained by tapping the return 
pipes. ($ 72) Cleaning mixture for glass.— 
Water (ordinary water may be used), 2000 cc. 
Potassium dichromate, 200 grams. 
Commercial sulphuric acid (strong),. 200 cc. 
Dissolve the potassium dichromate in the water and then add the 
sulphuric acid. This mixture will remove balsam, sealing mixtures, 
etc. A prolonged stay in it does not injure glass. 
($ 73) Normal fluid.—White of egg 15 cc. Water 200 cc. Mercuric 
chloride gram. Common salt (11a cl) 4 grams. This mixture 
should be thoroughly shaken in a bottle with bits of glass to mix the 
albumen with the water. It should then' be filtered, and kept in a 
cool place. The mercuric chloride (corrosive sublimate) retards de- 
composition without being injurious to white blood-corpuscles or 
ciliated cells. 
($ 74) Normal salt solution.—Water 100 cc. Common salt x%ths of 
a gram. 
(| 75) Sealing mixture for blood, etc.—Paraffin 50 grams. Vaseline 
50 grams. 
(g 76) Curara.—Curara of a gram. 95 per cent, alcohol 20 cc. 
Water 20 cc. Grind the curara with the alcohol and water in a mor- 
tar. Do not filter. Curara varies in strength. The proportions here 
given might be too strong in some cases and too weak in others. 
($ 77) Osmic acid solution.—Osmic acid 1 gram. Water 100 cc. 
The water should be distilled or freshly boiled snow or rain water. 
The bottle should be thoroughly cleaned with the cleaning mixture 
(| 72). Put the water into the bottle, break off one end of the sealed 
tube containing the osmic acid and then, being sure that the tube is 
clean, drop it into the bottle. It requires several hours for the osmic 
acid to dissolve. Cover the bottle with black paper or velveteen to 
exclude light. Osmic acid is very irritating to mucuous membranes, 
hence care should be taken to keep its fumes from the eyes, nose and 
mouth. 
(? 78) Nitric acid 20 per cent.—Concentrated nitric acid 77 cc. 
Water 23 cc. A 20 per cent, solution of hydrochloric acid also forms 
an excellent dissociating agent for muscular fiber-cells and cardiac 
muscle. 
(g 79) Acetic acid.—Glacial acetic acid from 1 to 2 cc. Water 99 or 
98 cc. 
(| 80) Glycerin.—Pure glycerin. This is often employed for objects 
that have been hardened. For fresh specimens and those dissociated 
in 35 per cent, alcohol, glycerin 75 cc, water 25 cc. is used. For 
objects stained in carmine, 1 cc. of acetic acid is sometimes added to 
each 100 cc. of glycerin. (2 8i) Alum carmine glycerin.—Glycerin 85 cc. Alum carmine 
(§ 87) 10 cc. Fluorescine ($ 89) 5 cc. 
(3 82) Fluorescine glycerin.—Glycerin 75 cc. Fluorescine 25 cc. 
(?, 83) Glycerin jelly.—Soak 25 grams best gelatin in water for half 
an hour or more ; pour olf the superfluous water and melt the gelatin 
in a porcelain or agate dish over a water bath. Add 10 cc. of the nor- 
mal fluid (§ 73) or a little egg albumen and heat the melted gelatin 
for half an hour or more to clarify it. Pour off the clear portion and 
add to it 5 grams of chloral hydrate. This clarified galatin is that to 
be used in making frozen sections with gelatin (§ 59). For glycerin 
jelly, mix equal parts of the clarified gelatin and glycerin, and shake 
thoroughly. Keep in a warm place for some time to get rid of air 
bubbles. “ When used, the jelly must be liquified by gentle warmth, 
and it is useful to warm both the slide and the cover-glass previously 
to mounting.” Care must be taken to avoid air bubbles as they do 
not disappear as with balsam. Seal as for balsam preparations (§ 27a) 
after the jelly is cold. 
($ 84) Shellac.—Shellac for sealing preparations is prepared by ad- 
ding shellac (either bleached or that in thin laminae) to 95 per cent, 
alcohol in sufficient quantity to make a solution of creamy consistency. 
This should stand in a warm place for the insoluble parts to settle. 
The clear part is poured off, and to each 50 cc. of shellac 1 cc. of cas- 
tor oil is added. This cement may be thinned at any time by adding 
95 per cent, alcohol. 
(§ 85) Gold-size.—This is best bought of dealers in microscopical 
supplies. It is considered to be one of the best cements for sealing. 
It hardens slowly. It should not be used for balsam preparations 
without first using shellac. It is well to add several coats of gold-size 
to all glycerin preparations sealed with shellac. 
($ 86) Liquid gelatin.—Gelatin or clear glue 75 to 100 grams. Com- 
mercial acetic acid (No. 8) 100 cc. Water 100 cc. 95 per cent, alcohol 
100 cc. Glycerin 15 to 30 cc. Crush the glue and put it into a bottle with • 
the acid, and set in a warm place, and shake occasionally. After three or 
more days add the other ingredients. This solution is excellent for fast- 
ening paper to glass, wood or paper. The brush must be mounted in a 
quill or wooden handle. For labels, it is best to use linen paper of 
moderate thickness. This should be coated with the liquid gelatin 
and allowed to dry. The labels may be cut of any desired size and 
attached by simply moistening them as in using postage stamps. 
