CORNELL UNIVERSITY

THE
Hlower Ueterinary Library
FOUNDED BY
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for the use of the

N. Y. STATE VETERINARY COLLEGE
1897

This Volume is the Gift of

Dr. V. A. Moore.

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LABORATORY GUIDE

FOR THE

BACTERLOLOGIsT

BY
LANGDON FROTHINGHAM, M.D. V.

ASSISTANT IN BACTERIOLOGY AND VETERINARY SCIENCE, SHEFFIELD
SCIENTIFIC SCHOOL, YALE UNIVERSITY.

ILLUSTRATED

PHILADELPHIA
W. B. SAUNDERS
925 Watnutr STREET..
1895.

CONTENTS.

PAGE
IPREPAGH «er Gee ck i edd Sit ue Bode Ros A Hoe ae we ak ve ce 5
1, BACTERIOLOGICAL TECHNIQUE ........-+..+..- Se etalk
2. Srarntne METHODS ... 1.2. eee eee eee 13

I. Staining Bacteria in Cover-glass Preparations with Watery

Solutions of Aniline Dyes... 2... 1... ee eee 13
II. Staining Bacteria in Sections with Watery Solutions of Ani-
Tine Dyes: si wt @, @ eS AO a ee ee ee ee le 17

III. Gram-Gunther Method of Staining Bacteria in Sections. . 19
IV. Gram Method of Staining Bacteria in Cover-glass Preparations 21
V. Ziehl-Gabbet Method of Staining Tubercle Bacilli in Cover-

glass Preparations... 1. ee eee ee ee 23
VI. Koch-Ehrlich Method of Staining Tubercle Bacilli in Cover-
glass Preparations. . © 6 6 ee ee eee ee te es 25 ©
VII. Koch-Ehrlich Method of Staining Tubercle Bacilli in Sections 27
VIII. Arens’ Method of Staining Tubercle Bacilli ........ 29
TX. Staining Tubercle Bacilli in Sections with Ziehl’s Carbol-
Fuchsin Solution... 1 6 6 ee ee ee ee ee es 31
Xx. Weigert’s Fibrin or Bacteria Stain in Sections ....... 33
XI. Staining the Glanders Bacillus... .-.. 1... eee 35
XII. Staining Spores, especially Anthrax Spores ........ 39
XIII. Moller’s Method of Staining Spores... ....-.-.-2.4- 41

XIV. Staining Sections with Bismarck-Brown, Loeffler’s Alkaline
Methylene-blue, or Watery Solutions of the Aniline Dyes . 43
XV. Double Staining of Sections with Hematoxylin and Picric

Acid: or Bosin® <c: ecg. ei ee eas res a 45
XVI. Typical Method of Staining Anthrax Bacilli from the Blood

in Cover-glass Preparations ...... 1... +e eee 49
8. PREPARATIONS OF Nutrient MEDIA... .... 22+ 5s 53

4, ImpeppinG TissvEs ror Curtine SECTIONS. ...-.-++- 59

PREFACE.

The following technical methods, arranged as simply
and concisely as possible, are especially intended for con-
venience in laboratory work. One constantly wishes and
uses the methods herein enumerated, and for the same
must refer to the large text-books. It is to avoid this
longer search, except for details, that the following scheme
is published—a scheme which the writer adopted some
years ago for his own convenience, and which he sincerely
hopes others may also find a useful desk companion.
Before describing the different methods, there are men-
tioned a few ways by which the work may be much
accelerated—ways probably known to and practised by
many, but not, perhaps, to all.

1. BACTERICLOGICAL TECHNIQUE.

Of paramount importance in bacteriological tech-
nique is rapid work associated with the best possible
results. In preparing, staining, and mounting cover-
glass preparations much valuable time is too often wasted,
and if this can be reduced to a minimum we are in many
ways the gainers. On account of this loss of time the
older method of making cover-glass preparations—by plac-
ing the material to be examined upon a cover-slip and
pressing another slip upon it, staining them in a watch-
glass with staining fluids made up when desired by adding
a few drops of a concentrated alcoholic solution of the
stain to be used to water or to aniline water, and mount-
ing at once in Canada balsam—has greatly fallen into
disuse.

By far the larger portion of cover-glass preparations
made, are prepared purely and simply for the purpose of
diagnosis; it is therefore ordinarily a useless waste of
cover-slips to make two preparations from the same ma-
terial, especially as such preparations are frequently much
too thick to be considered beautiful or even worthy of per-
manent mounting. Similarly it is evident that it is need-
less to mount at once in Canada balsam, for cover-glass
preparations are invariably thrown away after a short
microscopical examination, not because the preparations
are poor, but rather because they were made only for
diagnosis, which having been accomplished they are only
in the rarest cases of further use. Moreover, after mount-
ing in balsam one should properly wait until the balsam is
dry before examining in oil, or run the risk of making an
unsightly preparation; here again much time is lost.
Furthermore, the staining may be greatly accelerated. It
is seldom necessary to stain in the watch-glass or to make
up one’s staining fluids as required; the fluids for ordinary
staining should always be ready for use. The staining is
best and simplest performed upon the cover-glass, after
which the preparation may be mounted and at once ex-
amined in water. It is then a simple matter, if desired,

7

to remove the slip and to mount it permanently. It is
particularly in the line of work above noted that much
valuable time may be saved. The culture technique can-
not greatly be accelerated, excepting in a few instances.
The principal object, then, to be aimed at is to gain as
much time as possible, provided the best results are also
obtained. For the first purpose everything that is neces-
sary to work with must be within easy reach of the oper-
ator’s hands, and for ordinary work he should never be
obliged to leave his seat. The following description of his
desk may, therefore, not be inappropriate.
LABORATORY DESK.—It will be found an invalu-
able aid, in all microscopic work, to have the desk covered
with a large glass plate, perhaps 2 feet long by 17 inches
wide. It will be advantageous if the glass plate is let
into the woodwork of the desk. A working surface is
thus obtained which has many advantages, one of the
most important being its great cleanliness. This surface,
however, may vastly be improved if one-half of the glass
plate (from left to right) is painted jet-black, and the other
half pure white, the painted side, of course, being placed
downward. As it is sometimes difficult to obtain a pure
white paint a piece of white paper of suitable size may
be substituted. The advantage of a pure black-and-white
background are obvious to anyone who has done much
microscopic work. Unstained preparations, or sections,
for instance, in the watch-glass, or upon the slide, stand
out very distinctly upon the black side and stained ones
upon the white side; in the same way different colonies
upon the plate are seen to better advantage; the black
surface, again, is particularly useful in selecting the best
portions of tuberculous sputum for examination, etc. In
fact, it will be found exceedingly practical in innumerable
ways. ae
About 2 feet in front of the operator there should
be placed three vessels—tumblers, for instance. One of
these tumblers, containing a little cotton in the bot-
tom, is used principally for holding three platinum
needles, one straight, one with a large loop, and one with
a small loop—a hooked and a flat needle, which are often
useful, may be added if desired. This tumbler is a con-
venient place also to keep such articles as steel and fine
glass needles for handling sections, a pair of scissors,
forceps, etc. The other two tumblers are each filled
daily with fresh water, the use of which will be apparent

