Microscopical Diagnosis 
of Tuberculosis. 
Late Professor of Microscopy, Bacteriology, etc., and Director Laboratory of Pathology, Medical lW>artment, 
Missouri University; Director Laboratory of Hygiene, Battle Creek, Mich, AtMncan 
Microscopical Society, American Medical Association, American'•^jbli^*^ 
Health Association ; Editor “ Bacteriological World.” 
By PAUL PAQUIN, M. D., 
PUBLISHED BY 
LITTLE BLUE BOOK CO.. 
Battle Creek, Mich. Copyrighted, 1891, 
BY 
PAUL PAQUIN. TABLE OF CONTENTS. 
I. PAGE. 
Introduction ..... 5 
II. 
The Microscope and Other Instruments 7 
III. 
Principle, Object, and Effect of Staining 15 
IV. 
Mounting — General Idea . . . 17 
V. 
Collecting Sputum, Etc. . . .21 
VI. 
Staining Fluids, and Their Use . . 23 
VII. PAGE. 
Modus Operandi . . . . .27 
VIII. 
Staining Bacilli of Tuberculosis in Milk, 
Pus, Articular Secretions, Etc. . 83 
IX. 
How and What to See through a Micro- 
scope ...... 35 
X. 
The Bacilli of Tuberculosis . . 40 
XI. 
Remember ...... 43 
XII. 
Do n’ts ...... 46 BACILLI OF TUBERCULOSIS 
In sputum liquified with a solution of bi-borate of soda; stained with the author’s 
solutions No. 4, i. e. A and B of No. 3, modified, and C, as in No. 3. 
Appearance with objectives ranging' from J to J and with 
low-power eye-pieces. 
a, Foreign Bacilli — notoi Tuberculosis, stained green with background; b, Blackish 
Bodies, probably Cocci decolorized; they are not uncommon 
in sputum mounts. 
NOTE.—The color of bacilli may vary from pale red to a very dark shade,—almost purple. 
PLATE I, Microscopical Diagnosis of Tuberculosis. 
I.— INTRODUCTION. 
DURING the past six or seven years, the writer has been engaged very 
frequently in analyzing material of various kinds for bacillus tuberculosis. 
During his service at the University of Missouri, and since, in his new field of 
labor at the Laboratory of Hygiene of Battle Creek,1 he has had numerous 
opportunities to test the many methods of microscopical diagnosis of tuber- 
culosis, and to try new ones. His experience has also served in bringing 
forcibly to his mind the fact that most physicians and medical students suffer 
unnecessarily the loss of many of the unmistakable advantages which a micro- 
scope affords for diagnosis. They labor under the misapprehension that only 
experts can use this instrument with reliability, and with safety to the patients 
and practitioners. As a matter of fact, many cases of severe disease could be 
early diagnosed, and many lives saved, by any physician of earnestness, 
1 Battle Creek Sanitarium. MICROSCOPICAL DIAGNOSIS. 
who would use judgment, and would labor as earnestly for a few minutes with 
a microscope as he does in searching his text-books, or in attempts at physical 
diagnosis. 
And in no disease is this more true than in tuberculosis. Every doctor 
can learn to use the microscope with as positive results as this instrument can 
afford, and every practitioner owes it to his patients to take no chances with 
their lives ; to base a diagnosis only upon evidence, not upon opinions or 
guess-work. Indeed, in these days of progress and discoveries, a doctor 
who still guesses at the diagnosis or prognosis of a suspected consumptive, is 
doing the latter a heartless wrong, and is, in my judgment, guilty of malprac- 
tice. And yet we see this every day. 
Whatever relation one may think the bacillus of tuberculosis has to the 
fearful affliction of this name (consumption), one thing is certain : the bacillus 
is never found in any organ except in connection with this malady. There- 
fore, when it is present, it always indicates tuberculosis — danger ! 
In Plates I. and II., here presented, wrill be seen the appearance of the 
bacilli of tuberculosis when stained by the writer's method, which will be 
described farther on in these pages. Plate III. is very highly magnified, and 
is a rare appearance. BACILLI OF TUBERCULOSIS IN SPUTUM A FEW DAYS OLD 
As they appear under a 1-15 or 1-18 objective. 
The specimen had not been treated by any means to destroy the pus cells and mucus. The 
microbes are stained with the author’s solutions A and B, and the field (including 
pus cells and mucus) with C. 
«, Rod-Shape Bacillus; b, Pus Corpuscle; c, probably a Spore; d, a Bacillus, showing- dots 
which are probably Spores; e, Strings and Film of Mucus. 
PLATE II, LONGER BACILLI OF TUBERCULOSIS IN SPUTUM. 
(Largely magnified.) 
Borrowed from Cornil & Babes, “ Les Bacteries.” 
«, Isolated Bacillus; b, Bacilli in Epithelial Cell; c, Bacilli in a Pigmented Cell. 
(Uncommon appearances.) 
PLATE III, II.—THE MICROSCOPE AND OTHER INSTRUMENTS. 
FEW instruments, besides the microscope, are essential for the microscopical 
diagnosis of tuberculosis. Indeed, one may do the work with it and the 
few essential cover-glasses and glass slips. But a pair of small forceps and 
a needle — say a cambric needle fixed in a pen handle — are very useful, and 
scarcely to be dispensed with ; and something to rinse with, such as a wash- 
bottle or medicine dropper, is necessary. One also needs stains and reagents. 
It is not necessary to possess a costly microscope, but it is desirable to have 
a good solid stand, and a clear objective. 
The author could not do as well in describing the parts of a microscope as 
Dr. Whelpley in the following, quoted from Popular Science News} As this 
was written for pharmacists, only the portions essential to our object are 
quoted, and some other word is substituted for the word “pharmacist : ” — 
“ In selecting an instrument, the first thing for consideration is the stand. 
With a good stand for work, one is ready to add and make use of such quality 
1 Dr. H. M. Whelpley, F. R. M. S., a well-known scientist and microscopist of St. Louis, Mo. 8 
A/ICR OSCOPICAL DIA GNOSIS 
of optical parts as liis requirements may suggest and his means justify ; but 
with a poor stand, the possessor is always at a disadvantage, even with the 
best of optical parts. 
“The stand maybe defined as a com- 
pound microscope without optical parts. 