(| 87) Alum carmine (Grenacher's).—Saturated aqueous solution of 
alum 100 cc. Best carmine 1 gram. Boil 15 to 20 minutes. Finally 
add 2-4 grams of chloral hydrate. Sometimes a deposit is left in the 
tissue. If a small amount of normal fluid or egg albumen is shaken with the solution and then the bottle be placed where it will be heated 
to 70-90 C. for a few hours the defect will be remedied. This solution 
may be diluted with saturated alum solution or with water. 
(| 88) Haematoxylin.—Water 150 cc. Alum 5 grams. Haematoxylin 
crystals dissolved in 95 per cent, alcohol sufficient to give a deep pur- 
ple color. The solution improves by standing a few days. A fresh 
solution should be made about every three months. It is very diffi- 
cult to prepare a satisfactory haematoxylin dye unless crystal of 
haematoxylin are used. 
($89) Fluorescine.—Fluorescine x\, of a gram. Water icocc. Chlo- 
ral hydrate 2 grams. A fresh solution should be prepared every few 
weeks. Eosin gram, water 100 cc., may be used instead of fluo- 
rescine. 
(| 90) Picrocarmine.—Carmine 2.5 grams. Ammonia 5 to 10 cc. 
Picric acid, saturated aquepus solution, 100 cc. Chloral hydrate 5 
grams. Grind the carmine in water and place it in a tall jar contain- 
ing 3 to 400 cc. of water. Allow it to remain till the carmine forms a 
sediment on the bottom. Pour off the liquid and add the ammonia, 
then add the picric acid, and sufficient water to make 500 cc. Allow 
this to stand in a warm place (70 to 90 C.) till the liquid is nearly 
evaporated, then add water to make 500 cc. of solution. Add 10 to 20 
cc. of normal fluid (§73) and heat to 70 or 80 C. for a few hours. Fil- 
ter through filter paper and have absorbent cotton crowded into the 
neck of the funnel. Finally add the chloral hydrate. See W. 42 for 
Minot’s picrocarmine. 
(§91) Carmine.—Carmine 5 grams. Water 250 cc. Ammonia added 
drop by drop till the carmine is dissolved. Chloral hydrate 5 grams. 
The carmine is treated as for picrocarmine, and after the ammonia is 
added the jar is placed in a warm place till the odor of ammonia is 
scarcely perceptible. It is then placed in a bottle and the chloral 
added. Gierke recommends the addition of a small amount of ammo- 
nium carbonate [Z. w. m. I. 75]. Dilute solutions and a long time 
in the solution is the rule for staining with carmine. 
(| 92) Silver nitrate.—Silver nitrate 1 gram. Water 200 cc. 
(§ 93) Canada balsam.—Canada balsam (one tube) 25 cc. Chloro- 
form 2 cc. Clove oil 2 cc. Canada balsam so prepared clears any 
cloudiness that may arise in the collodion or celloidin used for fasten- 
ing sections to the slide. Canada balsam diluted with xylol, turpen- 
tine or chloroform is often used for mounting. Balsam thinned 
with xylol is best for mounting stained microbes, as the tubercle 
bacillus. 
(§ 94) Clearing mixture.—Carbolic acid (melted crystals) 20 cc. 
Turpentine (oleum terebinthincz rectification) 30 cc. (| 95) Chromium compounds.—Those most commonly employed in 
hardening tissues are : Chromic acid J to 1- per cent, aqueous solutions. 
Potassium dichromate 1 to 3 per cent, aqueous solutions. Ammonium 
dicliromate 2 to 5 per cent, aqueous solutions. It requires from 5 days 
to 5 months or more to harden, with a chromium compound. 
(| 96) Muller’s fluid.—Potassium dichroinate 25 grams. Sodium 
sulphate 10 grams. Water 1000 cc. This agent hardens any tissue 
fairly well ($ 37). 
(| 97) Picric acid.—Picric acid 1.5 grams. Water 50 cc. 95 per 
cent, alcohol 50 cc. A one per cent, aqueous solution is often em- 
ployed and a mixture of picric and sulphuric acids is excellent for 
many vegetable tissues (Kleinenberg’s solution : Saturated aqueous 
solution picric acid 100 cc. Concentrated sulphuric acid 2 cc. After 
filtering add 300 cc. of water). An alcoholic solution of picric acid is 
excellent for decalcifying, especially when it is necessary to preserve 
ciliated epithelium well. A stronger solution than that given above 
is preferable for this purpose : 95 per cent, alcohol 100 cc. picric acid 
2.5 grams is a good decalcifying agent. With it the bones of an adult 
frog’s head were decalcified in 28 days. The cilia and other soft 
structures were perfectly preserved. 
(| 98) Alcohol.—Ethyl alcohol of various percentages. From my 
own experience, alcohol of a higher percentage than 95 is unnecessary 
for any of the processes of histology. It is necessary to use plenty of 
alcohol and to use it but few times for dehydrating tissues. Com- 
mercial alcohol has, in many cases, an acid reaction. It may be ren- 
dered neutral by adding a small quantity of pure bicarbonate of soda. 
(| 99) Chloroform.—Pure anhydrous chloroform should be used. 
After it has been used for histological purposes it is good to kill ani- 
mals with. 