later.
8

e

On a line with the tumblers, or better still, on a small
slightly-raised shelf behind them, should be placed several
small bottles containing the fluids ordinarily used for
staining bacteria. There are now several bottles used
especially for this purpose, some being furnished with
pipettes of different forms, with other means for
transferring the stain to the cover-glass. Any small
(2-ounce), firmly-standing bottle, however, through the
cork of which passes a glass tube drawn out below, an-
swers every demand. For ordinary work there should
be five such bottles, each filled with one of the following
staining fluids: Methylene-blue, Gentian-violet, Fuchsin,
Ziehl’s carbol-fuchsin solution and Gabbet’s solution.
Although the latter often fails to stain well, it neverthe-
less is an excellent decolorizer, and in the hands of an
expert is admirable for making a rapid diagnosis of tuber-
culous sputum. To these stains may be added any other
desired stain, such as Bismarck-brown, Loeffler’s solution,
etc. The usual staining fluids (Methylene-blue, Gentian-
violet, Fuchsin) prepared as under I (foot-note 2 a) will not
only give excellent staining results, but will also keep in-
definitely.

Upon the desk there should also be a small bottle of
cedar-oil, from which a small drop may quickly be trans-
ferred.to the slide without necessitating the use of more
than one hand. Within easy reach may be hung a bunch
of filter-papers cut into strips, say 1.5 inches broad by 6
inches long. On a shelf above the desk may be placed

a large bottle of distilled water, from which a

rubber tube descends close to the hand; the tube

is closed near its lower end with a Mohr pinch-

cock (Fig. 1). This is an exceedingly convenient

arrangement also for the corrosive-sublimate

Fig.1. bottle. The desk is now in readiness, and should

always be so for ordinary work, which must necessarily

be much accelerated since everything that is desired is
within reach.

If there is any one thing the bacteriologist has to do
more than another it is perhaps to. make cover-glass prep-
arations, especially if he is working upon any special or-
ganism. Although it may seem a simple matter to make
such preparations, anyone who has had charge of students
knows some of the difficulties to be surmounted—that is,
if their results can be taken as criteria. Indeed, it is by
no means so seldom that men of experience have difficulty
in preparing what might be considered worthy specimens.

9

There will, therefore, be described rather minutely a well-
known, very simple, and rapid method of preparing, stain-
ing, and mount-

ing a cover-glass
preparation.
The well-cleaned

Fig. 2.—Cornet’s Double-spring Forcep. glass is held be-
tween the thumb and fore-finger of the left hand.
(A more convenient method is to use a Cornet
double-spring forcep (Fig. 2) especially made for hold-
ing the cover-glass while staining.) A small drop of water
from one of the tumblers is next placed in the centre of
the slip with the straight or small-looped needle. (This, of
course, is not always necessary, especially if the prepara-
tion is being made from a fluid.) The tube (agar we will
suppose), containing the culture it is desired to examine,
is held also in the left hand between the middle and “ring”
fingers, and a very minute portion of the culture is trans-
ferred to the drop of water with the sterilized straight
needle. Ifthe water becomes slightly turbid, or if a small
white speck is seen on the slip after touching the needle-
point to the drop, there is sufficient material and the
needle should again be sterilized. After cooling the needle
return it to the drop and spread the latter about upon the
slip. If the drop is small enough evaporation quickly takes
place during the spreading, but this may be hastened by
holding the glass high above the flame. When the evapo-
ration is complete the result is a scarcely visible, whitish
film upon the cover-glass. The slip is next grasped with
the forceps, held in the right hand, thoroughly dried high
above the flame, and then “fixed” as usual by passing the
slip three times through the flame. With the left hand
quickly lift a pipetteful of the desired stain and flood
the glass to its edges; return the pipette and put the cover-
glass for a moment in the lower part of the Bunsen flame
until a thin, white steam arises from the fluid; wash in one
of the above-mentioned tumblers of water, complete
washing in the second and at once invert upon the slide.
Pull a strip of filter-paper from the bunch, fold it from end
to end, and press it upon the cover-glass and slide, passing
the finger-tips over the paper several times. Superfluous
water is thus absorbed at once, excepting a very little
upon the surface of the cover-slip, which water may
quickly be removed with a corner of the folded paper,
resting the slide upon the index finger and holding the

slip in position with the thumb nail. Finally a drop of
10

cedar-oil completes the preparation, which is immediately
examined with the microscope. If the preparation has
been properly spread, nearly every field is good; there is
no massing of the organisms so as to make it almost im-
possible to study them carefully except in a few fields;
on the contrary, almost every organism stands out very
clearly. Such a preparation, including the cleaning of the
cover-glass, may be placed under the microscope in less
than three minutes. Usually a moment’s examination is
all that is necessary, but if one desires a longer study care
must be taken that the water beneath the slip does not
evaporate. Three or four such preparations, depending
upon the size of the cover-glasses, may be mounted on
one slide.

As before mentioned, by far the greater number of
cover-glass preparations are only made for diagnosis, and
are then thrown away; it is, therefore, absolutely unneces-
sary to mount at once in Canada balsam. If, after exami-
nation in water, it is desired to preserve any specimen, the
procedure is simply as follows: Again holding the slip
down with the thumb nail the oil is wiped off with a
piece of filter-paper or a cloth moistened with xylol (a
bottle of which should be within reach); a drop of water is
then placed at the edge of the cover-glass, which is slipped
off and dried between folds of filter-paper. After com-
pleting the drying over the flame, the preparation is
mounted in balsam. The specimen being already known
there is, therefore, no need of examining it again until the
balsam becomes dry.

A recent method of making and of staining the prepa-
ration upon the slide and examining at once in oil with-
out the intervention of a cover-glass, has some advantages
and it often saves time. The great advantage of this
method is, that four or five different preparations (made
exactly as are cover-glass preparations) may be made
upon one slide and be stained simultaneously. After wash-
ing and thoroughly drying the slide the preparations are
ready for examination upon the addition of a drop of oil.
Many cover-glasses are thus saved, the slides, which are
easily cleaned with a little alcohol, being ready for further
use. | The great disadvantage of this method is the im-
practicability of properly mounting permanently, if so
desired, any one or more of the specimens thus prepared.
If but one preparation is made upon a slide permanent
mounting is simple, but, as remarked above, the great
advantage of this method lies in the possibility of stain-

ing four or five preparations at one time.
1

An excellent fluid for cleaning slides and cover-
glasses, in fact, for cleaning any glassware, is the follow:
ing:

Water .2 ses ce we recneeeeniws 50 parts.
AlcOhOl vei sive.ceciawese ee oe a4 45 parts
AMMONIA ....... eee eee eee eee 5 parts

The writer has tried many methods and many fiuids
for cleaning up old balsam or other preparations, but he
has never found anything so satisfactory as the above.
A small covered vessel of this fluid may be kept upon the
desk, and any preparations that are further useless may
be placed therein and allowed to remain until one has a
leisure hour for cleaning them. In a few days the balsam
or the dried material upon the glass or the slide becomes
softened or loosened and is easily rubbed off with a cloth;
thus covers and slides may be used over and over again.

