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A LABORATORY GUIDE

PRACTICAL BACTERIOLOGY

WITH AN OUTLINE FOR THE CLINICAL EXAMINATION

OF THE URINE, BLOOD AND GASTRIC CONTENTS,

BY
W. T. CONNELL, M.D., M.R.C.S., ENG.

PROFESSOR OF PATHOLOGY AND BACTERIOLOGY, QUEEN’S UNIVERSITY.

PATHOLOGIST TO THE GENERAL HOSPITAL, KINGSTON.

KINGSTON, ONTARIO:
BRITISH WHIG, KING STREET,
1899,

\.\L4 20%

Entered according to Act of the Parliament of Canada, in the year
one thousand eight hundred and ninety nine, by W. T. Connell, M.D.,
at the Department of Agriculture.

PREFACE.

This book has been prepared for the guidance of
students taking the practical course in Bacteriology.
Much time and labor have hitherto been expended in
the writing out, both by the lecturer and the student,
of the daily work of the class, and to obviate this it
was thought best to place these notes in an accessible
and connected form. The usual course covers a period
of from eight to ten weeks, and comprises a series of
twenty demonstrations, of at least two hours each.

There are already several excellent handbooks for
the guidance of students, notably that of Kanthack
and Drysdale. As the author worked under the for-
mer’s guidance, their methods have been used as a
basis. Free use has also been made of methods advised
by, or acquired from others, which have been found
satisfactory.

In Part II the preparation of culture media, methods
of sterilization of bacteriological utensils, and the
methods followed in water and milk analysis are
considered.

The author has appended in Part III the plan of
work followed in the usual course in clinical microscopy
and diagnosis, covering that elementary work, with
which every physician should now not only be cogni-
zant, but employ.

W. T. ConneELL.
KINGSTON, ONTARIO,
May Ist, 1899.

TABLE OF CONTENTS.

PART I.
PRACTICAL BACTERIOLOGY.

DEMONSTRATION I.

Inoculation of Culture Media from Pure Cultures
of Bacteria,

Demonstration II.

Staining and Examination of Pure Cultures of
Bacteria from Liquid Media.

Demonstration ITI.
Examination of Pure Cultures of Bacteria grown
on Solid Media—Gram’s Method of Staining.
Demonstration IV.

Examination of Bacteria Unstained — Hanging
Drops—Hanging Drop Cultures.

Demonstration V.
Examination of Hanging Drop Cultures—Spores
and Spore Staining.
Demonstration VI.
Staining of Flagella—Cultures of Bacillus Anth-
racis. é ‘ , ‘ ‘ ‘
Demonstration VII.

Bacillus Anthracis in Pure Cultures—Tissues of
Animal Dead of Anthrax Septiceemia—Inocu-
lation of Animal with Bacillus Anthracis.

PAGE.

I

12

14

17

vi CONTENTS.

Demonstration VIII.
Examination of Guinea Pig dead of Anthrax Sep-
ticemia—Plate Cultivations—Cultures of the
Pyogenic Bacteria.
DeEmonstTRATION IX.

Impression Specimens of Bacillus Anthracis—Pyo-
genic Micrococci— Staining of Pus— Agar
Plates.

DEMONSTRATION X.
Examination of Plate Cultures—Gonorrhcal Pus—
Pus from Actinomycosis.
DEMONSTRATION XI.

Micrococcus Lanceolatus (Pneumococcus) in Spu-
tum—Bacillus Influenze in Sputum—Sections
of Pneumonic Lung—Tissues containing Pyo-
genic Micrococci.

DEMONSTRATION XII.
Pure Cultures of Tubercle Bacillus—Tubercular
Sputum. : : :
DEMONSTRATION XIII.

Sections of Tubercular Tissues—Leprosy Tissues—
Cultures of Tubercle Bacillus from an Inoculat-
ed Animal—Cultures of Bacillus of Diphtheria.

DEMONSTRATION XIV.

Diphtheria Bacillus—Cultures of Spirilla of Asiatic
Cholera, of Metchnikof, and of Finkler and
Prior—Inoculation of Animals with Diphtheria
Bacilli—Diphtheria Toxin—Antitoxin.

PAGE.

20

24

26

28

31

38

CONTENTS.

DEMONSTRATION XV.

Spirillum of Asiatic Cholera—Spirillum of Finkler
and Prior — Spirillum Metchnikovi — Indol
Test—Diphtheria Membrane—Intraperitoneal
Inoculation of Animals.

DEMONSTRATION XVI.

Examination of Animal Inoculated with Spirillum
Cholere Asiaticee—Cultures of Bacillus Typh-
osus and Bacillus Coli Communis—Typhoid
Tissues.

DemonsTRATION XVII.

Cultures of Bacillus Typhosus and Bacillus Coli
Communis — Serum Diagnosis of Typhoid
Fever—Cultures of Bacillus Pestis Bubonice,
Bacillus Icteroides and Bacillus Mallei. .

DeEmMoNSTRATION XVIII.

Bacillus Pestis Bubonice—Bacillus Icteroides—
Bacillus Mallei — Cultures of Tricophyton
Tonsurans and Achorion Schonleinii—Hair in
Tinea Tonsurans.

DEMONSTRATION XIX.

Anerobic Cultures— Bacillus Tetani— Bacillus
Cidematis Maligni—Bacillus Capsulatus Aero-
genes—Inoculation of Animals with Bacillus
Tetani, Bacillus Gidematis Maligni and Bacil-
lus Capsulates Aerogenes. : :

vii

PAGE.

40

42

44

47

48

viii CONTENTS.

PAGE.

DEMONSTRATION XX.

Bacillus Tetani—Bacillus idematis Maligni—Bacillus
Capsulatus Aerogenes—Examination of Ani-
mals Inoculated with Bacillus Cadematis Ma-
ligni and Bacillus Capsulatus Aerogenes. . 51

PART II.
BACTERIOLOGICAL ANALYSIS.

CuLtuRE Mepra—Broth, Glycerine Broth, Glucose
Broth, Gelatine, Agar-Agar, Blood Serum,

Peptone, Milk, Potatoes. ; j j . 58
PREPARATION OF UtTEnsiLs—Test Tubes, Slides,
Glassware, Instruments, Dressings. ; . 58
WatTER ANALYSIS. . . : ; ; : . 61
MiLk ANALYSIS. , ; ’ : : : . 65
PART III.
CLINICAL MICROSCOPY AND DIAGNOSIS.
Section I. The Urine. . ‘ ; : . 67
“II. The Blood. : . : ; . 74

fs IIL. The Gastric Contents. ; ; . 80

PART I.
PRACTICAL BACTERIOLOGY.

DEMONSTRATION I.

Inoculation of Culture Media from Pure Cultures of Bacteria.

Students must remember that they are to work with
many of the actual cdusative factors of disease—the
pathogenic bacteria; hence there is a liability to self-
infection or infection of the laboratory, unless care is
taken to develop a proper technique. The practical
work is started with non-pathogenic species, or with
species of low pathogenic power, so that such a tech-
nique may be acquired without danger.

The points to be noted by the student are:

(a) The character of the various culture media.

(b) The method of holding culture tubes, and re-
moving and replacing plugs during inoculation.

(c) The care, and particularly the sterilization, of
the platinum needle or loop, before and after use.

(d) That minute amounts of the bacterial culture
suffice for inoculation purposes.

(c) The proper labelling and placing in incubators
of the inoculated tubes.

1. Make two agar-agar slope cultures of Bacillus
prodigiosus.

Place one tube in the incubator at 37°C (98.6-F),
and the other at 20°C (69°F).

2 PRACTICAL BACTERIOLOGY.

Examine these tubes at the end of 24 and 48 hours.
Growth is more rapid at the higher temperature, but
the pigmentation is slight, while a more slowly appearing
growth, deeply pigmented, is noted in the other tube.

Pigmentation is in nearly all chromogenic species,
more marked at the lower temperature than at the
higher.

2. Make a gelatine slope culture of Bacillus cyano-
genus and place in the incubator at 20°C.

Examine each day and note the gradual intensifica-
tion of the pigmentation of the media, the growth itself
being uncolored:

3. Make two potato tube cultures of Bacillus ruber.

Keep one exposed freely to light in the room, and
place the second in the incubator (dark) at 20°C.
Growth and pigmentation will be more marked in the
latter tube.

4. Make gelatine stab cultures of (a) Bacillus
pyocyaneus, (b) Staphylococcus pyogenes aureus, (c)
Sarcina lutea.

Incubate at 20°C and examine daily. These bacteria
liquefy gelatine more or less rapidly.

5. Make broth cultures of (a) Torula rosea and (b)
Bacillus proteus mirabilis. Place in incubator at 20°C.

6. Make gelatine slope cultures of (a) Staphylococ-
cus cereus flavus, (b) Streptococcus pyogenes. Keep
in incubator at 20°C.