(§ 100) Ether.—Pure sulphuric ether should be used. 
(§ 101) Collodion.—Gun cotton (that used in photography) 1 gram. 
95 per cent, alcohol 50 cc. Ether 50 cc. This should be kept tightly 
corked and may be diluted by using equal parts of ether and alcohol 
if it becomes too thick. Only a thin coat should be painted on the 
slide, and the ether and alcohol added to fasten the sections should be 
allowed to entirely evaporate, or some staining agents will color the 
collodion. If ihe mixture of clove oil and collodion (see p. 26) is used 
for fastening the sections to the slide there is less danger of staining 
the collodion. If the sections are curled it is also preferable, as they 
unroll during the expulsion of the clove oil. 
(g 102) Xylol.—Pure xylol should be used. It burns with difficulty 
and hence is much safer than naptha, benzine, or turpentine. (I io3) Paraffin imbedding mass.—Hardest paraffin obtainable 90 
grams. Very soft paraffin 10 grams. These proportions have to be 
varied according to the temperature. 
(§ 104) Celloidin.—Two solutions are necessary: (A) Thin celloi- 
din. Dry celloidin 2 grams. Ether 50 cc. 95 per cent, alcohol 50 cc. 
(B) Thick celloidin. Dry celloidin 5 to 6 grams. Ether 50 cc. 95 
per cent, alcohol 50 cc. If gun cotton is used instead of celloidin the 
same proportions will answer. Sometimes the celloidin (§ 58) cannot 
be shaken out of the test tube after hardening in chloroform. A paper 
tube with a cork in one end may be used instead of the test-tube and 
as soon as a film is formed over the top it may be put into a vial of 
chloroform. The paper would best be removed on the second or third 
day of immersion. 80 per cent, alcohol may be used for hardening 
the celloidin instead of chloroform, but it renders the mass opaque 
and thus makes it impossible to see the object. 
(§ 106) Carmine, gelatin, inje<5ting mass for mammals.—Dry gela- 
tin 50 grams. Carmine (No. 40) 5 grams. Water 90 cc. Ammonia 
10 cc. Acetic acid q. s. Chloral hydrate 5 grams. Soak the gelatin 
in water until it is soft, then pour off the superfluous water and melt 
it in an agate or porcelain dish over a water bath. Grind the carmine 
to a paste with water, and add the ammonia and the water. This 
should be filtered through filter paper or absorbent cotton, warmed, 
and added to the warm gelatin. The acetic acid, mixed with an equal 
volume of water, should be added slowly while constantly stirring the 
mass, untill the mass smells very slightly of the acid. The mass 
should be filtered through fine flannel and finally the chloral should 
be added. The mass so prepared will not deteriorate for a long time, 
and is ready to use at any time by simply warming it over a water 
bath. 
(§ 107) Berlin-blue, gelatin, injecting mass for mammals.—Dry gel- 
atin 50 grams. Saturated aqueous solution of Berlin-blue 150 cc. 
Chloral hydrate 5 grams. Soak the gelatin in water for an hour or 
until it is soft, then pour off the superfluous water and melt in an 
agate or porcelain dish over a water bath. Heat the Berlin-blue to 
80 or 90 C. over a water bath and add it to the gelatin. Heat the 
mixture for 10 minutes or more and stir it occasionally. Filter it 
through fine flannel and add the chloral. This mass will last a long 
time without deterioration. It should be kept in a preserving jar and 
simply requires to be warmed before using. The mass for amphibia 
(§ 109) answers well for mammals also.* 
* Soluble Berlin-blue is now obtainable at almost any drug store. 
About i cc. of acetic acid should be added to each liter of the alcohol 
used in preserving specimens injected with Berlin-blue. (| 108) Carmine, gelatin, injecting mass for Amphibia and other 
cold-blooded animals.—Dry gelatin 20 grams. Carmine 5 grams. 
Water 100 cc. Ammonia 10 cc. Acetic acid q. s. Chloral hydrate 
5 grams. This should be prepared exactly as directed in $ 106. This 
mass melts at 20 to 25 C. and may be injected if the animal is at a 
temperature of 15 C. or above. It also answers fairly well for 
mammals. 
{$ 109) Berlin-blue, gelatin, injecting mass for Amphibia and other 
cold-blooded animals.—Dry gelatin 25 grams. Saturated aqueous 
solution of Berlin-blue 150 cc. Chloral hydrate 5 grams. Prepare as 
directed in \ 107. This mass is fluid at about 25 C. or less, and may 
be injected if the temperature of the animal is not below 15 C. 
References for $ 7/ to 109 : Bi.; Beh.; Car.; C.; Fi.; Fob; Gibbs,r 
H. & P.; Lee.; Prudden; R.; Robin; Ru.; Sat.; Si. &2; Seiler; St.; 
Tr.; W.; (A. m. m. j.); (A. n.); (A. s. m.); (Ar. m. a,); (J. r. m. s.); 
L.; M.; (M.z.s.n.); (Q.j.m.s.); (S. r.); (S.b.l.); (Z. a.); Z.w.m.)]. 
Nearly every original paper in histology and embryology introduces 
new reagents or special modifications of those already in use. A very 
full discussion of staining agents is given by Gierke [Z. w. m. 1884-5; 
translation in A. m. m. j. 1885-6]. 