Another place wherein a little time may be saved is
in making plate-cultures. Petri dishes have largely dis-
placed the old-fashioned plates, but the latter may still
often be employed to advantage, and much time may be
gained, by having a sufficiently large cooling-plate, upon
which, under three small bell-jars, the small plates can be
cooled. All three plates (for although as a rule only two
plates are preserved, it is often wise to keep all three)
are then flowed in rapid succession, and while the gela-
tin is cooling the moist chamber may be prepared. More-
over, it is a useless waste of time to label each tube in
writing as the dilutions are being made; it is sufficient
simply to mark each tube by twisting a small portion of
the cotton-plug into one or two small “ears.”

Again much time may be saved by not labelling every
tube-culture. It is customary to “carry over” each differ-
ent culture in the laboratory supply every month. In do-
ing this one generally makes several new cultures, for.
instance: Two or more cultures in gelatin; two or more
in or upon agar; on agar and glycerine; on potatoes, etc.
By this procedure one is apt not only to notice the vari-
ations of growth in the organism, but runs no risk of also
losing one or more altogether by the accidental entrance
of some mould or other foreign plant. These new cultures,
then, having been made are best placed in a tumbler hav-
ing a little cotton in the bottom to prevent breakage, plac-
ing only cultures of the same organism in one tumbler.
Tt is now absolutely useless to label each tube: writing
upon a piece of filter-paper and placing this writing out-

ward within the tumbler is all sufficient.
12

2. STAINING METHODS.

The following are a few simple methods, in constant
use, arranged with special attention to convenience in
laboratory work?:

f, STAINING BACTERIA IN COVER-GLASS PREPA-
RATIONS WITH WATERY SOLUTIONS OF
ANILINE DYES?

1. MAKING THE COVER-GLASS PREPARATIONS.

(a) From Fluid Parts of the Body.—With the steri-
lized straight or small looped platinum-needle place a
small drop of blood or of pus upon the well-cleaned cover-
glass and spread it out upon the latter in as even and as
thin a film as possible; or, with a sterilized knife, a scrap-
ing from the freshly-cut surface of the organ to be ex-
amined is spread out in a like manner; or, the cover-glass
is pressed directly against the freshly-cut surface of the
organ.

(Before cutting an organ burn the surface where the cut is to be
made with the flat side of a very hot knife.)

(b) From Pieces of Tissue.—With sterilized forceps
pull out a small particle of tissue from the freshly-cut sur-
face of the organ, crush between the forceps, and spread a
portion of this crushed tissue substance (with the plati-
num-needle or the points of the forceps) upon the cover-
glass as above directed. .

2. “FIXING” THE COVER-GLASS PREPARATION.

To fix the preparation the cover-slip is held in the
forceps high above the flame, “butter” side uppermost,

1For anthrax, symptomatic anthrax (black-leg), hog cholera, mouse
septicaemia, typhoid fever, gonococcus, tetanus, pus organisms, etc.,
and probably all saprophytic bacteria.

2 SOLUTIONS OF THE ANILINE DYHBS: (a) of the dye, 2
parts; water 85 parts; boil from five to ten minutes, and after cool-
ing add 15 parts of 90 per cent. alcohol; mix thoroughly and filter
twice. (b) Requisite amounts of concentrated alcoholic solutions of
the dyes are diluted ten times wih water.

until thoroughly iried. It is then passed three times
through the flame it intervals of about one second.

3. STAINING THE COVER-GLASS.

(a) Holding the cover-slip “butter” side up horizon-
tally in the forceps, the glass is flooded to its edges with
a pipetteful of the desired staining fluid.

(b) Allow the fiuid to remain from one-fourth to
one-half a minute upon the slip, or the staining may be
hastened by heating the slip until a light steam arises.

(c) Wash the slip in water. If gentian-violet and
fuchsin are used it is better if the wash water contains
from one-third per cent. to one-fourth per cent. of acetic
acid. Afterward, however, wash the slip thoroughly in
pure water.

4, EXAMINATION OF THE STAINED COVER-GLASS.

(a) Lay the just washed cover-glass upon a slide
(“butter”’ side down); absorb the superfluous water by
placing a folded strip of filter-paper upon both slip and
slide, and by lightly running the finger-tips over the same.
Holding the cover in place with the thumb nail, remove
all moisture from the surface of the cover with a corner
of the filter-paper. Examine with the microscope (placing
a small drop of cedar-oil in the centre of the cover if de-
sirous of using an oil-immersion lens). Or:

(b) Thoroughly dry the cover-glass first by pressing
it between two folds of filter-paper and then by holding
it above the flame. Place a small drop of Canada balsam,
thinned with xylol, in the centre of the slide and place
the cover-slip upon it. Preparations, examined first in
water, may then be thus mounted in balsam, after
first removing the cedar-oil with filter-paper moistened
with xylol, and then removing the cover-glass from the

slide.
15

Il. STAINING BACTERIA IN SECTIONS WITH WA-
TERY SOLUTIONS OF THE ANILINE DYES.

1. Section from alcohol into water, one minute.

2. In staining fluid,? two minutes to five minutes.

3. Decolorize in acidulated water (acetic acid 1:1000),
one minute.

4, Wash in eighty per cent. alcohol; then wash and
dehydrate in absolute alcohol until no more coloring mat-
ter comes away.

5. Clear in oil of cloves, cedar-oil, or oil of origanum.

6. Transfer to the slide, with a spatula or with cigar-
_ €tte paper, and absorb superfiuous oil with filter-paper.

7. Bring a drop of xylol-balsam upon the section and
place a cover-glass upon it.

1For the bacteria mentioned under I.

2See I, foot-note 2, a and b. a

Il. GRAM-GUNTHER METHOD OF STAINING BAC-
TERIA IN SECTIONS.’

(A) Without Previous Staining:

1. Section from alcohol into aniline- ‘gaten penta
violet solution,? from one to five minutes.

2. Transfer to iodine solution? two minutes. The
superfluous staining fluid may first be absorbed with
filter-paper.

3. Alcohol, one-half minute.

4, One per cent. hydrochloric acid alcohol, ten
seconds.

5. Wash in several watch-glasses of alcohol ‘until en
tirely decolorized and dehydrated.

6. Cedar-oil or clove-oil.

7. Balsam.

(B) Staining Afterward With a Contrast Color:

1. Section treated as above under A until after deco-
lorization with alcohol, then:

2. From one-fourth to one-half minute in watery Bis-
marck-brown or Eosin solution.

3. Wash and dehydrate in alcohol.

4, Oil.