STAINING OF PURE CULTURES. 3

DEMONSTRATION II.

* Staining and examination of pure cultures of Bacteria
from liquid media.

The stains which are ordinarily used are:

(a) Gentian violet. This stain is used in several
forms. Of these anilin water, gentian violet is most
used. It consists of,

Anilin oil . : ; : ‘ ; 4 ce.
Distilled water . 5 _ 100 ce.
Shake thoroughly, filter and add satan
ated alcoholic solution of gentian
violet. : : : 11 ce.

This stain ought to be Hee ee every two or

three weeks.

Gentian violet may also be used in a diluted alco-
holic solution, and in this form is one of the most
satisfactory stains for use in pure culture staining. It
is made at time of using by adding two or three drops
of filtered saturated alcoholic solution of gentian violet
to a watch glass of distilled water.

(b) Methylene blue. This may be used in several
forms. 'The stain most commonly employed is Loeffler’s
alkaline solution. Its composition is,

Saturated alcoholic solution of methy-
lee blue ; . ‘ ' 30 cc.
arbor caustic potash solution > «2 100 ce.

4 PRACTICAL BACTERIOLOGY.

(c) Fuchsin. Several staining solutions are used of
this dye, of these Ziehl’s carbol-fuchsin is most useful.
This stain is made by taking,

Saturated alcoholic solution of fuchsin
crystals . . ‘ i : j 1f cc.
5% carbolic acid solution : ; 89 cc.

Fuchsin can also be used as anilin water fuchsin,
or as diluted fuchsin. These are made of the same
strength as the like preparations of gentian violet.

All stains must be filtered before use.

Cover glasses should be kept in alcohol and handled
with forceps. When they are required for use, rub dry
with a clean cloth.

1. (a) Take a clean cover glass and sterilize it by
passing it quickly through the gas (or spirit lamp)
flame. Then with a platinum loop (using all the pre-
cautions of the first lesson), remove a drop of the lique-
fying gelatine culture of Staphylococcus pyogenes
aureus, and smear this over the cover glass.

(b) Allow the film to dry in the air, or dry it by
holding it high overthe flame. Fix the film by passing
it quickly three times through the flame.

(c) Clear away the gelatine by placing the cover
glass in 10% acetic acid for five minutes.

(d) Remove from the acid, rinse through water and
then dry.

(e) Float the cover glass, film surface down, in anilin
water gentian violet, or in diluted gentian violet, for

USE OF OIL-IMMERSION LENS. 5

one to two minutes. If desired, the stain can be
dropped on the cover glass.

(f) Remove and wash in water; dry thoroughly and
mount the cover glass in Canada balsam.

Examine the specimen with a +, oil-immersion lens,
using the plane mirror and open diaphragm, with the
Abbé condenser.

Use of Oil-Immersion Lens.

In using the oil-immersion lens, first place a drop of
immersion oil on the centre of the film to be examined,
and placing the preparation on the centre of the micro-
scopic stage, fix one end of the slide with a clip. Lower
the lens with the coarse adjustment, till it touches the
drop. With the eye to the ocular, in stained prepara-
tions, lower very slowly till the field becomes colored.
In unstained preparations always use the fine adjust-
ment after the lens touches the oil. Now, with the fine
adjustment carefully lower the lens till the field comes
clearly into view. The slide can be readily moved
about at the unclipped end. Before removing slide
always raise the objective well out of the oil with the
coarse adjustment.

2. Examine in like manner the liquefying gelatine
cultures of Bacillus pyocyaneus and of Sarcina lutea,
substituting Loeffler’s methylene blue as staining agent,
the films being left in the stain from two to three
minutes.

3. Examine in like manner the broth culture of
Torula rosea, using Loefiler’s methylene blue as stain.

6 PRACTICAL BACTERIOLOGY.

One-half to one minute will suffice for staining, as
torulae stain rapidly as a rule.

4. Prepare in like manner a cover glass film of the
broth culture of Bacillus proteus mirabilis. Stain with
carbol-fuchsin, one-quarter to one-half minute, wash,
dry and mount in Canada balsam.

GRAM’S METHOD, 7

DEMONSTRATION ITI.

Examination of pure cultures of Bacteria grown on solid media—
Gram’s ‘method of staining.

1. With a sterile platinum loop, place a drop of
sterile distilled water on a cover glass. Remove then,
with due precautions, a minute amount of Bacillus pro-
digiosus, and smear with the water, carefully over the
cover glass surface. Dry and pass three times through
the flame. Asa rule no clearing in acetic acid will be
needed in examining bacteria from solid media. Float
the cover glass in anilin gentian violet for one to two
minutes. Wash in water, dry and mount in Canada
balsam.

2. Examine in like manner the potato culture of
Bacillus ruber-and the gelatine culture of Bacillus
cyanogenus. Use either Loeffler’s methylene blue or
diluted gentian violet for staining.

Gram’s Method.

Gram’s method of staining is based on the fact that,
when some bacteria are stained with certain dyes (of
which anilin gentian violet is the principal), and then
treated with a solution of iodine, the iodine fixes the
stain so that it is not washed out by after treatment
with alcohol. It affords a good differential stain, as
some species stain, while others do not. Various modi-
fications will be noted in this method, for the staining
of pus and tissues.

8 PRACTICAL BACTERIOLOGY.

3. Prepare cover glass films from the gelatine culture
of Streptococcus pyogenes, and also from the liquefying
gelatine culture of Staphylococcus pyogenes aureus.
Stain them according to Gram’s method as follows:

(a) Place the films in alcohol one to two minutes.

(b) Without drying, transfer to anilin gentian violet
for two minutes.

(c) Rinse in water or .6% saline solution, and then
place for one-half to one minute in Gram’s iodine solu-
tion, (Iodine 1 part, Potassium iodide 2 parts, Water
300 parts).

(d) Rinse in water, end then in alcohol, till no more
stain comes away.

(e) Wash in water, dry and mount in Canada balsam.

HANGING DROPS. 9

DEMONSTRATION IV.

Examination of Bacteria unstained—Hanging Drops—
Hanging Drop Cultures.

1. With a sterile loop remove a drop of the liquefied
gelatine culture of Staphylococcus pyogenes aureus, and
place on a cover glass. Invert this cover glass on a
slide.

Examine with the J, oil-immersion lens, shutting
off nearly all the light; or examine, after removing the
Abbé condenser, using the concave mirror and partially
closed diaphragm.

2. Repeat this procedure, using a drop of the broth
culture of Bacillus proteus mirabilis. Slight motility
of the bacilli may be seen.

3. Examine in the same manner a drop of putrefy-
ing urine (48 hours old). Large numbers of bacteria,
usually of several species, are seen.

Hanging Drops.

Hanging drop preparations are made to study
motility of bacteria. Under the microscope we may
make out three kinds of movement: (a) Current move-
ment, where the bacteria move with the suspending
fluid. (6) Molecular or “Brownian” movement, where
the bacteria vibrate in the fluid, as do all solid particles
suspended in fluids. In this movement there is no
actual change in position of the bacterial cells. (c)

10 PRACTICAL BACTERIOLOGY.

Actual movement, where the bacteria swim or move
about in the suspending fluid. This last is what we
mean by motility of a bacterium.

4, Make a hanging drop preparation from the 18
hours, broth culture of Bacillus typhosus supplied.

(a) Transfer a drop of the culture toa clean cover
glass. (Never neglect aseptic precautions).

(b) Ring about with vaseline, the depression on a
hollow ground slide.

(c) Invert the cover glass over the cell and press
firmly into the ring of vaseline, so as to exclude the air.

In examining the preparation find the edge of the
drop with the low dry power, then fixing the slide,
examine with the oil-immersion lens, partially closing
the diaphragm.

5. In like manner examine the liquefied gelatine
culture of Staphylococcus pyogenes aureus.

No motility is present, but Brownian movement can
be noted.

Hanging Drop Cultures.

Hanging drop cultures are used tostudy the manner
of division, the process of spore formation and germina-
tion, and the motility of bacteria. The three former
require continual microscopic observation of the culture
drop, at certain periods.

6, Make hanging drop cultures of Bacillus anthracis
and of Streptococcus pyogenes,

(a) Place cleansed cover glasses, sterilized by pass-
ing through the flame, on a strip of wire gauze, sterilized

HANGING DROP CULTURES. 11

in like manner, and covered by a bell jar, sterilized by
washing in 1:1000 bichloride of mercury.

(b) With a sterile loop carefully place drops of
broth (sterile) on the cover glasses.

(c) Lightly inoculate these drops with the respective
bacteria.

(d) Invert these drops over the cell of a hollow
ground slide, previously sterilized, and having edge of
cell ringed with vaseline.