($ no) Alum cochineal.—Cochineal io grams. Calcined (burnt) 
alum io grams. Chloral hydrate io grams. Water 750 cc. Grind 
the cochineal (which would best be the whole insects), and the alum 
together, and add to the water. Boil till about 500 cc. remains, then 
after cooling filter and add the chloral. This dye may be used in- 
stead of alum carmine. It is cheaper, easier to prepare, and appar- 
ently just as good as alum carmine. [Ar. m. a. 1880, p. 413.] REFERENCE BOOKS AND PERIODICALS. 
The books and periodicals named below in alphabetical order, are 
in the laboratory or the University library. They pertain wholly or 
in part to the microscope, microscopical or histological methods, or to 
histology, or to all of these subjects. For the method of referring to 
these works and periodicals in the text, see the prefatory note, p. 1. 
For current microscopical and histological literature, the Journal of 
the Royal Microscopical Society,the Index Medicus, the Zoologischer 
Anzeiger, and the Zeitschrift fur wissenschaftliche Mikroscopie taken 
together furnish nearly a complete record. References to books and 
papers published in the past may be found in the periodicals just 
named, in the Index Catalogue of the Surgeon General’s library ; in 
the Royal Society’s Catalogue of Scientific Papers ; in the Zoological 
Record and in the bibliographical references given in special papers. 
BOOKS. 
Bal.—Balfour, F. M.—A Treatise on Comparative Embryology. 
Two vols., illustrated. Pp. 492 + 653. London, 1880. Structure. 
Bau.—Bausch, E.—Manipulation of the microscope. Pp. 95, illus- 
trated. Rochester, 1885. 
Bi.—Beale, L. S.—How to work with the microscope. 5th ed. 
Pp. 518, illustrated. London, 1880. Structure and methods. 
B2.—Beale, L. S-—The Microscope in Medicine. Pp. 539, 500 fig., 
4th ed. Philadelphia, 1878. 
B. et G.—Beauregard, H. et Gallippe, V.—Guide de et du 
praticien pour les travaux pratiques de micrographie, comprenant la 
technique et les applications du microscope a l’histologie vegetale, a 
la pliysiologie, a la clinique, a la hygiene et a la medecine legale. 
Pp. 904, 570 fig. Paris, 1880. 
Beh.—Behrens, J. W.—The microscope in botany. A guide for the 
microscopical investigations of vegetable substances. Translated and 
edited by Hervey and Ward. Pp. 466, illustrated. Boston, 1885. 
Bell, F. J.—Comparative Anatomy and Physiology. Pp. 555, illus- 
trated. London and Phila., 1885. Structure. 
Bernard, Cl.—Legons sur les phenomenes de la vie communs aux 
animaux et aux vegetaux. 2 vols., pp. 968, 50 fig., 4 plates. Paris, 
1878-1879. Structure. 
Bessey, C. E.—Botany for high schools and colleges. Pp. 611, illus- 
trated. New York, 1880. Structure and methods. 
B-S.—Bourdon-Sanderson (editor).—Hand-book for the physio- 
logical laboratory. Text, pp. 585; atlas, 123 plates. Phila., 1873. 
Reprint of the English edition, with slightly different paging. Struc- 
ture and methods. 50 
B. et et Jacob.—Traite complet de l’anatomie de 
I’homme, Paris, 1844. Vol. 8, Embryology, including Comparative 
Neurology and Histology, Structure. 
Bower and Vines.—A course of practical instruction in botany. 
Part I, pp. 226. London, 1885. 
Brass, A.— Kurzes Lehrbuch der normalen Histologie des Menschen 
und typischer Thierformen. Illustrated (incomplete). Cassel und 
Berlin, 1885. Structure. 
Buck, A. H. (editor).-—A reference hand-book of the medical sci- 
ences. Illustrated (incomplete). New York, 1885 + . Structure and 
methods. 
Car.—Carnoy, Le Chanoine.—La Biologic Cellulaire ; Etude com- 
paree de la cellule dans les deux regnes. Illustrated (incomplete). 
Paris, 1884. Structure and methods. 
C. —Carpenter, W. B.—The microscope and its revelations. 6th ed., 
pp. 882, illustrated. London and Phila., 1881. Methods and structure. 
Cooke, M. C.—One thousand objects for the microscope. Pp. 123. 
London, no date. 500 figures and brief descriptions of pretty objects 
for the microscope. 
C. and R.—Cornil, V., and Ranvier, L.—A manual of pathological 
histology, translated from the French, with notes and additions, by 
E- O. Shakespeare and J. H. C. Simes. Pp. 784, 360 fig. Philadel- 
phia, 1880. Structure, very few methods. 
Drysdale, J.—~The protoplasmic theory of life. Pp. 288. London, 
J874. 
Fol, H.—Lehrbuch der vergleichenden mikroskopischen Anatomie, 
mit Einschluss der vergleichenden Histologie und Histogenie. Illus- 
trated (incomplete). Leipzig, 1884. Methods and structure. 
F. and B.—Foster and Balfour.—The Elements of Embryology. 
Pp. 486, 2d ed., illustrated. Loudon, 1883. Structure and methods. 
F and L.—Foster and Langley.—A course of elementary instruction 
in practical physiology, 4th ed., pp. 276. London and New York, 1880. 