5. Balsam.

(C) Staining Previoustry With a Contrast Color:

1. Section from water to picrocarmine solution,‘
from twenty to thirty minutes.

2. Wash four or five times in fresh water.

3. Alcohol, ten minutes.

4, ~ame as above under A (1 to 7).

1Not appropriate for the bacilli of typhoid fever, for glanders,
malignant cedema, symptomatic anthrax, the gonococcus, etc., but
is appropriate for the bacilli of anthrax, for ‘mouse-septiceemia,
tetanus, the micrococcus ascoformans, ete.

2 (a) Aniline oil .............. See eon Keene ee ee 4 &
WAGER. oo 6.4 signed ae we ainda Maca). wtdud Sinveiave deena 100
Mix and filter through previously. moistened Hitespepey:
Concentrated ale. sol. gentian-violet.......... 11 ce

This solution remains useful for from three to four weeks.

(b) Saturated watery solution aniline oil, filter; add concentrated
alc. solution gentian-violet until a marked metallic film appears on
the surface of the fluid.

9 WOON: siacsis aes ee ide aay Caw AOS 2S4 Gea sae 4

Iodide of potassium. . :

ae wees Cr i ee ieee)

4 Picrocarmine sehition (Friedlander).

Carmine
AMMODIA Als. 6.45.0 2s wsiense oe awee4 elses aoe’ 1 part
Water. Oe cee eee eee e eee Cis 8 scans +s... 50 parte

To this wahadion add concentrated watery solution of picric acid
until the resulting precipitate no longer dissolves upon stirring. A
trace of ammonia added dissolves ithe precipitate,

IV. GRAM METHOD OF STAINING BACTERIA IN
COVER-GLASS PREPARATIONS.

1. Hold the cover-glass with forceps, and flood its
surface with aniline-water-gentian-violet solution (See ITI,
foot-note 2, a and b). Place glass in a flame until the fluid
boils, or stain in the cold from two to five minutes with
the same fluid in a watch-glass.

2. Iodine solution (See III, foot-note 3), two minutes.

3. Acid alcohol (HCI. one per cent.), ten seconds.

4, Alcohol until thoroughly decolorized.

5. Contrast stain with water solution of eosin or Bis-
marck-brown, one minute.

6. Wash in water.

7. Dry and mount.

‘1Boiling the fluid is only necessary when staining the bacillus
of tuberculosis. Pa

V. ZIEHL-GABBET METHOD OF STAINING TUBER-
CLE BACILLI IN COVHR-GLASS PREPARATIONS.

(a) 1. Spread a small portion of the material (sputum, |
cheesy. mass, mucus, etc.), that is most likely to con-
tain the bacilli, upon the cover-glass in'as thin a layer as
possible.

2. Dry and “fix.”

3. Flood the glass (held in the forceps) with Ziehl’s
carbol-fuchsin solution.t

4, Boil three or four times and allow the fluid to re-
main on the glass from one to five minutes. The boiling
is accomplished by holding the cover-glass in the lower
portion of the Bunsen flame until the staining fluid
steams; then slowly raise the glass to the top of the
flame and the fluid will boil; immediately lower again
and repeat.

5. Wash in water and partially dry the cover-glass
with filter-paper.

6. Flood the glass with Gabbet’s solution,? from one
to three minutes, according to the thickness of the prepa-
ration and to the intensity of the fuchsin stain.

7. Wash in water; place the cover-slip (“butter” side
down) on the slide; absorb the water with filter-paper, etc.,
and examine; or, to obtain a better contrast, stain (this is
no longer the method of Ziehl-Gabbet).

(b) 7. After 6, wash in water.

8. Stain as usual with 2 per cent. methylene-blue
(See 1, foot-note 2, a).

9. Wash in water.

10. Mount, ete., or, after 4: ,

(c) 5. Wash in water.

6. Decolorize in 25 per cent. nitric acid, from ten to
thirty seconds.

7. Complete decolorization in alcohol.

&. Wash in water.

9. Stain with methylene-blue, ete., as above (b).

1 Ziehl’s Carbol-fuchsin Solution:

MCNSIN iass occ ep HOS YS ARE Bore WES se 8 AD Ags
IV COHO]. esses ee wee and ad paene shia Bree Wiaiedeec dk ane 10 ce.
Carbolic Acid, 5 per cent. ..... 00... 00 2 sees 100 ce.

Warm to dissolve the fuchsin, and when cool filter.

2 Gabbet’s Solution:
Methylene-blue .......... cece eee eee 1to2 g.
Tigh g BO POP Cele 44s. aa mee dba wan eas + * 100 cc.
Filter.
23

VI.. KOCH-EHRLICH METHOD OF STAINING TU-

BERCLE BACILLI IN COVER-GLASS PREPARA-
. TIONS.

land 2 same as V (a).

3. Float the cover-glass (“butter” side down) upon
Koch-Ehrlich’s aniline-water-gentian-violet (methylene-
blue or fuchsin) solution, for from twelve to twenty-four
hours at room-temperature, or one hour if heated, until a
vapor arises. :

4. Decolorize in 25 per cent. nitric acid, from ten to
thirty seconds. ‘

5. Wash in alcohol until no more color comes away.

6. Contrast stain with a watery solution Bismarck-
brown (if fuchsin has been previously used with methy-
lene-blue) one or two minutes.

7. Wash in water and place upon slide, or

8. Dry and mount in balsam.

1For preparation of aniline-water-gentian-violet-fuchsin, or methy-
lene blue solutions (See ILI, ae aa 2, a and b)

VII. KOCH-EHRLICH METHOD OF STAINING TU-
BERCLE BACILLI IN SECTIONS.

1. Section from alcohol to freshly prepared aniline-
water-fuchsin solution,! for at least twelve hours at room
or incubator temperature.

2. Decolorize in 25 per cent. nitric acid from ten to
thirty seconds.

3. Continue decolorization in alcohol until section is
pinkish-white.

4. Contrast stain in watery methylene-blue solution
one or two minutes.

5. Dehydrate in alcohol.

6. Oil, balsam.

_1See III, foot-note 2, a and b. Instead of aniline-water-fuchsin so-
lution, aniline-water-gentian-violet, or methylene-blue, may be used,
but the latter stains do not seem to be retained so permanently.

27

VIII. ARENS’ METHOD OF STAINING TUBERCLE
BACILLI?

(A). IN COVER-GLASS PREPARATIONS.

1. Stain from four to six minutes in chloroform-
fuchsin solution?

2. Decolorize in 96 per cent. alcohol+-3 drops HCl to a
watch-glass. ;

3. Wash in water.

4, Contrast stain with methylene-blue, etc.

(B) IN SECTIONS.

1. Sections from alcohol to chloroform-fuchsin solu-
tion, four to six minutes.

2. Decolorize as under A 2.

3. Wash in water.

4. Contrast stain, ete.

JRecommended for staining these bacilli in fatty substances,
milk, ete.