(e), Place in the incubator at 87°C, on slide rack.

12 PRACTICAL BACTERIOLOGY.

DEMONSTRATION V.

Examination of Hanging Drop Cultures—Spores and
Spore Staining.

1. Examine the hanging drop culture of Streptococ-
cus pyogenes made last day, with /, oil-immersion lens;
at the margin of the drop long chains of streptococci
can be seen.

2. Stain the hanging drop culture of Streptococcus
pyogenes, just examined.

(a) Remove the oil carefully with blotting paper,
and then raising up the cover glass, clear away the
vaseline from its edges.

(b) Dry, and pass film three times through the flame.

(c) Stain the film by Gram’s method.

3. Examine the hanging drop culture of Bacillus
anthracis with the low and high dry lenses and the 7,
oil immersion.

Note the long segmented threads at margin of
culture drop, and the presence in the rods of oval refract-
ing bodies—spores.

4, Stain the hanging drop culture of Bacillus
anthracis with Loeffler’s methylene blue, removing the
cover glass as described above, and clearing the film
before staining in 10% acetic acid.

The spores remain unstained as oval refracting
bodies, both free and in the rods,

STAINING OF SPORES. 13

Staining of Spores.

Potato cultures of Bacillus anthracis and Bacillus
lactis viscosus are supplied. (B. megatherium, B. sub-
tilis or B. filamentosus might be employed.)

(a) Prepare thin cover glass films of these bacteria
in the usual manner, dry and fix in the flame.

(b) Float in Ziehl’s carbol-fuchsin, or in anilin water
fuchsin, for 20 to 30 minutes, keeping the staining fluid
warm.

(c) Wash the films in water and rinse for two to
three seconds in acid alcohol. (Alcohol 97 cc., Hydro-
chloric acid 8 cc.) At this stage the specimen may be
examined by mounting in a drop of water. The acid
decolorizes the rods, leaving the spores stained. If the
bodies of the bacilli are still red, the film must be again
washed through the acid alcohol. If the spores are not
stained place the film again in the carbol-fuchsin.

(d) If the spores are properly stained, place the film
for one to two minutes in Loeffler’s methylene blue to
stain the rods.

(e) Wash in water, dry thoroughly and mount in
Canada balsam.

The spores are stained red, rods blue.

14 PRACTICAL BACTERIOLOGY.

DEMONSTRATION VI.

Staining of Flagella—Cultures of Bacillus Anthracis.

Flagella Staining.

Ordinary methods of staining do not suffice to
demonstrate flagella, so that special staining processes
are required. Emulsions in sterile distilled water of 18
hour agar cultures of Baciilus typhosus and of Spirillum
cholerae asiaticae are furnished.

1. Muir’s modification of Pitfield’s method.

The following re-agents are required:

The mordant,
10% aqueous solution tannic acid, filtered 10 cc.
Saturated aqueous solution, bichloride

of mercury . , ‘ ‘ . 5ee.
Saturated aqueous cobain alum . . 5ee.
Ziehl’s carbol-fuchsin . ‘ : . 2ce.
This mordant will keep for one or two weeks.
The stain,
Saturated aqueous solution of alum,
filtered . ; ; ; ‘ . 10 ce.
Saturated alcoholic selukion of gentian
violet . ‘ : . 2ce.

This stain will keep two or ales cave
The cover glasses used must be thoroughly cleansed.
This may be easily effected by rubbing first with a clean
cloth, then washing in a mixture of equal parts of

FLAGELLA STAINING. 15

alcohol and ether, and while moist passing them through
the flame. Cover glasses may also be cleansed by the
method advised by van Ermenghem, as follows:

Boil the cover glasses for five minutes in the follow-
ing solution,

Potassium bichromate : . 6 grammes.
Sulphuric acid . F : . 6 grammes.
Water F ; ‘ ; . 100 ce.

From this solution wash through several dishes of
distilled water, and then place in alcohol. Before using
pass through the flame.

(a) Prepare thin films from the emulsions; dry and
fix in the flame.

(b) Cover the film (held in forceps) with the mordant,
and hold high over the flame till it steams gently for
one minute. (Or float the film in the warmed mordant
for the same period.)

(c) Wash thoroughly in large amounts of distilled
water.

(d) Pour on now some of the stain, and again heat
gently for one minute (or place in the warmed stain).

(e) Wash in water, dry and mount in Canada balsam.

Those bacteria which stain by Gram’s method can
be treated after the stain by iodine in the usual manner.
Fair results are usually obtained by this method.
Better microscopic pictures can be secured by van
Ermenghem’s method, but his method is longer and
usually requires considerable practice.

16 PRACTICAL BACTERIOLOGY.

Cultures of Bacillus Anthracis.
From the culture tube supplied inoculate,
(a) an agar-agar (slope) tube;
(b) a potato tube;
(c) a gelatine (stab) tube;
(d) a broth tube;

(e) a broth tube, to which carbolic acid has been
added in proportion of 1 to 1000;

(f) a litmus milk tube.

Place all except (c) in the incubator at 37°C, and
keep (c) at 20°C.

Examine these growths at the end of 24, 48 and 72.

hours, noting the characters of growth on the various
media.

BACILLUS ANTHRACIS, 17

DEMONSTRATION VII.

Bacillus Anthracis in pure cultures—Tissues of animal dead of
Anthrax Septicaemia—Inoculation of animal with
Bacillus Anthracis.

1. Make cover glass preparations from the agar, and
the broth cultures of Bacillus anthracis.

Stain with Loeffler’s methylene blue, diluted gentian
violet or by Gram’s method.

2. Make cover class preparations from the carbolized
broth culture of Bacillus anthracis.

Stain with lLoeffler’s methylene blue. No spores
are seen.

3. Inoculate two litmus milk tubes, one from the
ordinary broth culture and the second from the car-
bolized broth culture of Bacillus anthracis.

Incubate at 37°C and examine day by day. The
latter produces less acid. (By animal experiments it
could also be shown to be less virulent).

Tissues of Animal dead of Anthrax Septicaemia.
Sections from lung, liver and kidney are supplied.
4, Staining with Loeffler’s methylene blue.

(a) Place sections in the stain for 5 to 10 minutes.

(b) Remove excess of stain by washing in water.

(c) Place in 4 to 1% acetic acid for 10 to 20 seconds,
till the sections become a light blue.

18 PRACTICAL BACTERIOLOGY,

(d) Immediately wash in fresh water.
(e) Dehydrate in absolute alcohol for 1 to 2 minutes.
(f) Clear in xylol for 1 to 2 minutes.

(g) Transfer to slide with section lifter and mount
in Canada balsam.

Sections after dehydration may be transferred to
slide and cleared there with xylol before mounting, as
xylol causes at times considerable shrinkage and
curling.

Examine with the low and high dry lenses and the
zz oil immersion. Anthrax bacilli are seen in the
capillaries of the organs.

5. Staining by Gram’s method and eosin.

(a) Place the sections in alcohol for 1 minute.

(b) Transfer to anilin gentian violet, 5 to 10 minutes.

(c) Rinse the sections in distilled water, and then
place for 2 minutes in Gram’s iodine solution.

(d) Rinse in water, and then in alcohol for 4 minute.
(e) Place for 1 minute in eosin staining fluid,

Alcoholic eosin ‘ ; 3 . gramme,
Alcohol . i 3 ‘ : 10 cc.
Water : ; : ‘ : 30 ce.

(f) Remove excess of eosin by washing in water and
then in alcohol.

(g) Transfer to acid alcohol for 5 to 10 seconds, and
again wash in alcohol till the sections become red.

(h) Clear in xylol, transfer to slide, and mount in
Canada balsam.

BACILLUS ANTHRACIS. 19

6. Staining with picrocarmine and Gram’s method.

(a) Place the sections in picrocarmine 20 to 30
minutes.

(b) Wash in water to which a few drops of hydro-
chloric acid have been added.

Then pass the sections through the process described
under (5) with the exception of counterstaining with
eosin.

Inoculation of Mice or Guinea Pigs Subcutaneously
with Bacillus Anthracis. (Class Demonstration.)

The animal is placed in a holder or held by an
assistant. The hair is clipped from the point of inoc-
ulation, (root of tail in mouse, inner side of thigh in
guinea pig). The skin at this point is then washed
with 1:1000 solution of bichloride of mercury. Witha
sterile scissors a small snip is made through the skin.
With a platinum loop a drop of 48 hours broth culture
of Bacillus anthracis is introduced and pushed well up
under the skin. The point of introduction is then
lightly seared with a heated glass rod,

20 PRACTICAL BACTERIOLOGY.

DEMONSTRATION VIII.

Examination of guinea pig dead. of Anthrax Septicaemia—Plate
cultivations—Cultures of the Pyogenic Bacteria.