Since the optical parts are the only essen- 
tial portion of a microscope, and we can 
do no work whatever without them, this 
may seem like defining a gun as the por- 
tion of that firearm without lock, stock, 
or barrel; but such is not the case. The 
stand is devised solely for the purpose of 
using the optical parts to the best advan- 
tage. Therefore any one can readily see 
that the more perfect the stand, the 
greater the amount of work to be ac- 
complished with the complete instrument, 
and the more perfect and satisfactory it will be. In considering the various 
parts of the stand, the accompanying illustration will serve to make them clear. OF TUBERCULOSIS. 
9 
“The base, .or lower portion of the stand, first attracts our attention. In- 
struments can be found with bases of almost all conceivable forms. . . . 
“The feet forming the tripod may be disguised in the form of the base ; 
but that does not matter, so .long as there are three, and only three, points 
of support. This not only gives the greatest stability to the instrument, with 
the least tendency to vibrate when the table is jarred, but also has other 
advantages. . . . 
“Thopillar, or support, requires no special attention. With some instru- 
ments it is single, while others have a double support, so that the mirror bar 
swings between them. . . . 
‘‘ The joint seen at the upper end of the pillar is a feature of more impor- 
tance, and every one should see that his instrument can be inclined to any 
desired angle.1 
“The arm is not very prominent in our illustration. It is the portion 
above the joint, and bears the body. In some instruments it is prominent, 
and closely resembles in form the flexed human arm. If it supports the 
body firmly, that is all that is required. 
“The body is supported by the arm, and has attached directly to it the 
optical parts of the compound microscope. The body varies in size and length 
1A stiff microscope may do good work, but it is not so convenient. — P. 10 
MICROSCOPICAL DIAGNOSIS. 
in different instruments. The size is not of very much importance, but in 
length it should be what is known as ‘standard,’ or be so arranged that it 
can be lengthened out.1 . . . 
“The draw-tube is found only in the better class of instruments, and is a 
provision for adjusting the length of the body. With very higli-power object- 
ives it is very essential to have a draw-tube. It should be marked to indicate 
when the body is ‘ standard’ length. A great convenience is a ‘ society screw ’ 
in the lower end of the draw-tube.2 
“The collar is the ornamental ring, or projection, at the upper end of the 
draw-tube, or of the body when there is no draw-tube. 
“The nose-piece is the portion at the lower end of the body. It is provided 
with a female screw, into which the objective is fastened. By all means pur- 
chase a microscope with what is known as the ‘society screw,’ so that any 
ordinary objectives can be fitted to it. I have found that the English thread 
in the ‘ society screw ’ is not quite the same as the American, and I was 
obliged to get an adapter for the use of English objectives on an American 
stand. 
1 This is not an absolute necessity for our purpose, but is very useful.— P. 
2 To admit various objectives of different makes. OF TUBERCULOSIS. 
11 
“The stage is of importance. . . . It is an item to have a thin stage which 
admits of oblique illumination in the examination. The expensive mechanical 
stages are very convenient, but not essential. 
“The sub-stage must be so arranged that it will admit of the use of sub- 
stage condenser, polariscope, etc., if one expects to do much fine1 work. 
“ The diaphragm is a contrivance for regulating the volume of light which 
is admitted to the object. When a sub-stage is present, the diaphragm is 
adjusted to it; otherwise it is attached to the stage in place of the sub-stage. 
“The mirror bar and its arrangement is plainly shown in the illus- 
tration. . . . 
“The mirror, if single, must be a concave one. Where there are two, one 
is plain and the other concave. As far as the plain mirror is concerned, the 
size does not make much difference, but the larger the concave mirror the 
better.2 
“The clips are for holding the slide in position. 
“The coarse adjustment is found on all instruments. The rack and pinion 
arrangement is the best, and is the one used on the better class of instruments. 
1 The italics are the writer’s. —P. 
2 However, some microscopes with very small mirrors do very fine work.— P. 12 
MICROSCOPICAL DIAGNOSIS 
“The fine adjustment, or micrometer screw, is also a feature of the better 
instruments, and should be present on every one owned by any one who 
intends to do much work. It is much more convenient to have the tine adjust- 
ment near the coarse one.” 
To this description of Prof. Whelpley’s should be added the optical parts, 
that is, the eye-piece, or ocular, and the objective. The latter is the most im- 
portant part of the microscope. It constitutes the lens or series of lenses 
attached to the nose-piece, next to the object to be examined, by which the 
microscopic image is formed. The ocular is the lens or series of lenses next 
to the eye, which magnify the image formed by the objective, as a hand lens 
magnifies an image visible to the naked eye. 
The cheapest of the outfits generally used in our laboratory for the diag- 
nosis of tuberculosis, represents about the following : — 
Microscope stand, objectives, and eye-pieces $45 00 
Pincers 50 
Test-tubes and watch-glasses 60 
$46 10 
Stains and reagents, such as aniline oil, fuchsin, aniline blue or green, 
alcohol, nitric acid, acetic acid, carbolic acid, liquor ammonia, etc., etc., are OF TUBERCULOSIS. 
13 
needed according to the methods of staining adopted. These cost a few dol- 
lars. But one may purchase stains and reagents already prepared, and avoid 
the greater cost, and the trouble, and the failures attendant in case of attempts 
to prepare one’s chemicals without sufficient experience and without proper 
utensils, such as accurate scales, liquid measures, etc. For two or three dol- 
lars, one may purchase enough prepared mounting material, including glass 
slips, cover-glasses, and Canada balsam, to last a year in ordinary practice, 
while five to ten dollars would scarcely cover the cost of a first attempt to pre- 
pare everything one’s self. Furthermore, well-prepared mounting fluids, etc., 
from reliable houses, are usually made from chemically pure drugs, under the 
guidance of experts. 
A dollar Bunsen burner and two feet of rubber tubing may be added to 
this cost by the physician who may desire to use gas. An alcohol lamp, or 
even an oil lamp, will answer the purpose. Not much need be spent for a 
washing apparatus. If neither a medicine dropper nor the stream from a faucet 
or a dropper be desired, it is easy to improvise a wash-bottle. Every one remem- 
bers preparing these as a student, by fitting to a wide-mouthed bottle a cork with 
two holes holding a glass tube each,— one going to the bottom, and the external 
extremity bending outside at a sharp angle from the mouth of the bottle in 14 
MICROSCOPICAL DIAGNOSIS. 
a downward course; the other penetrating the cork only to the inside of the 
bottle, and the outward end projecting upward at a very slight angle. The 
former tube is to throw a jet of water out of the bottle when the mouth blows 
into the latter. This is used to wash the stain off the cover-glasses in mount- 
ing. A medicine dropper is slower, but works fairly well. Water may be 
used directly from a faucet allowed to run smoothly and thinly, but it is apt 
to break the glass if the force is great. 