2CHLOROFORM-FUCHSIN SOLUTION: -

(a) one or two crystals of fuchsin dissolved in from 2 to 3 co
chloroform, or (b) concentrated Beneol solution fuchsin, 3 to 4 drops.

IX. STAINING TUBERCLE BACILLI IN SECTIONS
WITH ZIBHL’S CARBOL-FUCHSIN SOLUTION.

(A) ALCOHOL SECTIONS.
1. Stain from ten to twenty minutes in Ziehl’s car-
bol-fuchsin solution (See V, foot-note 1).
2. Same as VII, 2 to 6.

(B) FROZEN SECTIONS.

(a) 1. Sections from salt-solution to 80 or 96 per cent.
alcohol for about five minutes to harden. That the sec-
tion may remain smooth, it is best to transfer it as fol-
lows: Lift from the salt-solution with a spatula; drain off
superfluous fluid; add 1 to 2 drops of alcohol with the
end of a glass rod, and after a moment slip off into alcohol.

2. Same as above under A, or
(b) 1. Sections from salt-solution to water.

2. Harden in corrosive sublimate (1:1000 to 1:500)
from one-half to one hour.

- 8. Wash in water and proceed as above under A.
31

X. WHIGERT’S FIBRIN OR BACTERIA STAIN IN
SECTIONS.

1. Transfer section from water or alcohol to the slide
and absorb all the moisture with filter-paper.

2. Flood the section (using a pipette) with aniline
water-gentian-violet solution (See III, foot-note 2, a and
b), from one to two minutes, and absorb the stain with
filter-paper.

3. Flood the section (using a pipette) with iodine solu-
tion (See IT, foot-note 3) two minutes, and absorb with
filter-paper.

4. Wash the section repeatedly with aniline-xylol!
until it is nearly decolorized.

5. Wash several times with xylol and as usual absorb
with filter-paper.

6. Balsam.

DOS VIN Caewu sd Paes a hee e Ne Paes Boum we ewes 1 part

XI, STAINING THE GLANDERS BACILLUS.

(A) IN COVER-GLASS PREPARATIONS.
a. LOEFFLER’S OLD METHOD.
1. Flood the cover-glass for three minutes with Loeff-
ler’s methylene-blue solution (See XIV, foot-note 2).
2. Heat in the flame until the fluid steams (do not
boil).
3. Wash in water.

4, Wash in acetic-acid water (1:100) from five to ten
seconds. ‘

5. Wash in water.
6. Mount, etc.

b. LOEFFLER’S NEW METHOD.
1. Flood the glass with alkaline aniline-water-gen-

tian-violet solution, five minutes.
2. Wash in acetic-acid water (1:100) rendered a Rhine-

wine-yellow color by the addition of a few drops of
tropaeolin (00) solution.
3. Wash in water, mount, etc.

ec. A SIMPLE METHOD.
1, Flood the cover-glass with 2 per cent. gentian-violet
solution (See under I, foot-note 2 a).
2. Heat in the flame until the fluid. steams well (do
not boil), and allow it to stand from one-quarter to one-
half minute.
3. Wash in water, mount, etc.

(B) IN SECTIONS.

a. LOEFFLER’S METHOD.
1.stain for a few minutes in Loeffler’s methylene-

blue solution (See XIV, foot-note 2).

2. Five seconds in the following solution: Aq. dest.,
10cc; concentrated H.SO, . 2 drops; 5 per cent. oxalic acid,
1 drop. Bye

3. Decolorize and dehydrate in absolute alcohol; oil;
balsam. — ae

: 1Alkaline aniline-water-gentian-violet solution (See III, foot-note
2, a, b) mixed with an equal amount of a potash solution (1:10,000),
or an equal amount of Liq. so, caustic, half per cent.

b. NONIEWICZ'S METHOD.

1. Section from alcohol to Loeffler’s solution (See XIV,
foot-note 2), from two to five minutes.

2. Wash in water and transfer to a, decolorizing fluid’,
in which thin sections are dipped once beneath the sur-
face, thicker sections remain from three to five seconds.

3. Wash in water.

4. Transfer section to slide and allow to dry thor-
oughly in the air, or carefully dry over the flame.

5. Balsam.

1Acetic acid, % per cent................4- 75 parts.
¥ per cent. ‘water Tenpscoline (00) solution. .25 parts.

XII. STAINING SPORES, ESPECIALLY ANTHRAX
SPORES.

To obtain very beautiful preparations of anthrax spores the fol-
lowing method is recommended: Make hanging-drop cultures in bouil-
lon from the heart’s blood of an anthrax mouse (or other experiment
animal), using a scarcely-visible amount of blood; place the incu-
‘bator at 35 to 37 degrees C.; in a few hours the well-known _beauti-
ful threads and ropes are formed, and after from twenty-four to
forty-eight hours these threads are usually filled with spores (Pl. I,
Figs. 1, 2). The cover-glasses are then removed from the slide (first
lifting one edge with a knife-blade), dried in the air, the vaseline
washed off with xylol, and stained as follows:

1. Fix the dried preparation.

2. Float “butter” side down, on the surface of aniline-
water-fuchsin-solution (See II], foot-note 2, a and b, sub-
stituting fuchsin for gentian-violet); heat until a delicate
vapor arises and continue this for from five to six hours
(if much evaporation occurs more aniline-water must be
added). It is better to leave the preparation in the dye
four or five hours after it has cooled.

3. Decolorize in 25 per cent. HNO; about five sec-
onds.

4, Carefully wash in alcohol until no more color comes
away, and place for a short time in distilled water.

5. Contrast stain in watery methylene-blue solution,
five minutes.

6. Wash carefully in water, mount, etc., and examine.
If the preparation is satisfactory:

7. Dry and mount in balsam.
39

. MOLLER’S METHOD OF STAINING SPORES.
Dry and fix the cover-glass preparation.

. In chloroform, two minutes.

Wash in water.

In 5 per cent. chromic acid, for a half to two min-

er:

utes.

5. Water.

6. Flood the glass with Ziehl’s carbol-fuchsin solu-
tion; boil and keep very hot for one minute.

7. Wash in water.

8. Decolorize in 5 per cent. H,SO, for from five to
ten seconds.

9. Wash in water.

10. Stain with methylene-blue.

11. Wash in water, mount, ete.
41

XIV. STAINING SECTIONS WITH BISMARCK-
BROWN, LOEFFLER’S ALKALINE METHYL-
ENE-BLUE, OR WATERY SOLUTIONS OF
THH ANILINE DYES.

1. Section from alcohol to water, one minute.

2. Stain with Bismarck-brown,! Loeffler’s alkaline
methylene-blue solution,” or with a watery solution of one
of the aniline dyes for ten minutes.