Methods and structure. 
Fi.—Frey, H.—The Microscope and Microscopical Technology. 
Translated and edited by G. R. Cutter. Pp. 624, illustrated. New 
York, 1880. Methods and structure. 
F2.—Frey, H.—Hand-book of the histology and histo-cliemistry 
of man. Translated by Barker. Illustrated. New York, 1875. 
Structure and chemistry. 
Gatngee, A.—A text-book of the physiological chemistry of the 
animal body. Part I. pp. 487, 63 fig. London and New York, 1880. 
Structure. 
Gibbs, H.—Practical histology and pathology. Pp. 107. Loudon, 
1880. Methods. 
Goodale, G. L.—Physiological botany. Pp. 499+36, illustrated. 
New York, 1885. Structure and methods. 
Green, T. H.—An introduction to pathology and morbid anatomy, 
2d American from the 3d English ed., pp. 316, in fig. Philadelphia, 
1876. 
Haeckel, E.—The natural history of creation, 2 vols., illustrated. 
ISew York, 1883. Also original, 1873. Structure and classification. H. and P.—Harris and Power.—Manual for the physiological lab- 
oratory, 2d ed., illustrated, pp. 213. London and New York, 1882. 
Histology, methods and physiological chemistry. 
Heitz.—Heitzmann, C.—Microscopical morphology of the animal 
body in health and disease. Pp. 849, illustrated. New York, 1883. 
Structure and methods. 
Hoffman, E.—Brouardel, P., translator.—Nouveaux Elements de 
medecine ldgale. Pp. 830, illustrated. Paris, 1881. 
H.—Hogg, J.—The microscope, its history, construction and appli- 
cation. New edition, illustrated. Pp. 764. London and New York, 
1883. 
Howes, G. B.—An atlas of practical elementary biology. Pp. 116. 
London, 1885. Figures, etc., of structures described in Huxley and 
Martin (q. v.) 
H. and M.—Huxley and Martin.—Elementary Biology, 2d revised 
edition, pp. 279. London and New York, 1876. Methods and 
structure. 
Kingsley, J. S.—The Naturalist’s Assistant. Pp. 228, illustrated. 
Boston, 1882. 
K.—Klein, E.—Elements of histology. Pp. 352, illustrated. Lon- 
don and Phila. 1883. Structure. 
K. and S.—Klein, E., and Smith, E. N.—Atlas of histology. The 
illustrations are by Mr. Smith, from the preparations of Dr. Klein, 
Text by Dr. Klein. Pp. 448, 48 colored quarto plates. London, 1880, 
Structure. 
Ki.—Kolliker, A.—Manual of human histology ; translated front 
the German by G. Busk and T. H. Huxley. Two vols.x pp. 932, 3x3 
fig. London, 1853-1854. Structure, and methods. 
K2.—Kolliker, A.—Entwickelungsgeschichte des Menschen und 
der hoheren Thiere. 2d edition. Pp. 1033. Leipzig, 1879. Structure. 
Lee, A. B.—The microtomist’s vade-mecum,. A hand-book of the 
methods of microscopic anatomy. Pp, 424, London, 1885. 
Lehman, C. G.—Physiological chemistry. 2 vols., pp. 648+ 547; 
illustrated. Philadelphia, 1855, 
Leidy, J.—Fresh-water Rhizopods of North America, Illustrated, 
Washington, 1879. Methods and descriptions. 
Lewis, IV. B.—The human brain-—histological and coarse methods 
of research. Pp, 163; illustrated. Loudon, 1882. 
Li.—Leydig, F.—Traite d’ histologic de l’homme et des animaux, 
Pp. 629, 270 fig. Paris, 1866. Structure. 
L2.—Leydig, F.—-Zelle und Gewebe. Neue Beitrage zur Histolo- 
gic des Thierkorpers. Pp. 219; illustrated. Bonn, 1885. Structure. 
Macdonald, J. D.~—A guide to the microscopical examination of 
drinking water. Illustrated, London, 1875. Methods and descrip- 
tions. 
M' Alpine, D. and A. N.—Biological atlas, a guide to the practical 
study of plants and animals. Edin. and London, 1880. Figures of 
many tissues. 
Marsh, S.—Section cutting; a practical guide to the preparation 
and mounting of sections for the microscope, special prominence being 
given to the subject of animal sections, 2d edition, illustrated, pp. 
156. London, 1882, Marshall, A. M.—The frog, an introduction to anatomy and histol- 
ogy. Pp. 86. London, 1882' 
Milne-Edwards, //.—Lemons sur la physiologie et l’anatomie de 
l’homme et des animaux. 14 vols. Paris, 1857-1880. Structure. 
Owen, R.—Comparative anatomy and physiology of vertebrates. 
Three vols., pp. 2155, 1471 fig. London, 1861-1868. Structure. 
Peaslee, E. R.—Human histology in its relations to descriptive an- 
atomy, physiology and pathology. Pp. 616, 434 fig. Philadelphia, 
1857- 
Phin, J.—Practical hints on the selection and use of the microscope 
for beginners. 3d edition, illustrated, pp. 231. New York, 1881. 
Prudden, T. M.—A manual of practical normal histology. Pp. 