A far better outfit, including a Lietz microscope, for instance, with a No. 
7 eye-piece corresponding to our ■§■, or say including one of our popular 
American or English makes, can be purchased now-a-days for little more 
than the cost of the above — between $50 and $60. But $30 to $35 will 
purchase a microscope that will diagnose tuberculosis positively. 
Note.—Before purchasing a microscope and other equipments, write to optical and surgical 
instrument houses for catalogues, and examine them closely. I think all of them give a discount for 
cash, varying from 15 to 25 per cent. See the advertisements in the Bacteriological World and Modern 
Medicine. III.—PRINCIPLE, OBJECT, AND EFFECT OF STAINING. 
THE knowledge of the presence of the bacillus of tuberculosis in an organ, 
is a positive indication of the dangerous nature of the case presenting it ; 
consequently, any analysis that may reveal it is to be sought, and should by all 
means be sought by every conscientious practitioner. The microscope is the 
instrument necessary for this analysis. 
In examining suspected matter in search of this bacillus, one may find nu- 
merous and different germs, particularly if the substance comes from the lungs 
or throat, as sputum, or from an open wound, as in tubercular joints, or from 
the fceces, as in intestinal tuberculosis. It is not always possible, then, by a 
mere glance, no matter how searching, to tell which is the bacillus of consump- 
tion and which is not. So investigators set to work to find differentiating 
methods of examination. As a result, numerous processes of staining this par- 
ticular germ were promulgated. Some of them are very slow ; others more 
rapid ; others, again, occupying only a very few minutes. In Chapter YI. will 
be found the most reliable and the safest formulas in vogue. These stains, 16 
.MICROSCOPICAL DIA GNOSIS. 
if applied as directed, will be found to stain the bacillus of tuberculosis only or 
mainly, and to stain the other germs as the background, or leave them as glis- 
tening, or yellowish, or brownish, or blackish bodies and debris. 
Some staining processes are termed “ double-staining,” for the reason that 
they stain the germs one color and the field another ; such a one was used in 
the mounts reproduced in our plates in this book. Thus, the object of staining 
material to see if any bacilli of tuberculosis exist, is to reveal their appearance 
in a striking, unmistakable manner, in contrast with the decolorized germs of 
other character that may be, and usually are, present ; or in contrast, some- 
times, with a background, and with foreign germs stained differently. The 
true germs of consumption are set off more or less boldly, in a distinct tint 
of their own, according to the methods used. Most staining methods now used 
reveal the bacillus in a shade of red on a background of different color, green, 
blue, etc. The author’s process always stains the bacilli red, and the field 
and sometimes some other germs a fair green, so that a mistake is not 
possible. IV.—MOUNTING —GENERAL IDEA. 
TO study any fine organism with a magnifying glass, it is not only neces- 
sary to increase its appearance in size, as one does letters by reading 
through an ordinary lens, but it is also necessary that light be reflected through 
it or through its immediate surroundings. . This is accomplished by the direc- 
tion of the rays of sunlight or lamplight through the object to be examined, 
by a little mirror attached to the microscope ; so the object must be very thin. 
In searching for the bacillus of tuberculosis for diagnostic purposes, in 
98 per cent of cases the material is fluid or nearly so, such as sputum, which 
furnishes at least 95 per cent of all specimens analyzed.1 A particle of this is 
spread thinly on a clean, thin cover-glass,— the cover-glass known as number 
1 or 2,— allowed to dry, then stained, washed, decolorized, and washed again, 
and then pressed, smeared surface downward, on a drop of water in the center 
of a clean glass slip, and examined at once with a microscope. Or, after 
washing it, the thin cover-glass may be dried by pressing it between two sheets 
1 I do not intend to go into details of analyzing the bacilli in tissue. This requires more skill and 
knowledge of histology and pathology than could he given here. It is rarely needed for diagnosis of 
tuberculosis 18 
MICROSCOPICAL DIAGNOSIS 
of clean, smooth blotting-paper or rice-paper (cigarette wrapper), then pressed 
on a small drop of Canada balsam instead of water, in the center of a glass slip. 
In the latter case, the mount should be held with the pincers, and heated slowly 
by passing the slip, cover-glass upward, to and fro over the blaze of a gas-jet or 
an alcohol lamp, or over a lamp chimney, until the balsam makes little bubbles 
by cooking between the slip and the cover. Then press the cover on the slip 
by gentle pressure with the pincers. Such a mount, when cold, will be glued 
tight,— cover-glass to slip,— and will be permanent. A water mount is tem- 
porary, but it ma*y be made permanent (if found good on examination) by 
abstracting the water from it with absorbent paper in the manner just 
explained, and then mounting in balsam. 
If the material be smeared too thickly, the stain will be so dense that light 
will not penetrate it well, and the mount will usually be found unsatisfactory. 
A few trials will indicate the amount of material required. In analyzing 
sputum, a little particle that will adhere to the point of a needle spread very 
thinly and as evenly as possible, will usually be found sufficient. A good- 
sized cambric needle, fixed at the end of a penholder or something of the kind, 
is very handy in taking up particles of sputum or any other such substances, 
and spreading them on the cover-glasses. Some prefer a very fine, smooth OF TUBERCULOSIS. 
19 
scalpel or knife-blade. Others prefer a special sputum spreader. Others, 
again, press a particle of specimen between two cover-glasses, held between 
the thumb and finger, and thus spread it over the two surfaces brought in 
contact, pulling them apart, after gentle friction for a moment. It matters 
not how the spreading is done ; the thing is to spread a thin film on the cover- 
glass. In other words, in the general process of mounting to analyze fluid or 
semi-liquid substances for tuberculosis,— 
First, clean the cover-glasses thoroughly. If they are cloudy, leave them 
in alcohol for a day or two, then very carefully wipe dry with a clean old 
cotton or silk cloth, or a thin piece of chamois skin, and inclose them in a 
clean box to use when needed. They may, however, be kept constantly in 
alcohol, and wiped clean before using. 