3. Decolorize in a watery solution of acetic acid
(1:1000), about one minute.

4. Thoroughly wash in 96 per cent. alcohol, using
fresh alcohol several times until no more colering matter
comes away.

5. Oil, balsam, ete.

1Bismarck-brown does not stain the bacteria at the same time
and is only used to bring out the tissue-structure.
2LOEFFLER’S ALKALINE METHYLENE-BLUE.
Concentrated alcohol solution methylene-blue ...80 ce
Watery solution potash (1:1000)...... .2.. .. 100 ce

3See I, foct-note 2, a and ,

Aw

XV. DOUBLE STAINING OF SECTIONS WITH HE-
MATOXYLIN AND PICRIC ACID OR HOSIN.

(Only for structure staining, or for staining sections containing
actinomyces or the zoogloza masses of the micrococcus ascoformans
found in the so-called. “‘scirrhous cord” of the horse).

1. Section from alcohol to Friedlander’s hematoxy-
lin! solution, from a half-hour to twenty-four hours, ac-
cording to the strength of the solution.

: (It is perhaps best to add to a watch-glass of water or of alum
solution (1:250) a suflicient quantity of Friedlander’s hematoxylin so-
lution to give the mixture a good red-wine color. In this mixture sec-
tions are stained, from one to two hours).

2. Decolorize in 96 per cent. alcohol, and if the sec-
_ tion remains too intensely stained, wash alternately in 1
per cent. hydrochloric acid alcohol, and water (or alcohol)

tr’ til the section becomes a delicate bluish-red color.

If a single hematoxylin stain only is desired the section is now
dehydrated in alcohol, cleared in oil, and mounted in balsam. [f,
however, a double stain is desired, proceed as follows:

3. DOUBLE STAINING WITH PICRIC ACID.

(a) Transfer the decolorized hematoxylin section to
a concentrated alcoholic (96 per cent.) solution of pic-
ric acid for a half minute; oil from two to five minutes;
balsam; or,

(b) Transfer the decolorized hematoxylin section
from alcohol to a watch-glass containing oil of cloves, to
which from 10 to 15 drops of a concentrated alcoholic so-
lution of picric acid has been added, five to six minutes; |
balsam.

4. DOUBLE STAINING WITH EOSIN.

(a) Transfer the decolorized hematoxylin section to
a watery solution of eosin,? one minute; dehydrate in 96
per cent. alcohol; oil; balsam; or

(b) Transfer the decolorized hematoxylin section
from alcohol to a watch-glass containing oil of cloves, to
which eight to ten drops of a concentrated alcoholic solu-
tion of eosin has been added; balsam.

1WRIEDLANDER’S HEMATOXYLIN SOLUTION.

Hematoxylin.

BAU BAe occas ae 6 bo 6 aE bok WWE WAS ee 2 parte
PINON scien 3k dew mah et WA Care PAE ing Dac Rea
Glycerin. .........e0e0e GsRNsribgh SRAM lehaueiie Soe) oeduietan eerevotees
AQ: Gest 94s idecsi weds aw iraeaewt ews eer -,100 parts

2 Add to a watch-glass of water a few drops of a cone. ale. so-
lution of eosin until a distinct eosin-red color is obtained; or mix
1 part of cone. ale. eosin eobation sini 100 parts of water.

IF THE SECTIONS CONTAIN ACTINOMYCES OR
THE ZOOGLOEA MASSES OF THE MICRO-
COCCUS ASCOFORMANS.
(SEE PL. I, FIG. 2).
the procedure is as follows:
(a) Stain from one to several hours in-a 1 to 2

per cent. watery solution of eosin.

(b) Wash in water and transfer to Friedlander’s he
matoxylin solution.

(c) Wash in water.

(d) In alcohol.

(e) Decolorize in 1 per iat: fivdeactitorie acid alcohol,
one or two seconds.

(f) Alcohol until dehydrated.

(g) Oil, ete.
47

XVI. TYPICAL METHOD OF STAINING ANTHRAX
BACILLI FROM THE BLOOD IN COVER-GLASS
PREPARATIONS.

According to Johne the anthrax bacillus in the blood has a typi-
cal structure which no other bacillus presents under similar condi-
tions. By this peculiar structure the anthrax becillus can quickly
and absolutely be diagnosed from other large cadaver bacilli often
found in the bJood of animals from ten to fifteen hours after death
and with which bacilli the anthrax bacillus is often confounded.

It is well known that the many organisms are surrounded by
a colorless, gelatinous sheath or membrane (often overlooked even
when present) similar to the membrane, for instance, frequently
seen in some of the higher plants, such as the unicellular alge. Dif-
ferent énvironments alter the appearance of this membrane, it
some times being very evident and at others scarcely visible or absent
in the same organism. For instance, there are several pathogenic
bacteria, such as the micrococcus tetragenus, micrococcus as-
coformans, and Friedlander’s capsule-coccus of pneumonia,
which within the body shows a well-defined membrane or capsule,
while if grown upon an artificial media this capsule is lost. The
same is true of the anthrax bacillus. :

If a cover-glass preparation is made from the blood
or from the organs of an anthrax animal and is intensely
stained with gentian-violet or with fuchsin, there will
generally be noticed enormous bacilli of different lengths;
with irregular outlines and often with prolonged, rounded
swellings at the ends. In such cases it is only with
difficulty that the separate cells can be _ dis-
tinguished. If now this preparation is. decol-
orized in 1 to 2 per cent. acetic acid and again be
examined a marked alteration will be observed. The
bacilli are now seen occuring singly, in pairs, or in distinct
chains of varying lengths, still well stained but much
smaller than before; their outlines are perfectly regular,
except where one is undergoing the process of division,
and the ends of the rods are square or more often slightly
convex 2c never concave —1= Surrounding the
bacilli, however, is observed a perfectly distinct, colorless
membrane. This membrane is apt to be irregular in out-
line, and its ends are often swollen; it is now very evi-
dent that it was this membrane (stained in the first prep-
aration) which caused the apparent size and irregular
shapes of the organisms.

It has been suggested that this capsule-like mem-
brane is nothing more than serum clinging to the organ-
isms. This theory cannot, however, be accepted, for not
only would such serum be washed away from the bacilli

if diluted many times with water (which is not the case),
49

but serum would also not stain in such a definite man-
ner; moreover, a similar membrane, though much less
marked, has been observed in cultures of this organism
upon some of our media. We must, therefore, consider this
membrane to be a true portion of the bacillus itself and it
gives the organism, in the blood of anthrax animals,a very
characteristic appearances (Pl. I, Fig. 1), which may be
brought out by staining, as follows:

1. Make the cover-glass preparation from the blood,
splenic-pulp, ete.

2. Dry and fix.

3. Flood the glass (held in the forceps) with 2 per
cent. gentian-violet solution (See I, foot-note 2 a) and stain
from a quarter to a half minute.

4, Wash in water.

5. Decolorize in 1 to 2 per cent. acetic acid from five
to twenty seconds.

6. Wash in water.

7. Mount, etc.
; 51

8. PREPARATION OF NUTRIENT MEDIA.

A. BOUILLON.

1. Add one pound of finely-chopped lean meat to 1000
ce of water, and allow this to stand from twelve to twen-
ty-four hours in a cool place.