265. New York, 1881. Methods and structure. 
Quain.—Elements of anatomy, 9th edition. Edited by Allen 
Thompson, E. A. Schafer and G. D. Thane. Two vols.. pp. 1694, 
illustrated. London and New York, 1882. Structure. 
R.—Ranvier, L.—Traite technique d’histologie. Pp. 976, illus- 
trated. Paris, 1875-1882 (incomplete). Structure and methods. 
Richardson, J. G.—A hand-book of medical microscopy. Pp. 333, 
illustrated. Phila., 1871. Methods and descriptions. 
Robin, Ch.—Traite du microscope et des injections, 2d ed. Pp. 
noi, illustrated. Paris, 1877. Methods and structure. 
Ru.—Rutherford, W.—Outlines of practical histology. 2d edition, 
illustrated, pp. 194. London and Phila., 1876. Methods and struct- 
ure. 
Sachs, J.—Text-book of Botany ; 2d English, from the 4th German 
edition. Pp. 980, illustrated. London, 1882. Structure. 
Sat.—Satterthwaite, F. E. (editor).—A manual of histology. Pp. 
478, illustrated. New York, 1881. Structure and methods. 
51. —Schafer, E. A.—A course of practical histology, being an in- 
troduction to the use of the microscope. Pp. 304, 40 fig. Philadel- 
phia, 1877. Methods. 
52. —Schafer, E. A,—The essentials of histology, descriptive and 
practical for the use of students. Pp. 245, illustrated. London and 
Philadelphia, 1885. 
Sc.—Schwalbe, G.—Lehrbuch der Neurologie. Pp. 739, illustrated. 
Erlangen, 1881. Structure. 
Schwann, T.—Mikroskopische Untersuchungen fiber die Ueber- 
einstimmung in der Structur und dem Wachsthum der Thiere und 
Pflanzen. 4 Tafelu. Berlin, 1839. Structure. Cell doctrine. See 
below. 
Schwann, T.—Microscopical researches, also Schleiden’s contri' 
butions to phytogenesis. Illustrated, pp. 263. English translation 
by H. Smith. London, 1847. Cell doctrine in plants and animals. 
See above. 
Seiler, C—Compendium of microscopical technology. A guide to 
physicians and students in the preparation of histological and patho- 
logical specimens. Pp. 130, illustrated. New York, 1881. 
St.—Stowell, Chas. H.—The students’ manual of histology, for the 
use of students, practitioners and microscopists. 3d ed. Pp. 368, il- 
lustrated. Ann Arbor, 1884. Structure and methods. 
Stowell, Chas. H. and L. R.—Microscopical Diagnosis. Illus- 
trated. Detroit, 1882. Structure and methods. Strcisburger, E.—Das botanische Practicum. Anleitung zum. 
Selbststudium der mikroskopischen Botanik, fiir Anfanger und Fort- 
geschrittuere. Pp. 664, illustrated. Structure and methods. 
Str.—Strieker, S.—A manual of histology. American translation 
edited by A. H. Buck. Pp. 1106, illustrated. New York, 1872. Struct- 
ure and methods. 
Suffolk, IV. T.—On microscopical manipulation. 2d edition, pp. 
227, illustrated. London, 1875. 
Taylor, A. S.—A manual of medical jurisprudence, 8th ed. Pp. 
937, illustrated. Pliila., 1883. 
Taylor, A. S.—Principles and practice of medical jurisprudence, 
2d ed. Two vols. Pp. 723 + 672, illustrated. London and Phila., 1873. 
Todd, R. E.—Cyclopaedia of Anatomy and Physiology, 5 vols., il- 
lustrated. London, 1835-1859. Structure. 
Tr.—Trelease, Wm. (Ed.)—Poulsen’s botanical micro-chemistry, an 
introduction to the study of vegetable histology. Pp. 118. Boston, 
1884. Methods. 
Tyson, J.—Cell doctrine ; its history and present state, with a copi- 
ous bibliography of the subject; 2d ed., illustrated. Philadelphia, 
1881. 
Tyson, J.—A guide to the practical examination of the urine, 4th 
edition, pp. 196. Phila. 1883. 
Virchow, R.—Cellular pathology as based upon physiological and 
pathological histology. Twenty lectures delivered before the Patho- 
logical Institute of Berlin, 1858. Translated from the German by F. 
Chance ; pp. 554, 144 fig. New York, 1866. Structure. 
W.—Whitman, C. O.—Methods of research in microscopic anato- 
my and embryology. Pp. 255, illustrated. Boston, 1885. 
W. and G.— Wilder and Gage.—Anatomical technology as applied 
to the domestic cat; an introduction to human, veterinary, and com- 
parative anatomy. 2d edition. Pp. 575, 130 fig., 4 plates. New York 
and Chicago, 1886. 
Wood, J. G.—Common objects for the microscope. Pp. 132. Lon- 
don, no date. Upwards of 400 figures of pretty objects for the micro- 
scope, also brief descriptions and directions for preparation. 
Woodhead, G. S.—Practical Pathology. Pp. 484, illustrated. Edin- 
burgh, 1883. Structure and methods. 
Wormley T. G.—The micro-chemistry of poisons. 2d ed., pp. 742, 
illustrated. Phila., 1885. 
Wythe, J. H.—The microscopist, a manual of microscopy and a 
compendium of microscopical science, 4th ed., pp. 434, 252 fig. Phil- 
adelphia, 1880. 