Guinea Pig dead of Anthrax.

1. (a) Carefully examine the animal, noting the
swelling extending from the groin, to a variable extent
over the abdomen. Then stretch out the animal by
tacking down the legs to a board, moistened with
1: 1000 bichloride of mercury solution.

(b) Lay the hair of the animal by moistening with
methylated spirit.

(c) With a sterilized scissors carefully cut through
the skin along the median line and reflect it from over
the thorax, abdomen and thighs.

(d) Note the situation and character of the cedema.
From the cedema fluid, smear cover glass films for after
staining. (Cultures might also be made.)

(e) Sterilize the abdominal wall in the midline and
the thorax at the rib-costal junctions, with a flat-bladed
knife, heated to redness.

(f) With sterilized forceps and scissors open into
the abdomen along the midline. With fresh forceps
remove the spleen and break a portion of it upin a
sterile capsule. From the: broken pulp, make cover
glass preparations by placing a drop of pulp on one
cover glass and dropping a second cover glass upon this,

ANTHRAX SEPTICAEMIA. 21

spread out the drop into a thin film, and carefully slide
the two cover glasses apart.

(g) Open carefully the thorax, cutting through the
costal cartilages, and expose the heart.

(h) Sterilize a portion of the exposed heart wall by
searing it with a heated glass rod. Push through this
part, a sterile pipette and withdraw the blood of the
chambers. Several pipettes may be filled in this man-
ner, from one of which cover glass preparations can be
made as described in (f) for staining. On withdrawing
pipettes fuse the open ends carefully in the flame.
Preserve the pipettes for the making of plate cultiva-
tions.

The remaining viscera of the animal may be removed
and preserved in 4% formalin for the after study of
sections. :

Staining of the Cover Glass Films.

Fix the films after drying by passing through the
flame. Better results are secured in staining the pre-
parations from the spleen pulp and the blood by fixing
the films in a 10% solution of formalin in 90% alcohol,
for one minute.

2. Staining with eosin and methylene blue.
(a) Place the fixed films in eosin 4 to 1 minute.

(b) Wash in water and then dry.

(c) Place now in Loeffler’s methylene blue for 1 to
2 minutes.

(d) Wash in water, dry and mount in Canada balsam.

22 PRACTICAL BACTERIOLOGY.

Examine with the dry and oil-immersion lenses.
Anthrax bacilli and nuclei are stained blue, the red
corpuscles and eosinophile granules of leucocytes, pink.

Spores are not seen in the animal body.

3. Staining by Gram’s method and eosin.

(a) If films have been fixed by heat place in alcohol
first for 1 minute; if by formalin place at once in anilin
gentian violet for 2 to 3 minutes.

(b) Rinse in water and transfer to Gram’s iodine for
1 minute.

(c) Rinse again in water and then in alcohol till
stain ceases to come away.

(d) Place in eosin, 4 to 1 minute.

(e) Wash in water, dry and mount in Canada balsam.

Plate Cultures.

4. Make plate cultivations from blood of heart of
guinea pig, dead of Anthrax septicaemia.

(a) Liquefy two or three tubes of gelatine in water
bath, or incubator at 37°C.

(6) Observing all aseptic precautions, with a steril-
ized forceps break off the fused end of pipette contain-
ing blood from the heart, and by carefully applying
heat to bulb of pipette, allow one drop of blood to fall
into one of the liquefied gelatine tubes.

Label this tube No. 1.
(c) From tube No. 1, after careful admixture of the

blood drop, transfer three loops to a second liquefied
tube (No. 2). If microscopic examination has shown a

PLATE CULTURES. 23

farge number of bacilli to be present, a further dilution
can be made from tube No. 2.

(d) Heat the tops of the inoculated tubes to assure
thorough sterilization, and then permit them to cool.

(e) Remove the plugs and carefully pour the gela-
tine into sterilized Petri dishes, causing the gelatine to
spread thoroughly over the plate surface.

(f) Set the gelatine by placing the plates on blotting
paper moistened with cold water, and then place in
incubator at 20°C.

Examine at the end of 24, 48 and 72 hours, noting
the character and number of colonies, and the presence
of other species of bacteria.

Esmarch roll tubes may be prepared instead of plates,
by spreading the liquefied gelatine over the sides of the
tubes and setting the gelatine rapidly by revolving the
tubes on ice.

5. Make cultures of Staphylococcus pyogenes aureus,
Staphylococcus pyogenes albus and Bacillus pyocyaneus
in agar, potato and gelatine (stab) tubes.

6.: Make cultures of Streptococcus pyogenes in agar,
gelatine (slope) and broth tubes.

Examine these cultures day by day, noting the gen-
eral characters of growth.

24 PRACTICAL BACTERIOLOGY.

DEMONSTRATION IX.

Impression Specimens of Bacillus Anthracis—Pyogenic Micrococci—
Staining of Pus—Agar Plates.

1. Impression specimens of Bacillus anthracis.

(a) Examine the Anthrax plate cultures and when
colonies appear, gently press over a part showing them
a sterile cover glass.

(b) Carefully raise the cover glass, dry by holding
high over the flame, and pass three times through the
flame.

(c) Stain the film in Loeffler’s methylene blue, or by
Gram’s method.

A stained impression of the colonies will be obtained.

2. Prepare cover glass films from the agar or potato

cultures of Staphylococcus pyogenes aureus, albus, and
Bacillus pyocyaneus.

Stain with anilin gentian violet, or Gram’s method.
(B. pyocyaneus does not stain by Gram’s method.)

3. Prepare cover glass films from the agar and the
broth cultures of Streptococcus pyogenes.

Stain with anilin gentian violet, or by Gram’s
method. Contrast the appearances microscopically.

4, Staining of pus from an abscess.

(a) Make cover glass films by placing a drop of the
pus (obtained after the first gush, from an opened

AGAR PLATES FROM PUS. 25

abscess) on a cover glass, dropping upon this a second
cover glass and sliding the two apart.
(b) Dry and fix by passing through the flame.
(c) Stain these films,
(1) with eosin and methylene blue (page 21);
(2) with diluted gentian violet;
(3) by Gram’s method and eosin (page 18).

Agar Plates from Pus.

5. (a) Liquefy two agar tubes by heating to 100°C.
for several minutes. Permit these tubes to cool in
water. (Agar sets at 38°C.)

(b) Inoculate a few ce. of sterile water or a broth
tube with two loops of the pus furnished.

(c) When the agar tubes have cooled to 42°C. rapidly
inoculate a tube with three loops from the inoculated
water or broth tube.

(d) From this tube inoculate the second tube, using
three loops. Place the tubes in water at 42°C.

Sterilize the tops of the tubes, and when cooled pour
into Petri dishes in the same manner as described in
making gelatine plates.

Incubate at 37°C., examining after 24 and 48 hours.

The use of plates in ordinary practice is not neces-
sary, as tubes can be smeared either directly from the
pus, using minute amounts, or better, can be smeared
from the dilution in sterile water or broth.

26 PRACTICAL BACTERIOLOGY.

DEMONSTRATION X.

Examination of Plate cultures—Gonorrheal pus—Pus from
Actinomycosis.

1, Examine the plate cultures from pus, made last
day. Make agar and gelatine sub-cultures of any differ-
ent varieties of bacteria that may be present.

Examine the cultures daily, noting characters of
growth, so as to aid identity of the species.

2. Make cover glass preparations from any different
varieties of bacteria developing on plates.

Stain with anilin gentian violet, and also by Gram’s
method.

Staining of Gonorrhceal Pus.

In the usual manner prepare cover glass films of the
pus. It is best to make these directly from the dis-
charges.

3. Stain the dried and fixed films in eosin and
methylene blue.

Note the relationship of the Gonococci to the pus cells.

4, Stain other films in anilin gentian violet for one
minute. Mount these films in water and examine for
Gonococci. If present, remove film from slide and wash
rapidly several times through alcohol. Then wash in
water, dry and mount in the usual manner. The
Gonococcus is decolorized. A second film may be
treated by Gram’s method. The Gonococcus does not
retain the stain, when so treated.-

PUS FROM ACTINOMYCOSIS. 27

Pus from Actinomycosis.
Note the minute greyish yellow granules in the pus.
5. Place on a slide a drop of the pus, containing
one or more granules. Treat it with several drops
10% caustic potash solution and press on a cover glass.
Examine with low and high dry lenses. Radiating

masses of the fungus and separate threads can be readily
detected.

6. Prepare cover glass films from the pus, and stain
by Weigert’s modification of Gram’s method.

(a) Place the films in alcohol 1 to 2 minutes.

(b) Transfer to anilin gentian violet, 10 to 15
minutes.