2, Strain through cheese-cloth, or a coarse towel, and
squeeze in a meat-press, or by twisting the ends of the
cloth, until 1000 cc of so-called “meat-” or “flesh-water,”
are obtained. If less than 1,000 cc are thus obtained,
make up the deficiency with fresh water.

3. Add 5g. common salt and Gissolve.

4. Add 10g. dried peptone and dissolve.

5. Boil one-quarter hour, either in steam or over an
open flame.

6. Make the solution slightly alkaline by the addi-
tion of sodium carbonate (saturated solution).

7. Boil from three-quarters to one hour, filter; the
filtrate should be clear, if not, add the whites of two eggs,
mix rapidly, boil from a quarter to half hour and again
filter. ;

B. BOUILLON (PETRI AND MAASZEN METHOD).
_ 1. Mix meat and water, as above, under A 1, and let
it stand for one hour.

2. Heat in water-bath to about 60 degrees C. for three

3. Boil one-half hour.

4, Filter and when cool make slightly alkaline.
5. Add 5g. salt and dissolve.

6. Add 10g. dried Peptone and dissolve.

7. Boil one-quarter hour (best over a free flame).
8. Add glycerine if desired.

9. Filter.

1In the preparation of all the nentonized media, it is often the ease
that the final filtration is not sufficiently clear. This cloudiness is gen-
erally due to the fine particles of matter suspended in the fluid. If egg
albumen is now added. and the solution is again boiled, these particles
are generally caught in the coagulum. and they ean he removed with
the filter. It is. perhaps, a good practice always to add the eggs he-
fore the last boiling, thus avoiding a second filtration. Mven after every
precaution has heen taken, it sometimes seems impossible to obtain a
perfectly clear filtrate; in such cases, the repeated addition of eggs
and boiling are recommended: in the ease of houillon, the narticles
will sink to the bottom on cooling, and, as a rule, may then be ‘fil-
tered off. Pe

C. GELATIN.

1. Make meat-water as above, under A 1 and 2.

2. Salt, 5g. .

8. Peptone, 10g.

4. Gelatin, 100g.

5. Heat in water-bath to 50 degrees until the gela-
tin is dissolved.

6. Make slightly alkline.

7. Boil three-quarters to one hour.

8. Filter.

7 D. AGAR-AGAR.
1. Make bouillon (See A and B).
2. Add 10 to 20 g. agar-agar, finely cut up.
3. Boil until the agar is dissolved (five to elght
hours).

4. Neutralize if necessary and add glycerin if de-
sired.

5. Boil from three-quarters to one hour.

6. Filter (filtration is often rapid at room-tempera-
ture, but if it should occur slowly the agar must be

filtered in steam or in a hot-water filter).
E. AGAR-AGAR GELATIN.

This medium is prepared as indicated above, using,
however, only 7.5g. of agar, and, when this is dissolved,
adding 50g. of gelatin.

F. PREPARING THE PEPTONE MEDIA WITH
LIEBIG’S MEAT EXTRACT.

Instead of using chopped meat, Liebig’s Meat Extract —
may be substituted in the proportion of from 38 to 5
grammes to the litre of water.

G. BLOOD-SERUM.

(Ascitic ond hydrocele fluid often contain too little albturen to ad-
rait of coagulation. Stich fluids should therefore be tested by boil-
ing before tubing.)

1. Collect the blood in large jars, which can be closed
tightly, and close the jars.

2. In about fifteen minutes, or after clotting has
begun, pass a sterilized glass rod around the clot, be-
tween its surface and the wall of the jar, thus breaking
up the glass adhesions, which prevent the clot from
sinking.

. 8. Again close the jars and place them in an ice
chest, for from twenty-four to forty-eight hours.

1One must be cautioned against too long boiling of the solution,
since this lowers the solidifying point of the gelatin.

ets

4. Draw off serum with sterilized pipette and run
into test-tubes. ,

5. Place tubes in blood-serum sterilizer and heat to
68 or 70 degrees C. for one hour on five successive days.

6. Place tubes in the apparatus for solidifying blood-
serum and heat to 75 or 80 degrees C., until the serum
is coagulated.

H. BLOOD-SERUM (QUICK METHOD.)

(Serum made in the following manner is, as a rule, mere opaque
than that prepared as indicated above; but for all practical purposes
it is admirable. Much time and annoyance are saved, and imper-
fect sterilization, by means common by the other method, is
avoided).

1-4 See G.

5. Place the tubes in the serum sterilizer and heat
at once to 90 or 95 degrees C. for one hour or more.

6. Place the tubes (in an inclined position) in the

steam sterilizer for one hour or more.

(It is advisable not to allow the temperature in the steam sterili-
zer to run too high or bubbles may result in the solid serum.
With the Arnold steam sterilizer, the inside cover may be left off,
or with other sterilizers, full gas pressure need not be used.)

7. Place the tubes in baskets as usual, and steam for

from fifteen to thirty minutes on two successive days.
ST

4, IMBEDDING TISSUES FOR CUTTING SECTIONS.

A. IMBEDDING IN CELLOIDIN.

1. Transfer the hardened tissue from alcohol to equal
parts of alcohol and ether, twelve hours or longer.

2. Into thin celloidin, twelve hours or longer.

3. Into thicker celloidin, thirteen hours or longer.

4, Into thick celloidin (that is, so thick that the
specimens but slowly sink to the bottom), twelve hours or
longer.

5. Place specimen with as much celloidin as possible
clinging to it upon cork, a small block of wood, or, best, a
block of vulcanized fibre* With a glass rod add several
layers of the thick celloidin until the specimen is quite
surrounded, especially toward its base, with perhaps one-
eighth inch of celloidin. Allow it to remain in the air
until a sufficiently thick film forms on the outside, then

6. Into 50 to 75 per cent. alcohol (the celloidin must
be beneath the surface of the fluid) to harden; or harden
quickly by dropping it into chloroform, and after a few
minutes place in alcohol (75 per cent.).

7. Cut in alcohol.

B. IMBEDDING IN CELLOIDIN AND PARAFFIN.
1—4, See above under A.
5. Drop into chloroform and keep immersed (by means
of a small net loosely attached to a wire frame, for in- -
stance) from six to eight hours.

6. Place in melted paraffin’, one to two days; or
better, place in a bottle of melted paraffin kept at 55 to

«8 degrees C. in a water-bath, attach a suction pump? and
pump until a few, or no more, bubbles come away from

the tissues, from one to three hours.

_1This may be obtained from the Vulcanized Fibre Co., Wil-
mington, Del., and, when cut into small blocks, is admirable for
mounting tissues imbedded in celloidin. One may write with a
pencil upon the blocks and they readily sink in fluids, thus saving
much trouble.

2Hard and soft paraffin are mixed in such proportion that the
melting point of ihe combined mass is about 52 degrees C.