Secondly, clean glass slips or slides likewise, and inclose them in a box, 
or wrap in paper to use when needed. 
Thirdly, when a specimen is on hand, smear a very thin layer over clean 
cover-glass, and let it dry face upward, and pass once or twice rapidly over a 
blaze to coagulate the matter. 
Fourthly, stain as per directions to be found elsewhere in this book. 
Fifthly, decolorize in order to get out all the excess of stain possible from 20 
MIC.R O SCO PIC A L D/A GNOSIS. 
the background, and even from the germs themselves, as is practicable by the 
use of special agents spoken of elsewhere ; also wash in water. 
Sixthly, if you wish (it is not indispensable), you may clarify the field by 
putting a drop of clove oil on the stained surface, after drying it well, and 
before mounting in balsam. 
Seventhly, mount on the glass slip in a small drop of Canada balsam. 
If a water mount is desired, put the cover-glass, after the fifth process, 
directly on a little drop of water on a glass slip, and examine. V.—COLLECTING SPUTUM, ETC. 
DR. SEVELIEFF recommends mixing the fresh sputum with 95 per cent of 
alcohol in order to preserve it. He suggests that expectoration be made 
directly into the alcohol. This, in fact, does preserve specimens, but it coagu- 
lates the albumen,— a condition which necessitates dissolution, with a two per 
cent solution of caustic potash, before spreading over a cover-glass for the pur- 
pose of staining and mounting. The author prefers to mix the sputum with 
a saturated solution of borax, which is recommended by Kuhne to liquefy the 
mucus before examination. A small, wide-mouthed bottle — say a quinine 
vial — is cleansed, filled about one third or one fourth with the solution, and 
well corked. It is then left with the patient with the request to expectorate 
directly into the vial, the first and second expectorations in the morning. The 
bottles distributed among one’s patients ought to be labeled, numbered, and 
recorded in a book ; and when the analysis is made, the results may be written 
opposite the right name. One could thus watch the weekly increase or 
decrease of bacilli under a particular mode of treatment. 22 
MICROSCOPICAL DIAGNOSIS. 
The method of collecting sputum in paper boxes, envelopes, pieces of 
paper, and mailing the same to experts for examination, is a dangerous prac- 
tice, which no conscientious physician should be guilty of. The writer has 
received such samples ; oh being opened, the dried sputum broke or crumbled, 
and flew in a powder sometimes, thus endangering all in the immediate neigh- 
borhood. 
The remainder of specimens which have been sufficiently analyzed, should 
be burned, or immersed in a solution of corrosive sublimate 1-1,000 for a 
few hours. 
Other tuberculous matters may be collected the same as sputum, but chiefly 
in alcohol, and should be treated accordingly before examining. VI.—STAINING FLUIDS, AND THEIR USE. 
IN order to appreciate and differentiate the bacilli of tuberculosis, various 
staining solutions and methods of staining have been recommended. Unfor- 
tunately, most of them are better for experts than for busy practitioners. 
No. 1. — Koch’s Original Stain. 
Concentrated alcoholic solution of methylene-blue, 1 part ; 10 per cent potash sol., 
2 parts ; distilled water, 200 parts. 
Float on, or immerse the preparation in, this stain, cold, 2d hours, or 
1 hour at 104° F. (40° C.) Rinse in water; immerse in a watery solution of 
vesuvin for two minutes ; rinse again in water, put on glass slip (slide), and 
examine. Or, treat with alcohol and clove oil, and mount in Canada balsam, as 
indicated elsewhere. 
No. 2. — Gibbes’s Double Stain. 
Rosaniline hydrochlorate, 2 parts ; methylene-blue, 1 part; triturate in a glass mortar. 
Dissolve 3 parts aniline oil in 15 parts rectified spirit, and add slowly to the above. 
Lastly, slowly add 15 parts distilled water. Keep in stoppered bottle. 24 
MICROSCOPICAL DIAGNOSIS 
Heat a little of the solution in a test-tube1 until the steam rises, pour into 
a watch-glass, drop the cover-glass preparation in it, and allow it to remain for 
five minutes. Wash in methylated spirit till no color comes away. Dry in the 
air or over a spirit-lamp, and mount in Canada balsam. 
No. 3. — Paquin’s Three Solutions. 
A. Fuchsin, 15 parts (or 15 grains); absolute alcohol, 12(J parts (or 2 drachms); water, 
distilled, 480 parts (or 1 ounce). Dissolve the fuchsin in the alcohol and add 
the water. 
B. Distilled water, 480 parts (or 1 ounce) ; liquor ammonia, 2 to 3 parts (or 2 to 3 
minims). Mix and shake well. 
C. Alcohol, 720 parts (or \\ ounces); water, 360 parts (or 6 drachms); nitric acid, 30 
parts (or £ drachm) , aniline, green, to saturation. Dissolve the green in the alco- 
hol, stir, add water, and then acid. 
These solutions, which have been evolved from several formulas and from 
personal experience, — principally the formula of Pittion and Roux,— though 
they consist of several ingredients, are more easy and simple to use than any 
1 To heat stain in a test-tube, pass the tube in and out, and revolve it in the blaze of a gas-jet or an 
alcohol lamp, or over a lamp chimney. It may be held with the fingers, near the mouth. OF TUBERCULOSIS. 
with which the writer is acquainted. Once made, which takes but a few min- 
utes,1 they fill every want in the staining process. No alcohol, no nitric acid, 
no ammonia, no other reagent of any kind is needed ; nor is one obliged to 
make up stains and counter-stains every time sputum is to be examined as in 
certain processes. It stains admirably. 
To use, it is only necessary to put about equal parts of solutions A 
and B (say a half teaspoonful of each) in a test-tube, heat it over a lamp 
till it boils or till vapor rises, empty in a watch-glass, drop the cover-glass 
preparation in it, and in five or six minutes — while the glass slip is 
being cleaned and prepared — it is sufficiently stained. Then rinse it in 
water, put a drop of solution C on it, rinse again ; put on another drop of C 
if the mount is not a distinct green ; wash again, and it is done. Put on a 
glass slip, and examine directly ; or mount in balsam. These stains have 
revealed bacilli to mq many times when all other methods had failed. They 
are very reliable, and usually give such a clear field that a £ objective, or a 
No. 7 of Leitz, is sufficient for diagnosis ; whereas, by the other methods 
such a low power is not always reliable. 