Ziegler, is.—General and special pathological anatomy, translated 
by D. Macallister: 2 vol., pp. 360+371, illustrated. Methods in ap- 
pendix separately printed. 
Information relating to the microscope, histology, pathology, etc., 
may also be found in the library or laboratory, in : The Encyclopaedia 
Britannica, Appleton’s Cyclopaedia and the yearly supplements ; John- 
ston’s and the Penny Cyclopaedia ; Ashhurst’s International Cyclopae- 
dia of Surgery ; Quain’s dictionary of medicine ; Littre’s dictionnaire 
de medecine, etc. ; Reynold’s, and Flint’s systems of medicine ; Watt’s Chemical Dictionary ; Wurtz’s Dictionnaire de Chemie ; Coat’s man- 
ual of pathology ; Mittheilungen aus dem Kaiserlichen Gesundheit. 
samte, 1884 : Reports of the U. S. bureau of animal industry. The 
publications of the Smithsonian Institution ; Publications of the mu- 
seum of comparative zoology. 
Human Anatomy : Allen, Gray,Henle, Hoffmann, Sappey, Gegen- 
baur, etc. In Physiology: Carpenter, Dalton, Flint, Foster, Martin, 
Yeo, Briicke, Hermann, etc. In Comparative Anatomy: Huxley, 
Owen, Gegenbaur, Bronn’s Thier-reich, Brass, Wiedersheim. In 
Zoology: Brooks and Claus. In Physiological, pathological and 
Medical Chemistry; Hoffman’s Zoocliemie; Hoppe-Seyler’s Physio- 
logische Chemie ; Naquet’s legal chemistry ; Neubauer and Vogel’s 
analysis of the Urine ; Ralfe’s physiological and pathological chemis- 
try ; Vaughn’s handbook of clinical physiology and pathology ; 
Wheeler’s medical chemistry; Blytli, Foods, their composition, an- 
alysis, adulteration, etc. In Geology: Credner’s Elemente der Geolo- 
gie; Geike’s text-book of geology; Zirkel’s microscopical petrography; 
Teall’s British petrography. 
PERIODICALS * 
A. J. m. s.—The American Journal of microscopy and popular 
science. New York, 1876-1881. Illustrated. Methods and structure. 
A. m. m. j.—The American monthly microscopical Journal. Illus- 
trated. 1880+ . Methods, and structure. 
A. n.—American naturalist. Illustrated. Salem and Pliila. 1867 + . 
Methods and structure. 
A. q. m j.—American quarterly microscopical journal, containing 
the transactions of the New York microscopical society. Illustrated. 
New York, 1878. Structure and methods. 
A. s. m.—American society of microscopists. Proceedings. Illus- 
trated. Methods and structure. 
A. s. n.—Anuales des sciences naturelles. Illustrated. Paris, 
1824 + . Structure. 
Ar. a. e.—Archiv. fur Anatomieund Entwickelungsgeschichte. Il- 
lustrated. 1877+ (1795 + ) Structure. 
Ar. b.—Archives de biologie. Illustrated. Paris, 1880+. Structure. 
Ar. i. b.—Archives italienues de biologie. Illustrated. Turin, 1882 + . 
Structure. 
Ar. m. a.—Archiv fur mikroscopische Anatomie. Illustrated. Bonn, 
1865 + . Structure and methods. 
Ar. p. a.—Archiv fiir pathologische Anatomie und Physiologie uud 
fur klinische Medicin. Berlin, 1847 + . Structure. 
*NoTE.—When a periodical is no longer published, the dates of the 
first and last volumes are given ; but if still being published, the date 
of the first volume is followed by a plus sign. Ar. z.—Archives de zoologie experimentale et geuerale. Illustrated. 
Paris, 1883 + . Structure. 
I. m.—Index Medicus. New York, 1879 + . Bibliography, including 
histology and microscopy. 
J. a. p.—Journal of anatomy and physiology. Illustrated. Lon- 
don and Cambridge, 1867 + . Structure and methods. 
J. d. a. p.—Journal de l’Anatomie et de la physiologie normales et 
pathologiques de l’homme et des animaux. Illustrated. Paris, 1864+. 
J. d. m.—Journal de micrographie. Illustrated. Paris, 1877 + . 
Methods, and structure. 
J. n-y. m. s.—Journal of the New York microscopical society. Il- 
lustrated. New York, 1885 + . Methods and structure. 
Jr. a. p.—Jahresberichte iiber die fortschritte derAnatomie und Phy- 
siologie. Leipzig, 1873 + . Bibliography and brief summaries. 
J. r. m. s.—Journal of the royal microscopical society. Illustrated. 
London, 1878 + . Bibliography of works and papers relating to the 
microscope, microscopical methods and histology. It also includes a 
summary of many of the papers. 
J. z. n.—Jenaische Zeitschrift fur Naturwissenschaft. Illustrated. 
Leipzig, 1864+. 
L. —The Lens, a quarterly journal of microscopy and the allied 
natural sciences, with the transactions of the state microscopical so- 
ciety of Illinois. Illustrated. Chicago, 1872-1873. Methods and 
structure. 