(c) Rinse in water and then transfer for 2 to 3
minutes to Weigert’s iodine solution,

Todine : : ; : : 1 gramme.
Potassium iodide. : : 2 grammes.
Water ; : ; : . 100 ce.

(d) Rinse again in water. Counterstain in eosin
for 1 minute.

(e) Wash in water and remove excess of water with
blotting paper.

(f) Dehydrate and decolorize in a solution of anilin
oil 2 parts, xylol 1 part, till violet color has almost
disappeared.

(g) Wash in xylol to remove all anilin oil and then
mount in Canada balsam.

Examine with the low and high dry lenses and with
the oil-immersion lens.

28 PRACTICAL BACTERIOLOGY.

DEMONSTRATION XI.

Micrococcus Lanceolatus (Pneumococcus) in Sputum—Bacillus In-
fluenzez in Sputum—Sections of Pneumonic Lung—
Tissues containing Pyogenic Micrococci.

Pneumonic Sputum.

Fresh sputum is furnished when possible, otherwise
sputum preserved in 5% carbolic acid solution is used.
Films prepared from the preserved sputum have to be
cleared, after fixation in the flame, by placing in a
mixture of equal parts alcohol and ether for 1 to 2
minutes.

Prepare cover glass films from the sputum by placing
asmall portion on a cover glass and, after pressing on
a second cover glass, sliding the two apart.

1. Stain the films so prepared, by Gram’s method,
counterstaining with eosin.

This method stains the capsule fairly well.

2. Stain other prepared films of the pneumonic
sputum in eosin and methylene blue.

3. For staining the Pneumococcus and its capsule
in fresh sputum, use the following method:

(a) Prepare films in the usual manner, dry and fix
in the flame.

(b) Pass the films quickly through glacial acetic
acid.

(c) Without washing, place the film in anilin gen-
tian violet for 5 to 6 minutes.

STAINING OF PNEUMONIC LUNG. 29

(d) Rinse in water, dry and mount in Canada
balsam.

4. Prepare cover glass films from the sputum, (pre-
served in 5% carbolic acid), of a patient with Influenza
Bronchitis. Dry and pass through the flame. Clear
for 1 to 2 minutes in the alcohol-ether mixture.

Stain in Loeffier’s methylene blue or in anilin gen-
tian violet in the usual manner.

The Influenza bacilli appear as fine short rods, at
times forming fine segmented threads. Many lie in pus
cells.

Staining of Pneumonic Lung (red hepatization),
for Fibrin and Pneumococci.

5. Owing to the fragile nature of the sections, the
staining processes are best carried out on aslide. The
use of such fixing fluids as the glycerin albumin mix-
ture, should be avoided, owing to the precipitation of
the stains. The sections are given out in alcohol.

(a) With a section lifter remove a section to a slide,
and remove excess of alcohol with blotting paper.

(b) Cover the specimen with anilin gentian violet for
15 minutes, protecting from dust by placing under a
bell jar.

(c) Remove the stain by blotting paper and carefully
wash with water. _

(d) Treat the sections with Weigert’s iodine for 2 to
3 minutes.

(e) Remove the iodine solution and wash in water.

30 PRACTICAL BACTERIOLOGY.

(f) Cover the section with eosin for 1 minute, and
then wash off with water.

(g) Remove the water with blotting paper, and treat
with the anilin oil—xylol mixture, till no more blue stain
comes away.

(h) Wash several times in xylol and mount in Can-
ada balsam.

. The section might be previously stained in picro
carmine, and then put through this process, with
the exception of the eosin staining. Hither method
usually shows both the fibrin net work and Pneumoccocci
well, when examined with the 54, oil immersion lens.

Staining of Tissues for Pyogenic Micrococci.

6. Stain sections of erysipelas of rabbit’s ear.

(a) By Gram’s method, counterstaining in eosin
(page 18).

(b) By Gram’s method, previously staining in picro-
carmine (page 19).

7. Stain the sections of kidney, from acute suppura-
tive nephritis. (Staphyloocus pyogenes aureus).

(a) With eosin and methylene blue (page 21).

(b), By Gram’s method, previously staining with
picrocarmine or counterstaining in eosin.

These methods may be employed for other tissues,
containing the pyogenic micro-organisms.

TUBERCULAR SPUTUM. 31

DEMONSTRATION XII.

Pure Cultures of Tubercle Bacillus—Tubercular Sputum.

1. From the glycerine agar cultures of the Tubercle
bacillus prepare cover glass preparations, and stain in
the following manner :

(a) Float in Ziehl’s carbol fuchsin for 5 minutes.

(b) Wash in water and then rinse quickly through
334% nitric acid solution.

(c) Wash immediately in'70 % alcohol and then in
water.

(d) Dry and mount in Canada balsam.

2. Prepare other cover glass films and stain in
sudan III. (Dorset).

Sudan III staining fluid consists of a saturated
solution of sudan IIT in 80% alcohol.

(a) Place the prepared films in the stain for 5
minutes.

(b) Wash the films in 70% alcohol and then in
water, dry and mount in Canada balsam.

Sudan ITI has a selective affinity for fat, which is
present in large amount in the bodies of Tubercle
bacilli.

Examination of Sputum for Tubercle Bacilli.

Pour a thin layer of sputum into a glass capsule
and pick out for examination a yellowish (caseous)
particle if present; otherwise choose a purulent part.

32 PRACTICAL BACTERIOLOGY.

Prepare cover glass films by pressing out the particles
between two cover glasses, drying and fixing in the
flame.

3. Staining by Ziehl-Neelsen’s method.

(a) Float the films in Ziehl’s carbol-fuchsin for 5
to 10 minutes, keeping the stain warm. (Do not boil).

(b) Wash off the excess of stain in water.

(c) Rinse quickly through 334% nitric acid. The
red color disappears at once.

(d) Wash immediately in 70% alcohol for 1 to 2
minutes. If the red color which re-appears is persist-
ent, agein pass the film into the acid and rinse in the
alcohol.

(e) Wash the film in water and dry.

(f) Counterstain in Loeffler’s methylene blue for 4
to1 minute. Wash in water, dry and mount as usual.

Tubercle bacilli appear as red rods; other bacteria
and the nuclei of cells are stained blue.

This method is decidedly the most accurate for
routine examination work.

4, Staining by Gabbett’s method.
The same method of procedure is followed till after

the removal of the film from the carbol-fuchsin.. It is
then placed for 830 seconds in Gabbett’s solution:

Methylene blue : 5 : 2 grammes.
Sulphuric acid : : é 25 cc.
Water ; : : , 15 ce.

The film is then washed in water, dried and mounted
in the usual manner.

TUBERCULAR SPUTUM. 33

This method is shorter but it has not proven so
accurate, nor does it give as clear a film as the Ziehl-
Neelsen process.

5. Staining in sudan III.

(a) Place the prepared films in sudan III for 5
minutes.

(b) Wash for 5 minutes in 70% alcohol.

(c) Wash in water and then dry.

(d) Counterstain for 1 minute in Loeffler’s methy-
lene blue. Wash in water, dry and mount in the usual
manner.

Examination of Sputum or Fluids containing
but few Tubercle Bacilli.

When Tubercle bacilli are suspected but cannot be
demonstrated by ordinary examinations, several methods
may be adopted for further microscopic investigation.

(a) Van Ketel’s method. Add to the sputum about
three or four times its volume of 5% carbolic acid. If
the sputum is watery, add pure carbolic acid to make
a5% solution. This is particularly applicable when
examining urine. Shake the mixture thoroughly, for
at least five minutes, to break up sputum particles.
Pour into a conical urine glass and allow to stand 12 to
24 hours. With a pipette, withdraw the lowest portion
of sediment for examination by any of the methods
already given. Films must be cleared in the alcohol-
ether mixture, before staining.

(0) A more rapid method is to add to the sputum
sufficient 10% caustic potash solution, to liquefy it.

34 PRACTICAL BACTERIOLOGY.

This liquefied fluid is allowed to stand for 12 to 24
hours in a conical urine glass, and the sediment exam-
ined. Or a portion of the liquefied sputum may be
centrifuged and the sediment examined at once by the
usual methods. In urine, smegma baciili give staining
re-actions allied to those of the bacillus of Tuberculosis,
If the Ziehl-Neelsen process is accurately carried
through, only rarely will there be danger of mistaking
smegma bacilli for Tubercle bacilli. Sudan III is
however, a positive differential stain.

When microscopic methods fail, inoculation of a
guinea pig subcutaneously or intraperitoneally, will lead
to the development of Tuberculosis, if any living bacilli
are inoculated.

TUBERCLE BACILLI IN TISSUES. 35

DEMONSTRATION XIII.

Sections of Tubercular Tissues—Leprosy Tissues—Cultures of
Tubercle Bacillus from an Inoculated Animal—
Cultures of Bacillus of Diphtheria.