1 They may perhaps be purchased already prepared and tested more cheaply than one can make them. 26 
MICROSCOPICAL DIA GNOSIS. 
No. 4. — Paquin’s Two Solutions (a modification of No. 3). 
A and B of the above combined and modified thus : Fuchsin, 20 parts (or 20 grains) ; 
carbolic acid, 6 parts (or six minims); liquor ammonia, 2 parts (or 2 minims); 
glycerine, 15 parts (or 15 minims); alcohol, 120 parts (or 2 drachms); water, dis- 
tilled, 960 parts (or 2 ounces). Dissolve the fuchsin in the alcohol ; add the water 
and the other ingredients while stirring. To use, heat about a half teaspoonful in 
a test-tube until steam rises, put in a watch-glass, drop the preparation in it, and 
after five or ten minutes wash in water, decolorize, and color the field green in solu- 
tion C of No. 3, as explained on the preceding page. 
The following chapter gives the modus operandi of the writer in his daily 
examinations of sputum. It would take too much space to explain all the de- 
tails, but it really takes less than ten minutes to do the work. It can be ac- 
complished in five or six. VII.—MODUS OPERANDI. 
THE methods which the author has evolved out of several processes of 
staining the bacillus of tuberculosis for diagnostic purposes, give the 
greatest satisfaction. It is a fact that most all processes in vogue, though 
excellent when once well understood, and used by one who has had more or 
less experience, are very frequently disappointing to the beginner and the 
busy practitioner. Try as he may, he often finds no bacilli where an expert 
succeeds. Indeed, nearly all are better adapted for the trained microscopist 
or bacteriologist than for the busy man or student, because they usually 
give satisfactory results only on condition of tedious and exceedingly great 
care in details, and of knowledge of the minute but important changes in 
staining, decolorizing, etc. The following process avoids all these difficul- 
ties ; it stains the bacilli red and the field green readily, without any expert- 
ness ; in fact, one might say, despite carelessness (which, however, should be 
foreign to the character of a diagnostician). 28 
MICROSC O PIC A L DIA GNOSIS 
1. If the sputum is dry, wet it. Then if it is gluey, or thick, or stringy, 
— conditions which preclude the possibility of spreading a very thin layer of 
it on a cover-glass, — mix a portion or all the specimen with about equal parts 
of a saturated solution of bi-borate of soda (borax), and shake well to make 
a uniform semi-liquid mass. This can be done in a test-tube (small preferred), 
or one may crush and mash a particle of sputum, and mix it well enough 
in a watch-glass, by the aid of a glass rod or something of the kind. 
The use of this solution is always a wise precaution, to avoid errors of 
diagnosis, which are common with thick mounts. In case of nummular 
(cheesy) matter, it may be treated in like manner with a saturated solution of 
carbonate of ammonia. If the sputum is putrefied, it is liquid enough, and 
needs no such treatment, though, possibly, it may be unsatisfactory in other 
respects. If the specimen was preserved in alcohol, take a small lump of the 
clots formed, and liquefy it in a drop or two of a two per cent solution of 
caustic potash, either in a watch-glass or directly on a cover-glass. 
2. Hold cover-glass between the thumb and index-finger of the left hand, 
spread a thin layer of specimen over its surface in the manner indicated in a 
preceding chapter, take hold of the cover-glass with fine forceps or pincers in OF TUBERCULOSIS. 
29 
the right hand, and pass it rapidly over a blaze to and fro once or twice, to 
coagulate and dry the material on. 
3. Put in a test-tube about equal parts, say a half teaspoonful, of solutions 
A and B of stain No. 3, or only of A of No. 4, of the foregoing list of stains, 
and heat by moving it over the blaze of a gas-jet or alcohol lamp, or the 
mouth of a lamp chimney, until it boils, or vapors rise. Then empty the 
solution into a watch-glass, and drop the cover-glass, smeared face downward, 
in it, and leave it there at least five minutes. 
4. While it stains, clean a glass slip thoroughly, and holding it horizontally 
between the thumb and index-finger of the left hand, deposit a small drop of 
Canada balsam in the center, on one side, by pressing it out of the balsam tube 
gently with the right hand, and lay down the slip, gummed face downward, 
letting one end rest on something, so that the slip will make an inclined 
plane, and the balsam will not touch the table. By keeping this gummy surface 
under, one avoids the gathering of dust thereto. 
5. Now take the cover-glass with pincers from the stain, and wash under 
a stream of water (from wash-bottle, faucet, or something else) until the water 
falls free from red tint. Then drop on it (the preparation), with a medicine 30 
MICROSCOPICAL DIAGNOSIS 
dropper, sufficient of solution 0 to cover the smeared surface, hold a few 
seconds, wash again, and if the tint of mount is still reddish or violet, even in 
spots or streaks, put on more of solution C, and again wash under a light stream 
of water. Continue this operation until the water comes off clear, and the 
smeared surface is green. The thinner the preparation, the sooner this will be 
accomplished; the thicker it is, the greener and darker the background, and 
the harder to get a clear field. As a rule, two successive treatments with solu- 
tion C, and as many immediate washings, are sufficient. This need not take 
longer than a minute or two. 
6. Hold the cover-glass, edge downward, on absorbent paper to drain, 
then put it on a clean sheet of such paper (or cigarette-paper, say), stained face 
upward, and dry it with a fold of the same leaf, or with another, in the manner 
of using a blotter on an ink stain. After this, if the preparation is too dark 
and thick, it may be clarified by the addition of a drop of clove oil on its 
stained face, this being expunged afterward, as if it were water, by the use of 
bibulous paper. The use of clove oil is not very safe, as it sometimes fades 
the stain of bacilli a little, and may cause errors. Use it quickly. 
7. Now lay the cover-glass, stained surface upward, on a sheet of white 
paper (say a dry corner of the same bibulous paper used, if you please), take OF TUBERCULOSIS. 