8A Chapman water-pump is the simplest and best to use for this
purpose.
59

7. Imbed in paraffin.’

8. Cut with a Minot microtome. Such sections may
be placed in a box and they will keep indefinitely. Be-
fore staining—

9. Sections into chloroform or benzole to dissolve
paraffin, using two watch-glasses of the fluid.

10. Into absolute alcohol, using two watch-glasses of
the fluid.

11. Stain.

1For this purpose cheap and very satisfactory paraffin moulds
may be made frcm four pieces of heavy type-metal (brass is
in commoner use, but is more expensive). These pieces
need not be fastened together, thus permitting moulds
of different sizes to be formed. Small pasteboard boxes
may also be used. These pieces of metal are placed upon a glass
plate, a mould of the desired size is formed and filled with melted
paraffin, first, however, smearing the surfaces, which are later to
come in contact with the paraffin, with the glycerin. Soon a thin,
semi-solid Jayer of paraffin forms at the bottom of the mould, and
the material to be cut is then placed into and held in any position de-
sired by this layer. When the paraffin has cooled so that a thin film
or crust has formed on its surface, immerse the mould and all in
eold water. The block of paraffin is thus quickly hardened and the
mould can almost immediately ga es

PLATE I.

Fig. 1.—Anthrax bacilli; hanging-drop culture from blood, showing threads and spores
(x Ca. 700).

Fig. 2,—Anthrax from hanging-drop culture from blood, showing threads and ropes (x 130).

PLATE II.

Fic. 2.—Section of ‘‘ myko-fibrom”’ from the spermatic cord of a horse (x ca 50): @, grape-
like clusters of zodgloea (containing cocci not visible) ; 4, cellular centre, consisting of pus and some
lymphoid cells ; ¢, space from which cells have fallen out ; d, fibrous stroma (stained by method xv. 5).

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NOSE AND THROAT. By D. Brapen Kyte, M. D,, Chief Laryngologist
of St. Agnes’ Hospital, Philadelphia; Instructor in Clinical Microscopy,
and Assistant Demonstrator of Pathology in the Jefferson Medical College,
etc. Price, $1.25 net.

*,* There will be published in the same series, at close intervals, carefully-pre-
pared works on the subjects of Anatomy, Gynecology, Pathology, Hygiene, etc., by

prominent specialists. ms

PLATE I.

Fic. 1.—Anthrax bacilli; hanging-drop culture from blood, showing threads and spores
(x ca. 700).

PLATE II.

Fic. 2.—Section of ‘‘ myko-fibrom”’ from the spermatic cord of a horse (x ca 50): @, grape-
like clusters of zodgloea (containing cocci not visible) ; 4, cellular centre, consisting of pus and some

lymphoid cells ; ¢, space from which cells have fallen out ; d, fibrous stroma (stained by method xv. 5).

MR. SAUNDERS is pleased to announce as in active prepara-

~~ NEW AID SERIES OF MANUALS

Students and Practitioners. .

S publisher of the ‘Standard Series of Question Com-
pends,” and through intimate relations with leading members -

of the medical profession, Mr. Saunders has been enabled to study
progressively the essential desideratum in practical “self-helps” for

students and physicians. ;
This study has manifested that, while the published ‘“ Question

Compends” earn the highest appreciation of students, whom they serve
in reviewing their studies preparatory to examination, there is special
need of thoroughly reliable hand-books on the leading branches of Medi-
cine and Surgery, each subject being compactly and authoritatively
written, and exhaustive in detail, without the introduction of cases
and foreign subject-matter which so largely expand ordinary text-
books.

The Saunders Aid Series will not merely be condensations
from wpresent literature, but will be ably written by well-known
authors and practitioners, most of them being teachers in rep-
resentative American Colleges. This new series, therefore, will
form an admirable collection of advanced lectures, which will be invalu-
able aids to students in reading and in comprehending the contents of
“recommended” works.

Each Manual wili further be distinguished by the beauty of the new
type; by the quality of the paper and printing; by the copious use of
illustrations ; by the attractive binding in cloth; and by the extremely
low price, which wiil uniformly be $1.25 per volume.

For further particulars, address

W. B. SAUNDERS, Publisher,
925 WALNUT ST., PHILADELPHIA.

SAUNDERS’ NEw AID SERIES OF MANUALS.
VOLUMES NOW READPY.

PHYSIOLOGY. By JosepH Howarp Raymonp, A. M., M. D., Professor of
Physiology and Hygiene, and Lecturer on Gynecology in the Long Island
College Hospital, etc. Price, $1.25 net.

SURGERY, General and Operative. By Joun Cuatmers DaCosta,
M. D., Demonstrator of Surgery, Jefferson Medical College, Philadelphia,
etc. Double number. Price, $2.50 net.

DOSE-BOOK AND MANUAL OF PRESCRIPTION-WRITING.
By E. Q. THornton, M. D., Demonstrator of Therapeutics, Jefferson Med-
ical College, Philadelphia. Price, $1.25 net.

MEDICAL JURISPRUDENCE. By Henry C. Cuapman, M.D., Pro-
fessor of Institutes of Medicine and Medical Jurisprudence in the Jefferson
Medical College of Philadelphia, etc. Price, $1.25 net.

SURGICAL ASEPSIS. By Cari Becx, M.D., Surgeon to St. Mark’s
Hospital; the German Poliklinik; Instructor in Surgery, New York Post-
Graduate Medical School, etc. Price, $1.25 net.

VOLUMES IN PREPARATION FOR EARLY PUBLICATION.

OBSTETRICS. By W. A. Newman Dortanp, M. D., Demonstrator of
Obstetrics, University of Pennsylvania; Chief of Gynecological Dispensary,
Pennsylvania Hospital; Member of Philadelphia Obstetrical Society, etc.
Price, $1.25 net. ,

MATERIA MEDICA AND THERAPEUTICS. By Henry A. Grir-
rin, A. B., M. D., Assistant Physician to the Roosevelt Hospital, Out-patient
Department, New York City. Price, $1.25 net.

SYPHILIS AND THE VENEREAL DISEASES. By James Nevins
Hype, M. D., Professor of Skin and Venereal Diseases in Rush Medical Col-
lege, Chicago. Double number. Price, $2.50 net.

NERVOUS DISEASES. By Cuarues W. Burr, M. D., Clinical Professor
of Nervous Diseases, Medico-Chirurgical College, Philadelphia, etc. Price,
$1.25 net.

PRACTICE OF MEDICINE. By Grorcrt Roe Locxwoop, M.D., Pro-
fessor of Practice of the Woman’s Medical College and of the New York
Infirmary, etc. Double number. Price, $2.50 net.

NOSE AND THROAT. By D. Brapen Ky ez, M. D., Chief Laryngologist
of St. Agnes’ Hospital, Philadelphia; Instructor in Clinical Microscopy,
and Assistant Demonstrator of Pathology in the Jefferson Medical College,
etc. Price, $1.25 net.

*,* There will be published in the same series, at close intervals, carefully-pre-
pared works on the subjects of Anatomy, Gynecology, Pathology, Hygiene, etc., by

prominent specialists. os