Tubercle Bacilli in Tissues.

Sections are furnished (in water), from acute miliary
Tuberculosis of lung, and also of Tuberculosis of lung
of cattle.

1. Staining in fuchsin solutions.

(a) Place the sections in carbol-fuchsin or in anilin
fuchsin for from 30 minutes to 24 hours, keeping the
stain covered in the incubator at 37°C. Better results
are obtained by leaving in stain for 24 hours.

(b) Rinse the sections in water, and then transfer
for a few seconds to 334% nitric acid.

(c) Wash at once in 70% alcohol till no more red
stain comes away. If the section is still red, wash
again through the acid and then in the alcohol.

(d) Rinse in water and place in Loeffler’s methylene
blue for 1 minute.

(e) Rinse again in water, dehydrate in absolute alco-
hol, clear in xylol and mount on slide in usual manner.

Sections from paraffin blocks, received into water at
48°C, may be fixed on cover glasses and stained as cover
glass films in the following manner:

Float a section on acover-glass and removing from
water, blot firmly and evenly with several layers of

36 PRACTICAL BACTERIOLOGY.

blotting paper. Heat the section carefully to melt the
paraffin, rinse through xylol to free the section from
the paraffin, then in alcohol to free from xylol. From
the alcohol place directly into the carbol-fuchsin.

2. Staining in sudan III solution..

(a) Place the sections for 10 minutes in the sudan
III stain.

(b) Wash for 5 minutes in 70% alcohol.

(c) Rinse in water and transfer for 1 minute to
Leoffler’s methylene blue.

(d) Rinse in water, dehydrate in absolute alcohol,
clear in xylol and mount in Canada balsam.

Leprosy Tissues.

Bacillus leprae stains like the Tubercle bacillus, but
more readily, and is more rapidly decolorized. It does
not stain with sudan ITI.

3. Stain sections by the following methods:

(a) Carbol-fuchsin for 20 to 80 minutes, treating
afterwards as for tuberculous tissues.

(b) Weigert’s method and eosin.
The Leprosy bacilli are always present in large

numbers in the leprous nodules, and the discharges in
ulcerating tubercular leprosy.

Cultures of Tubercle Bacilli from Guinea Pig.

The guinea pig has been inoculated three or four
weeks previously with sputum from pulmonary Tuber-
culosis, and has been killed by chloroforming.

TUBERCLE BACILLI FROM GUINEA PIG. 37

The animal is opened in the usual manner, and the
enlarged inguinal and iliac glands are removed with a
sterile forceps, and broken up in sterile capsules.

4. From the pulp in the capsules inoculate freely,
(a) 6 blood serum tubes;

(b) 6 glycerine broth tubes;

(c) 6 glycerine agar tubes.

After inoculation seal the tubes with ordinary wax
or paraffin, so as to prevent evaporation.

Incubate at 87°C. and examine at the end of 7, 10,
14, and 21 days.

5. From the culture of Bacillus diphtheria furnished
inoculate,

(a) a blood serum tube;
(b) a glycerine agar tube (slope);
(c) a broth tube.

Place in the incubator at 87°C. and examine at end
of 18, 24 and 48 hours.

38 PRACTICAL BACTERIOLOGY.

DEMONSTRATION XIV.
Diphtheria Bacillus—Cultures of Spirilla of Asiatic Cholera, of
Metchnikof, and of Finkler and Prior—Inoculation
of Animals with Diphtheria Bacilli—Diph-
theria Toxin—Antitoxin.

1. Make cover glass preparations from the colonies
of Bacillus diphtheriz on the blood serum and glycerine
agar tubes.

Stain with Loeffler’s methylene blue, and also by
Gram’s method. Note the variable morphology of the
bacilli, and the differences between the growth on the
different culture media.

Clinically, cultures are made on blood serum or
glycerine agar, from suspected throats, and after incu-
bation for 16 to 20 hours, the tubes are examined. The
material is best removed from the throat by a small
swab, prepared by wrapping a fragment of absorbent
cotton about the end of a fairly rigid wire, which is
then placed in a plugged test tube and sterilized in the
hot air sterilizer. The swab is placed against the mem-
brane, and firmly pressed upon it with a slight twirling
motion, taking care not to touch other parts. The
swab is then lightly rubbed over one or more culture
tubes. Immediate staining and microscopic examina-
tion might be made of the material on. the swab, but
owing to the presence of numerous saphrophytic bac-
teria, and of bacteria which somewhat closely resemble
Diphtheria bacilli in morphology, it is best to depend
on cultural examination.

TOXINS AND ANTITOXINS. 39

2. Make inoculations of Spirillum cholere asiatice,
Spirillum Metchnikovi, and Spirillum of Finkler and
Prior on,

(a) agar tubes (slope);
(b) gelatine tubes (stab);
(c) peptone tubes.
Toxins and Antitoxins. (Class Demonstration).

(a) A medium sized guinea pig (about 400 grammes)
is inoculated subcutaneously with .5 cc. of a 24 hours
broth culture Bacillus diphtherie (virulent). The ani-
mal usually dies in from 24 to 36 hours.

Broth cultures of Bacillu sdiphtherie are filtered
through porcelain (Chamberland’s candles), to obtain
the toxins free from the bacilli. Guinea pigs are inocu-
lated and the maximum fatal dose learned and the toxin
is then standardized. (.01 cc. of standard toxin kills a
medium sized pig in 34 days).

(b) A guinea pig is inoculated with the minimum
dose of the toxin (death in 3 days).

(c) A guinea pig is inoculated with 100 times the
minimum fatal dose, plus one unit of some standard
antitoxin. The animal recovers.

(d) A guinea pig is inoculated with 100 times the
minimum fatal dose. 15 minutes afterward one unit of
antitoxin is inoculated. The animal dies.

40 PRACTICAL BACTERIOLOGY.

DEMONSTRATION XV.

Spirillum of Asiatic Cholera—Spirillum of Finkler and Prior—Spiril-
lum Metchnikovi—Indo] Test—Diphtheria Membrane— :
Intraperitoneal Inoculation of Animals.

1. Examine hanging drop preparations from the
peptone cultures, of each of the spirilla.

2. Make cover glass preparations from the agar and
the peptone cultures, of each of the spirilla.

Stain with anilin fuchsin for 830 seconds and mount
in the usual manner.

Contrast the preparations from the different culture
media.

3. Make and stain cover glass preparations from the
10 days old, potato cultures of the Cholera spirillum.

Many involution forms will be found.

4. To the peptone cultures of each of the spirilla
add carefully, with a pipette, 10 drops of sulphuric acid.

A rose red coloration appears with Spirillum
choleree asiaticee and Spirillum Metchnikovi, but not
with the Spirillum of Finkler and Prior. This is due
to the formation by the two former of indol and nitrites.
Indol only gives this reaction in the presence of
nitrites. If no reaction follows the addition of the acid,
add 1 ce. of a .01% solution of potassium nitrite, freshly
prepared. If the reaction does not then develop, indol
is certainly absent.

INTRAPERITONEAL INOCULATION. 41

Stain the sections of diphtheria membrane furnished.

(a) By Gram’s or Weigert’s method, counterstain-
ing with eosin.

Carry through the staining processes on the slide.

(b) In Czinzinski’s solution,

Alcoholic eosin—.5% solution in 70% alcohol 20 ce.
Saturated aqueous solution, methylene blue 40 cc.
Water ‘ : ‘ ; : ‘ : 40 ce.

Place the sections in this stain for 6 to 24 hours.
Wash thoroughly in water till all blue color is removed,
that will come away. Dehydrate in absolute alcohol,
clear in xylol and mount in usual manner.

Intraperitoneal Inoculation of Guinea Pigs.
(Class Demonstration.)

A medium sized guinea pig is chosen. The hair is
removed from the median line of abdomen and the skin
is washed with 1 in 1000 bichloride of mercury solution.
With a sterilized hypodermic syringe .5 cc. of a 24
hour broth culture of moderately virulent Spirillum
cholere asiaticze is thrown into abdomen, the needle
being entered about the centre of the mid-line. The
animal usually dies in from 18 to 36 hours.

42 PRACTICAL BACTERIOLOGY.

DEMONSTRATION XVI.

Examination of animal inoculated with Spirillum Cholere Asiatice—
Cultures of Bacillus Typhosus and Bacillus Coli
Communis—Typhoid Tissues.

Examination of animal inoculated with the Spirillum
of Cholera.

Open up the abdominal cavity of the guinea pig,
using all aseptic precautions (page 20).

Note the condition of the peritoneum and character
of the exudate.

1. With a sterile loop, or capillary pipette, transfer
a drop of the peritoneal exudate to a liquefied gelatine
tube. From this tube inoculate a second.