31 
the glass slip (slide) containing the drop of Canada balsam, and apply this 
gum directly on the stained face, so that the smeared surface of the cover-glass 
will come in contact with the drop of balsam, in juxtaposition in the center of 
the slip, and turn it over so as to bring cover-glass upward.1 
8. Take the glass slip with pincers by one end, hold it with cover-glass 
upward, pass it to and fro, rapidly at first, and then slower and slower, over a 
blaze from gas or lamp, in order to melt down the thick balsam. Heat until it 
begins to boil, and little bubbles begin to raise the cover-glass from the slip. 
Then put the slide down (not on paint), cover-glass upward, and press the lat- 
ter down with the pincers. It is then ready for examination. Canada balsam 
thus cooked becomes hard immediately after cooling. 
9. If on examination it is found interesting or useful, it may be labeled 
and stored away as part of a collection. 
It will be observed by some, that several of the details of this technique 
1 In case only a temporary mount is wanted, No. 7 is omitted, and the cover-glass is put, smeared 
face down, directly on a drop of water in the center of a glass slip, and examined at once. In case clove 
oil is used, one cannot thus mount in water. If one forgets which side the cover-glass and the material 
is on, scratch both sides of a colored spot with the point of a needle or pin or penknife blade. This will 
make a streak on the right side, and nothing on the smooth side. 32 
MICROSCOPICAL DIAGNOSIS. 
are not strictly orthodox, that it is not in every respect just as many of us were 
taught. This is true ; but it has the advantage of being simple and accessible 
to all. It removes many of the usual dangers of dirt, and soiling with dust and 
various kinds of flying particles ; it stains rapidly, is reliable for diagnostic 
purposes, and finally it gives one a permanent mount within seven to ten 
minutes. 
However, the most reliable, striking, and beautiful mounts are obtained 
by leaving the preparation in the red stain about ten minutes. VIII.—STAINING BACILLI OF TUBERCULOSIS IN MILK, 
PUS, ARTICULAR SECRETIONS, ETC. 
MANY fail in this because of bad methods. A writer unknown to the 
author, suggested, in the Monitore dei Farmacisti, the saponification of 
the fat globules as follows : — 
“A drop of the milk is placed on a glass slip, and two or three times its 
volume of a one per cent solution of sodium carbonate is added, and the fluids 
mixed with the aid of a platinum wire. The slip is then cautiously held over 
the flame of an alcohol lamp, or over the chimney of a kerosene lamp, and the 
liquid slowly evaporated to dryness. During the evaporation, the butter parti- 
cles are saponified, leaving a thin layer of desiccated soap on the slip.” 
After this, stain by the method recommended in the preceding chapter for 
the staining of tuberculosis bacilli in sputum. 
None of the processes recommended to precipitate the casein in milk 
before staining and mounting, are satisfactory or reliable. As to bacilli in 
pus, sticky or viscous or thick secretions from articulations, etc., various 34 
MICROSCOPICAL DIAGNOSIS. 
methods should be resorted to before forming an opinion, when the trials are 
negative. One should treat these matters as sputum or milk, with alcohol, or 
a borax solution, or a caustic potash solution, or with bicarbonate of soda, 
using these ingredients in the proportions and manner indicated elsewhere. 
The final object is to dissolve or liquefy the substance, and free the germs, so 
that they will take stain and reveal themselves. 
Matter from an ulcer, such as scrapings from a suspected lupus, may be 
treated with a two per cent solution of caustic potash, and mashed thoroughly 
in it with a pestle in a small mortar, or with a glass rod in a watch-glass. The 
former is preferable. If one succeeds in getting a semi-liquid mass, a particle 
of it may be mounted as sputum. Bowel discharges need to be diluted, if too 
solid. The traces of mucus, or streaks of blood or pus, or spots containing 
viscous matter, should be chosen. If too thick, treat with some or other of the 
substances mentioned for the liquefaction of material, as explained for other 
substances, until the desired results are obtained, remembering that caustic 
potash liquefies coagulated albuminoids, and bi-borate of soda emulsifies, as it 
were, the fresh mucous matters. 
Thin fluids, as urine and pleuritic effusions, need no special preliminary 
treatment. IX. —HOW AND WHAT TO SEE THROUGH A 
MICROSCOPE.1 
EVERYTHING has a beginning, and so many students, and even physicians, 
have yet to take their first peep through a magnifying tube such as is used 
in medical practice. To these a word on the subject may not be amiss. 
In the first place, it is well to bear in mind that the higher the lens is 
capable of magnifying, the closer to the object it must be adjusted. So a lens 
known as a £ is a low power as compared with the lens known as a Tx¥, and the 
latter needs to be approached to within a much shorter distance of the object 
than the former. This is the focal distance ; and the longer it is, the easier it 
is to focus properly. So one must act cautiously, according to the power used ; 
for a careless or rapid screwing down or pushing of the tube to a focus, may 
strike the object and glass slip on which it is fixed, and possibly ruin it and 
the lens at the same time. 
We will suppose that the microscope represented by the accompanying cut 
is now before you. A mounted slip, stained, is placed on the stage (platform) 
1 With special reference to tuberculosis. 36 
MICROSCOPICAL DIAGNOSIS 
beneath the lower point of the tube (objective), one eye looks through from 
above the eye-piece, the mirror underneath is revolved until the light1 strikes 
the object fully, and the tube is slowly pushed down 
with the right hand, or screwed down, as it were, ac- 
cording to whether the microscope has a rack and 
pinion or not (this cut has them). At last the color of 
the field will appear. At this moment, stop lowering 
the tube with the coarse adjustment, and instead con- 
tinue the focusing by revolving the fine adjustment 
screw — the small one 2— now to the right, then to the 
left, if the vision indicates it, until a perfect outline of 
the object or objects sought is discernible. 
If one is looking for the bacillus of tuberculosis 
with any objective, many other things than this will 
appear first. Perhaps dirt or spots of stain on the 
outside of the cover-glass will first be seen. The layer spread on the glass, 
1 Bright daylight is the best. A northern window is preferable. Lamplight may be used to 
advantage, and will reveal fine bacilli better than indifferent daylight. 