M. —The microscope. Illustrated. 1881+ . Methods and structure. 
M. z. s. n.—Mittheilungen aus der zoologischen Station zu Neapel, 
zugleicli ein Repertorium fiir Mittelmeerkunde. Illustrated. Leip- 
zig, 1878 + . Methods and structure. 
M. —Morphologisches Jahrbuch. Bine Zeitschrift fiir Anatomie und 
Entwicklungsgecliichte. Illustrated. Leipzig, 1875 + . 
N. —Nature. Illustrated. London, 1869 + . Structure. 
P. B. s. n. h.—Proceedings of the Boston society of natural history. 
Illustrated. Boston, 1841 + . 
P. r. s.—Proceedings of the royal society. London, 1854.+. 
P. T.—Philosophical Transactions of the royal society of London. 
Illustrated. London, 1665 +. 
Q. j. m. s.—Quarterly Journal of microscopical science. Illustra- 
ted. London, 1853 + . Structure and methods. 
R . z. s.—Recueil zoologique suisse. Illustrated. Geneve, 1884+. 
Structure. 
S.—Science. Illustrated. Cambridge and New York, 1883 + . 
Structure. 
S. r.—Science Record. Boston, 1883-4. Methods and structure. 
S. b. 1.—Studies from the biological laboratory of Johns Hop- 
kins University. Baltimore, 1879 + . Illustrated. Structure and 
methods. 
Z. a.—Zoologischer Anzeiger. Leipzig, 1878 + . A weekly bibliog- 
raphy of current zoological literature, including animal histology. 
Z. b.—Zeitschrift fiir Biologie. Illustrated. Miinchen, 1865 + . 
Structure. 56 
Z. w. m.—Zeitschrift fur wissenschaftliche Mikroskopie und fiir 
mikroskopische Technik. Illustrated. Braunsch. 1885 + . Methods 
and bibliography of same. 
Z. w. z.—Zeitschrift fiir wissenschaftliche Zoologie. Illustrated. 
Leipzig, 1849+. Structure. 
Besides the list of periodicals just given, the library contains the 
following in which papers relating to histology and the microscope 
are sometimes found :— 
American academy of arts and sciences, Memoirs and proceedings ; 
American association for the advancement of science, Proceedings; 
American journal of neurology and psychiatry ; American journal of 
science; American philosophical society, Proceedings; Annals and 
magazine of natural history ; Archiv fiir die gesammte, Physiologie ; 
Archiv fur Psychiatrie und Nervenkrankheiten ; Archives de physi- 
ologie normale et pathologique; Biologisches Centralblatt; Brain ; 
Comptes Rendus de l’acad£mie des sciences; Journal of Physi- 
ology; Journal of Science; Linnaean soc., transactions; London 
Edin. and Dublin Philos. Mag. ; Neurologisches Centralblatt; Phil- 
adelphia academy of natural sciences, Journal and proceedings ; Pop- 
ular science monthly; Revue scientifique; Royal Irish academy, 
Transactions; Royal soc. of Edinburgh, Transactions ; Zoological 
Record ; Zoological society of London, Proceedings and transactions. 
Zeitschrift fiir Instrumentenkunde. Pringsheim’s Jahrbiicher fur 
wissenschaftliche Botanik ; Just’s botanischer Jaliresberichte; Bulletin 
of the Torrey botanical club ; Botanical gazette ; Botanische Zeitung ; 
Scientific American ; Bulletin de l’academie imp6riale des sciences de 
St. Petersbourg; Zeitschrift fiir physiologische Chemie ; Archiv fiir 
physiologie. X^GEO/T 
Corrections. 
P. 3, 8th line from the top, for “ three ways of varyi ug the magnifica- 
tion of,” read : three methods of micrometry wrttrr-- 
P. 3, last line, for “ I-VIII,” read : 49-56. 
P. 6, g 8, 6tli line, for “ floculent matrials,” read : flocculent materials. 
9th line, same correction. 
P. 9, 9th line from the top, for “ matrial,” read : material. 
P. 14, g 23, 3d line, for “moke,” read : make ; g 24, for “mamalian,” 
read : mammalian. 
P. 17, g 27, 20th line, for 11 tliorougness,” read: thoroughness; 31st 
line, for “ fluoriscine,” read: fluorescine. 
P. 18, g 28, 15th line, for “fill,” read : fill. 
P. 20, g 32, 13th line, for “ connula,” read : cannula. 
P. 25, for “ g 41,” read: g 41-42. 
P. 27, g 47, 6th line for “fluoriscine,” read : fluorescine. 
P. 28, g 50, 4th line, for “ transactions,” read : transectious. 
P. 31, 2d line of *, for “ quite as celloidin,” read : quite as good as 
celloidin ; 3d line from the bottom, for “tranferred,” read: 
transferred. 
P. 35, 25th line, for “mesentric,” read : mesenteric. 
P. 36, g 64, 7th line, for “witth,” read : with. 
P. 38, g 69 (B), 2d line, for “Kleinberg’s,” read : Kleineuberg’s. 
P. 40, (c), for “ C. 95, 97, 184, 7x3,” read : C. 111, 207, 786. 
P. 41, g 71, 1st line, for “ microscopial,” read : microscopical. 
P. 42, g 77, 8th line, for “mucuous,” read : mucous. 
P. 43, g 83, 6th line, for “ galatin,” read : gelatin. 
P. 45, g 101, 7th line, for “ihe,” read : the.