Pour plates in the usual manner.

Incubate at 20°C. and examine carefully on succes-
sive days.

2. With a sterilized loop, smear a series of cover
glasses, with the peritoneal exudate.

Stain some films with eosin and methylene blue;
others may be stained with anilin gentian violet or
carbol fuchsin after clearing in 10% acetic acid.

3. Carefully open the thorax, sterilize a portion of
the exposed heart surface, and with a capillary pipette,
withdraw blood from the chambers.

From this blood make cover glass preparations, fix
in formalin solution, and stain with eosin and methylene

blue.

BACILLUS TYPHOSUS. 43

4, Make inoculations of Bacillus typhosus and Ba-
cillus coli communis on,

(a) agar-agar tubes;

(6) gelatine (slope) tubes;

(c) liquefied grape sugar gelatine;

(d) potato tubes;

(e€) peptone tubes;

(f) litmus milk tubes.

Incubate all except the gelatine tubes at 37°C. Ex.
amine and contrast the growths from day to day.

5. Sections of mesenteric gland, spleen and liver
from Typhoid fever cadaver.

In the tissues Bacillus typhosus is found scattered
in clumps, so that a number of sections may have to be
examined before finding any bacilli. At least six sec-
tions of each of the tissues should be stained.

(a) Stain in Loeffler’s methylene blue, leaving in the
stain from 30 minutes to 2 hours. Rinse in water
and then in .1% acetic acid for 2 or 3 minutes; dehy-
drate in ebsolute alcohol, clear in xylol and mount in
balsam.

(b) Stain in Stirling’s gentian violet solution,

Gentian violet : ; : . 5 grammes.
Alcohol . ‘ ; F : 10 ce.
Anilin oil . : ‘ : : 2 ce.
Water. j : ; . 88 cc.

Leave sections in this iat for 10 minutes. Rinse
in water and then in .1% acetic acid for 2 or 3 minutes;
dehydrate in alcohol, clear first with oil of cloves and
then wash several times with xylol before mounting in
balsam.

44 PRACTICAL BACTERIOLOGY.

DEMONSTRATION XVII.

Cultures of Bacillus Typhosus and Bacillus Coli Communis—Serum
Diagnosis of Typhoid Fever—Cultures of Bacillus Pestis
Bubonice, Bacillus Icteroides and
Bacillus Mallei.

1. Make and compare hanging drop preparations
from the peptone tubes of Bacillus typhosus and Bacil-
lus coli communis,

2. Examine the peptone cultures of Bacillus typhos-
us and Bacillus coli communis for indol, adding to each
tube 1 cc. of a .01% fresh solution of potassium nitrite,
before the addition of the sulphuric acid.

Bacillus coli communis gives the indol reaction.

3. Make cover glass preparations of Bacillus
typhosus and Bacillus coli communis. Stain in anilin
gentian violet or in Loefiler’s methylene blue.

The serum diagnosis of Typhoid Fever and
Typhoid Bacilli.

In the blood serum of animals immunized against
Bacillus typhosus, and also, at an earlier or later date,
in the blood of persons with Typhoid fever, there are
present certain constituents which possess the specific
property of agglutinating liquid cultures of Typhoid
bacilli. The phenomena noted microscopically are loss
of motility of the bacilli and their aggregation in
clumps. In tube experiments, we have precipitation of
the contained bacilli. For the proper application of

SERUM DIAGNOSIS OF TYPHOID. 45

this test, an 18 to 24 hour broth culture, or suspension,
of moderately virulent Typhoid bacilli, and certain
dilutions of the serum used, are required.

-A dilution of at least 1 part of serum in 30 is neces-
sary. The time limit for the reaction should be set at
2 hours. Usually the phenomena are noted within a
few minutes.

Blood, from a fatal case of Typhoid fever, dried on
sterile thin filter sheets and cut into squares of 1 cc. is
furnished. (Blood may also be preserved in standardized
capillary pipettes).

4. With sterile forceps place a dried blood square
in a sterile glass capsule. With a medicine dropper
carefully add 15 drops of sterile distilled water, so as
to extract the agglutinating constituents.

On a sterile cover glass place one drop of this ex-
tract, and add one drop of the 18 hour broth culture of
Bacillus typhosus furnished. This makes a dilution of
about 1 in 30.

Examine in hanging drop, under the microscope, and
note the characteristic reaction.

5. Repeat this procedure, using 1 drop of the extract
to 3 drops of the broth culture of Bacillus typhosus, giv-
ing a dilution of about 1 in 60. Examineas before and
note results.

6. Repeat this procedure using 1 drop of the extract,
and 1 drop of a broth culture of Bacillus coli communis.
No clumping occurs.

For clinical examination in cases of Typhoid fever,
blood may be taken from the lobe of the ear or the fin-

46 PRACTICAL BACTERIOLOGY.

ger, after thorough cleansing of the parts, and allowed
to dry on firm white paper or on a slide. The above
method can then be carried out.

Cabot advises an excellent method for bedside
examination. With a medicine dropper, one drop of
blood from the patient is added to 80 (or more) drops
of broth culture, or emulsion of Typhoid bacilli. Drops
from this dilution are then examined in hanging drop
preparations.

7. Make inoculations of Bacillus pestis bubonicze
and Bacillus icteroides on,

(a) agar tubes;

(6) gelatine (slope) tubes;

(c) broth tubes.

Inoculate with Bacillus mallei,

(a) an agar tube;
(b) a potato tube.

Examine these tubes from day to day, noting char-
acters of growth.

BACILLUS PESTIS BUBONICE, 47

DEMONSTRATION XVIII.

Bacillus Pestis Bubonice—Bacillus Icteroides--Bacillus Mallei—
Cultures of Tricophyton Tonsurans and Achorion
Schonleinii—Hair in Tinea Tonsurans.

1. Prepare cover glass films from the agar cultures
of Bacillus pestis bubonice, Bacillus icteroides and
Bacillus mallei. Stain in anilin gentian violet or in
anilin fuchsin.

2. Remove in the usual manner a small portion of
the growth of Tricophyton tonsurans from the cultures
furnished. Tease out on a slide after treatment with
10% caustic potash solution. Examine with the low
and high dry lenses.

3. In like manner examine a preparation from the
culture of Achorion Schonleinii (Favus).

Compare this fungus with Tricophyton tonsurans.

4, Treat in the same manner the hairs furnished
from Tinea tonsurans. The fungus threads and spores
are seen lying in the root sheath and also in the hair
shaft.

This method of examination may be adopted for the
detection of the fungus elements in Ringworm else-
where (Tinea barbs, Tinea circinate), and also in
Favus and Tinea versicolor.

48 PRACTICAL BACTERIOLOGY.

DEMONSTRATION XIX.

Anaerobic Cultures—Bacillus Tetani— Bacillus CEdematis Maligni—
Bacillus Capsulatus Aerogenes—Inoculation of Animals with
Bacillus Tetani, Bacillus Gidematis Maligni and
Bacillus Capsulatus Aerogenes.

1. Make inoculations in the usual manner of Bacillus
tetani, Bacillus ceedematis maligni and Bacillus capsu-
latus aerogenes on,

(a) agar tubes (stab) to which .5% formate of soda
has been added;

(b) grape sugar gelatine tubes (stab);

(c) grape sugar broth tubes.

Place the broth cultures in Novy’s jar, and treat the
agar and gelatine cultures by Buchner’s method, as de-
scribed below.

Anaerobic Culture Methods.

Anaerobiosis may be obtained in several ways. One
of the most commonly employed methods, particularly
when working on a large scale, is to replace the oxygen
(air) by hydrogen gas. For culture tubes and plates
Novy’s jars are very convenient for use with hydrogen.

2. The cultures being placed in the jar, hydrogen
from a generating apparatus (Kipp’s) is allowed to
stream into the jar for one hour. The hydrogen is
usually purified by passing through a series of wash
bottles containing lead nitrate in the first, silver nitrate
in second and freshly prepared pyrogallate of potash in

ANAEROBIC CULTURE METHODS. 49

the third. At the end of one hour turn the stopper
of the jar, which automatically seals it. The same
process can be carried out by using an ordinary large
tube with a rubber stopper perforated with two holes
for glass tubing. After the hydrogen has been passed
in for an hour, seal in the flame the glass tubing, clos-
ing first the exit tube:

Place the jar in the incubator at 87°C.

Another method of producing anaerobiosis, which is
much employed is that of Buchner. It consists in the
absorption of the oxygen by the use of freshly prepared
pyrogallate of potash. This may be carried out in
large jars, or in large test tubes.

3. Treat the agar and gelatine cultures made, by
Buchner’s method.

(a) In the bottom of the large test tubes furnished,
place one-half inch layer of sand, and then add 1
gramme pyrogallic acid.