3 It is underneath in this cut. OF TUBERCULOSIS. 
37 
no matter how thin, when measured microscopically, has a certain relative 
thickness, and the objects nearest to the lens will be revealed first ; if the tube 
be lowered a trace more, the things directly underneath the first ones seen will 
appear. So in moving the tube the hundredth or fiftieth part of an inch say, up 
and down, after the glass is focused, one may perceive different things lying 
superposed in the thickness of the layer of substance examined. Besides 
this the microscope will reveal coarse and comparatively large objects, such as 
specks of dirt for instance, which may appear like dirty rocks ; or a cotton 
thread, which may look like a broad, ragged tape, much sooner than the in- 
comparably smaller objects, such as the bacillus of tuberculosis. With a low 
power, one must look sharply to see these. 
Sometimes the outside surface of cover-glass is not clean, or is stained a 
little ; when the lens comes to it, the beginner stops ; he is disappointed to find 
only blurred, dirty spot crystals, cotton threads, or unstained or stained 
patches of dirt, and perhaps even germs (of the air or substance analyzed). 
These are outside the cover. Always be sure that the mount to be examined is 
first wiped with a clean cloth. If the dirt will not rub off, dip a kerchief in 
alcohol, and gently wash it from the cover-glass, at least. 
Having focused the lens properly, look sharply, coolly, intently, over 38 
MICROSCOPICAL DIAGNOSIS 
the field, then deliberately and slowly in each doubtful spot. If no bacilli are 
visible after a good survey of the field, move the slide very, very, VERY 
slightly with the left hand while focusing up and down with the fine screw or 
tube adjustment with the right.1 At first, the unsteadiness of the hand moving 
the slide, may jerk the field clear out of sight, and cause a feeling of nervous- 
ness, impatience, and even anger to arise in the heart. I have seen students 
utter imprecations at the unsteadiness of their own hand, when a beautiful field 
was suddenly removed from their gaze, not to be found again until after hours 
of search. But this only spoils the nerves, and makes matters worse. An 
hour of practice in moving a focused object under a microscope, while looking 
at it, will make one master of it sufficiently for practical purposes, and every 
examination adds to the skill in manipulations. A smeared cover-glass of 
sputum may be only a half inch in diameter; but when it is under the micro- 
scope, it is a vast field, and one should not weary or be at once discouraged 
if the object of search is not found. One must look carefully all over the field 
by moving the slide as methodically as possible, first one way, then another, 
with the fingers (or a mechanical stage such as some microscopes are provided 
with) until the whole is surveyed, if necessary. If one, two, or even three 
1 Moye the slide with the right if it is easier, and focus with the left. OF TUBERCULOSIS. 
39 
mounts of a given specimen fail to reveal the germ, it may be wise to make 
still another and another, and examine carefully. As a rule, in cases where 
one fails to find the bacillus the first time, another specimen is examined a day 
or two later ; and if this reveals nothing, examine another, and then another, 
at various intervals (of days or weeks), until the parasite is found, or the indi- 
cations point to its absence. 
The pus cells, mucous debris, dirt, cotton fibers from the cloth or kerchief 
used, blurred patches, etc,, need not be considered, except as a curiosity, to 
indicate improvement in mounting. Other germs should be left entirely un- 
noticed, at least until the particular one sought is discovered or believed to be 
absent. The knowledge of the presence of these other microbes, has no positive 
value, from a diagnostic point of view, in tuberculosis. X.—THE BACILLI OF TUBERCULOSIS. 
'T'HESE little rod-shaped parasites vary between 2 and 6 p1 in length, ac- 
1 cording to special conditions of their life and o fi 3 to o ft 5 in width. Their 
average length is about 3 /*, and the average width about They may 
be straight, or slightly curved, or irregular in their aspect. They appear 
either as homogeneous little rods, or as finely dotted bacilli ; these dots are 
due to the presence of fine ovoid or spherical bodies placed end to end like a 
very minute string of beads. These germs can scarcely be seen without color- 
ing reagents, except with liigh-power lens, and then the dots, or spherical 
bodies, which are looked upon as spores, do not appear. The organisms 
unstained, particularly if treated with potassa, have the appearance of hyaline, 
or motionless rods. 
The stained bacilli of tuberculosis can be appreciated when magnified 350 
to 400 diameters, but it is better to examine them under greater increase, say 
1 The [M (mikron), a unit in certain microscopic measurements, means micromillimeter, and is equal 
to one-thousandth of a millimeter. A millimeter is, as the word implies, the one-thousandth of a meter, 
and a meter is equal to 39.37 inches. A millimeter, then, is nearly 1-25 part of an inch and fj. (mikron) 
is the 1-1,000 part of 1-25 of an inch. MICROSCOPICAL DIAGNOSIS. 
41 
480, and it is still better to see them at 700 or even 800 if possible. This high 
increase is not necessary for diagnosis, if good stains are used. 
The bacilli may appear shorter or longer in the same specimen, according 
to their age. For instance, fresh sputum may at first show very fine short rods. 
Let it stand a day or two, and if it does not desiccate, the forms will be found 
more clearly dotted and longer. After standing for days or weeks, many fine, 
loose dots—spores doubtless — may be observed, and long bacilli formed by 
them, disposed end to end. In patients having large lung cavities, the bacilli 
are sometimes longer, and the loose spores more numerous, than in milder 
cases. 
The quantity of bacilli in sputum, and in all fluids, varies greatly ; it 
depends on the extent and age of the lesions. They are usually much more 
numerous in extensive cavities than in secretions from small lesions resulting 
from slight tubercles. A given mount may contain only a few — half a dozen 
or less, and the next, hundreds. 
Sometimes we find bacilli in a large cell, a giant cell, indicating an attempt, 
perhaps, on the part of nature, to destroy the germs by cellular digestion — 
phagocytosis. 
Usually specimens to be analyzed, particularly sputum, contain numerous 42 
MICROSCOPICAL DIAGNOSIS. 
other germs which have no direct relation to the disease so far as the cause is 
concerned, but are doubtless very harmful complications ; for they must be, as 
in wounds, dangerous and most potent factors in the destruction of tissue, in 
producing pus cavities, and in creating very damaging ptomaines. 
The good influence of high altitudes on consumptives, is due not a little to 
the fact, I imagine, that pus or decomposition germs are comparatively rare in 
high altitudes, and complications such as almost always exist in other atmos- 
pheric conditions in tubercular lesions opened to the air, find there no destruc- 
tive agent of the class of ptomaine makers and the like. These foreign germs, 
in making analysis, can be made to appear unstained at the side of stained 
tubercle bacilli, or may be stained as the background, and may therefore be 
readily distinguished. 
Tubercle bacilli may be found in sputum, milk, urine, tubercular abscess of 
the skin, joints, etc., and also in intestinal discharges. In cases of these dis- 
charges, one may discover the bacilli in analyzing the liquid of an evacuation 
obtained by a clyster, given a few minutes after a thorough washing of the 
intestines by careful but extensive enema. At other times it is necessary to 
analyze much of the faecal matters, and frequently to repeat the operation. 
Hundreds of other germs may be found in these matters. XI.—REMEMBER: 
THAT the presence of the bacillus of tuberculosis in any organ always indi- 
cates the presence of a field in which it grew — a lesion of some sort, 
considered justly a symptom of consumption, whatever name man may give 
to this disease ; but — 
That the absence of the bacillus from sputum, milk, urine, and faeces, is 
not always an indication of the absence of the disease, as it is necessary that a 
lesion be broken open, and that a secretion come from it, before it is possible 
that bacilli be expelled. 
That in the earliest stages of lung tuberculosis, cough and mucus from the 
bronchials, and even from points irritated by the presence and action of bacilli, 
may precede the expulsion of the latter by many days, weeks, and even months. 
That when coughing or other symptoms of pulmonary consumption are 
present, analysis of the sputum should be made daily, or at least two or three 
times a week, until a satisfactory diagnosis can be arrived at. 44 
MICE OSCOPICAL DIA GNOSIS 
That tuberculosis should be considered transmissible, and the specimens 
after testing should be burned, and never allowed to desiccate in a vessel or 
anything else. Nor should instruments used, be used again without steriliza- 
tion, i. e., cleaning by steam, or washing in boiling water, or heating over 
the blaze of an alcohol lamp or some other fire. 
That it is a grave responsibility that a physician has, in handling cases of 
consumption or suspects thereof, and microscopy is often the most reliable 
means of diagnosis. 
That every other means of diagnosis, such as family history, patient’s 
history, etc., should be considered in evidence, and the microscope used as 
an expert witness. 
That in doubtful cases, a few rabbits or guinea-pigs may be inoculated 
hypodermically, and in the peritoneum, with fresh sputum, and thus all 
doubts be removed by the production of the disease, or the reverse result. 
By inoculation of virus in this way, quantities of sputum thousands of times 
larger than can be seen under the microscope by a hundred analyses, may be 
used, and several bacilli or spores that the microscope might not have come 
across, be inoculated. OF TUBERCULOSIS. 
45 
That an expert may readily see the bacilli of tuberculosis in sputum with 
a objective, and that a beginner may fail to see them, even with a -fa. 
Therefore the eye should be trained by exercise. 
That a sub-stage condensor attached to a microscope is an admirable aid 
in concentrating the light to study fine objects, such as bacteria. 
That abscesses produced by inoculation of sputum, arising within a week 
or so, followed by more or less pus and perhaps suppuration, is not of itself 
an indication of tuberculosis. The characteristic tubercles must be found, and 
also the germs, to conclude affirmatively. 
That one may often mash and macerate a meaty tubercle (thereby avoid- 
ing sections), and by mounting the pulp as sputum, find the bacilli, even in 
cases artificially produced in animals. XII.—DO NTS. 
DON’T hasten in mounting. You will waste stain, break glass, stain badly, 
and perhaps injure some instrument. 
Don’t jump at conclusions in the negative or affirmative, when the revela- 
tions of the microscope are not clear cut and positive. 
Do n’t guess at a diagnosis, in a matter of such grave responsibility and 
great magnitude. It is wrong. A mistake may send a father, mother, or 
beloved child to the grave. 
Do n’t neglect or discard other means of diagnosis because you use a 
microscope. This instrument is an expert witness, whose affirmative evidence 
always indicates consumption, but whose failure to reveal the germ does not 
in all cases — though it does in the majority, after repeated tests — indicate 
the opposite. 
Do n’t forget that the determining cause of tuberculosis is doubtless the 
bacillus, but that bronchitis, La Grippe, so-called bad colds, etc., may prepare 
the field, or may be, in other words, the exciting cause. 
Do n’t accept, for the bacillus of tuberculosis, any germ having its appear- MICROSCOPICAL DIAGNOSIS. 
47 
ance, unless it clearly presents itself in the proper shade and form, as indi- 
cated by the particular staining method used. For instance, if the author’s 
process is used (see Nos. 3 and 4, staining methods), the background should be 
a good green, the bacilli red, and the other germs unstained or pale, or stained 
a faint green, or indistinctly blurred with shades bordering on these tints, such 
as purple. 
Do n’t forget that these staining processes are all differentiating in their 
effect, and should stain distinctly only the bacillus of tuberculosis. 
Do n’t forget, either, that a beginner may fail to get, at first, uniformly 
distinct shades between germs and background, because of insufficient decolor- 
izing or washing, or a too thick layer of material on the cover-glass. In such 
cases, the thinnest parts will have a fair appearance, but the thicker spots will 
appear somewhat blurred, violet or purple or dark reddish, and in them the 
differentiation of germs is not satisfactory. Such spots are unreliable points 
of study. 
Do n’t be puzzled when bacilli of tuberculosis in one case are very large 
and plain, and in the next so fine and thin as to be barely discernible with 
certainty ; such is often the case. It is only a difference in size, etc., due to a 
better or poorer soil. In this respect, it has valuable indications. 48 
MICE OSCOPICAL DIA GNOSIS. 
Do n’t dismiss the thought of tuberculosis when you find only red spots or 
specks on a different colored background,— say green,— for they may be 
groups of bacilli or spores. Search farther ; make more mounts. 
Don’t pay attention to bacilli stained the same color as the background ; 
they are not bacilli of tuberculosis. 
Don’t forget that spores in fresh sputum are exceedingly rare; in old 
sputum they may be very numerous. 
Don’t forget that one may search daily for months in the sputum of one 
individual, and find no bacilli of tuberculosis, and then suddenly find a perfect 
mass of them. This is due to a sudden breaking of a tubercle or several of 
them.1 
1 The writer once examined very closely, two or three times a week, during two months, the 
sputum of a patient, for bacilli, and failed to find any. Two weeks after he stopped, another person 
made a single examination, and found several. The author then made another examination, and also 
found several.