(b) Now place in this test tube one of the inoculated
tubes and add 10cc. of 1% caustic potash solution,
taking care not to soil the inoculated tube.

(c) Immediately plug firmly the large test tube with
a rubber cork. Pour melted wax or paraffin about the

»edges of the cork, to secure perfect sealing.

The oxygen is quickly absorbed and the bacteria

develop in an atmosphere of nitrogen.

Place the agar tubes in the incubator at 37°C. and
keep the gelatine tubes at 20°C.

50 PRACTICAL BACTERIOLOGY.

Class Demonstration.

(a) Inoculate a guinea pig subcutaneously with .25
ce. of a 72 hour broth culture of Bacillus tetani, tearing
the tissues slightly at point of inoculation.

(b) Inoculate a guinea pig subcutaneously with .5 cc.
of a broth culture of Bacillus cedematis maligni.

(c) Inoculate .5 cc. of a broth culture of Bacillus
capsulatus aerogenes into the posterior auricular vein
of a rabbit. Five minutes after the inoculation, kill the
rabbit by a sharp blow on the back of.the neck. Keep
the body in a room at a temperature of 20°C and exam-
ine after 18 to 24 hours. Gas formation is seen in the
veins everywhere, the viscera will be “foaming,” and
the animal generally emphysematous.

BACILLUS TETANI, al

DEMONSTRATION XX.

Bacillus Tetani—Bacillus (idematis Maligni—Bacillus Capsulatus
Aerogenes—Examination of Animals Inoculated with Bacillus
(Edematis Maligni and Bacillus Capsulatus Aerogenes.

1. Make hanging drop preparations from the broth
culture of Bacillus tetani.

Slight motility can usually be detected.

2. Make cover glass films from the broth cultures of
Bacillus tetani, Bacillus cedematis maligni and Bacillus
capsulatus aerogenes.

After clearing in 10% acetic acid, stain in anilin
gentian violet. Note the presence and position of the
spores in the two former bacilli.

3. Examine carefully the guinea pig inoculated with
Bacillus cedematis maligni, noting the extent and char-
acters of the cedema. Reflect the skin in the usual
manner. Make cover glass smears from the cedema

fluid.

Open up the abdomen, and removing spleen, make
cover glass preparations of the splenic pulp, as de-
scribed under anthrax. In like manner make cover
glass preparations from the heart’s blood.

After drying fix these cover glass films in the form-
alin fluid for 1 minute.

Stain the films with eosin and methylene blue.

52 PRACTICAL BACTERIOLOGY.

Examine the animal inoculated with Bacillus capsu-
latus aerogenes, and then killed and kept 18 to 24
hours.

Make cover glass preparations from the blood of the
various organs. Stain with eosin and methylene blue.
Capsules are seen surrounding the bacilli. These may
be better demonstrated by staining in the manner de-
scribed for staining the capsule of the Pneumococcus.
(Page 28).

PART II.
BACTERIOLOGICAL ANALYSIS.

Culture Media.

For a study of the general biological characters of
bacteria, growths in pure culture are required. For
this growth certain nutrient materials are necessary, the
nutrients varying more or less for the various species of
bacteria. For the pathogenic bacteria, media approxi-
mating to the nutrients of the body are most employed.
Desirable characters in culture media are clearness and
ease of preparation and sterilization. The methods of
preparation of the culture media in common use only,
will be given.

Broth or Bouillon is much employed itself and as
the basis for nutrient jellies, as agar-agar and gelatine.

One pound of lean beef-steak, freed from all fat and
connective tissue bands, is chopped up in a sausage
machine. 1,000 cc. of distilled water are added and the
meat is lightly boiled in a covered sauce-pan for ? to 1
hour, occasionally being stirred. The mixture is filtered
through several layers of moistened filter paper and dis-
tilled water added to make up to 1,000 cc. Place this
in a flask containing 10 grammes peptone (Witte’s or
Fairchild’s), and 5 grammes of common salt, and steam
in sterilizer for 4 hour.

54 BACTERIOLOGICAL ANALYSIS.

Add a saturated solution of sodium carbonate, drop
by drop, until the reaction becomes faintly alkaline,
using litmus paper as an indicator.

(For greater accuracy in neutralization, phenolph-
thalein in 1% alcoholic solution may be used as an
indicator. To 10 cc. of the broth, add 2 drops of the
indicator and then drop in from a burette a diluted
solution of sodium carbonate, till the indicator shows
alkalinity. A ready calculation can then be made as to
the amount of the sodium carbonate solution it will be
necessary to add to the broth).

Steam again in the sterilizer for 4 hour and then fil-
ter into a flask.

Steam this flask in the sterilizer for 20 minutes, on
3 successive days.

Instead of using beef steak, Liebig’s meat extract
may be employed, 3 grammes of this extract being dis-
solved in 1,000 cc. of water, with 10 grammes of peptone
and 5 grammes of salt. The after treatment is the
same as before described, except that it is better not to
filter for the last time, till the broth is cold.

Glycerine Broth is ordinary broth to which 3 to 6%
of pure glycerine is added, before sterilization.

Grape Sugar Broth or glucose broth is ordinary
broth to which 1 to 2% of grape sugar has been added.

In Making Tubes place the broth (or liquefied jelly)
in a covered funnel with stop cock or clip attachments.
Add from 6 to 10 cc. to each test tube. The tubes are
then re-plugged, covered with tin foil and sterilized in
the steamer for 15 to 20 minutes on 3 successive days.

CULTURE MEDIA. 55

Gelatine. To 1,000 cc. of the stock broth add 120
grammes of gelatine (“gold label”) cut up into small
pieces. Dissolve carefully over a water bath, and neut-
ralize if necessary with sodium carbonate. Place in a
flask, cool down to 60°C and add the whites of two eggs.
Steam for 4 hour in the sterilizer and filter through
moistened filter sheets, using a warming funnel.
Steam in the sterilizer for 20 minutes on 3 successive
days. Tubes are treated in same manner as broth
tubes.

Grape Sugar Gelatine is stock gelatine to which 1
to 2% of grape sugar has been added.

Agar-Agar. Cut up 15 grammes of agar-agar fibre
fine, and allow to’ soak in .5% acetic acid for 2
hours. Then wash repeatedly in water to free from
all traces of the acid. Place in a flask with 1,000
ce. of stock broth, and dissolve by heating in the auto-
clave, up to 120°C. After removal from the autoclave,
neutralize if necessary with sodium carbonate, cool to
60°C, and add the whites of two eggs dissolved in 50 cc.
of water. Cook in the autoclave for 10 minutes at
110°C, and filter through moistened filter sheets, using
a warming funnel. As a rule the agar runs through
rapidly. If it tends to clog, immediately replace by
fresh paper, keeping the unfiltered solution hot. Steri-
lize the filtered agar in the autoclave for 20 minutes at
120°C,

Another method which is very satisfactory is that of
Ravenel, viz.,

56 BACTERIOLOGICAL ANALYSIS.

(a) Liebig’s meat extract : 3 to 4 grammes.
Peptone , ’ : . 10 grammes.
Salt. ‘ F ; ; 5 grammes.
Water (distilled) . ‘ . 500 ce.

Boil up together for 15 minutes.

(b) Agar (prepared as above described) 15 grammes.
Distilled water ; : 7 500 ce.

These are placed in a flask in the autoclave, and the
temperature run up to 134°C.

Mix the two solutions, neutralize, cool to 60°C. add
the whites of two eggs, and proceed as before described.

Blood Serum. Hither the serum which separates
from the clot or the fluids which are effused in Pleurisy
or Ascites, may be. employed in the making of this
nutrient medium. It is one of the most difficult culture
materials to handle satisfactorily. Blood is collected at
the slaughter house, from cattle or sheep, in well stop-
pered sterile litre jars. The blood is collected after it
has run for some time from the cut vessels, as it lessens
the danger of contamination. The jars are then set on
ice for 24 to 48 hours, and the serum which collects is
syphoned off into sterile flasks, with a sterile pipette.

Several culture media are prepared from this serum.

(a) Loeffler’s serum mixture. To 3 parts of the
serum add 1 part of 1% grape sugar broth. Run into
sterile test tubes and then place these tubes properly
slanted in the serum coagulator, and expose them for
24 to 3 hours to a carefully regulated temperature of
from 90° to 95°C. This firmly sets the tubes. After-

CULTURE MEDIA. 57

wards they may be steamed for 15 to 20 minutes in the
sterilizer on 3 successive days.

(b) Lorrain Smith’s serum. To each 100 cc. of
serum add 1 to 1.5 cc of a 10 % solution of caustic soda.
Place in test tubes and treat as for Loeffler’s serum.
Or the tubes may be slanted, covered with tin foil and
placed in the steamer with the lid raised and exposed to
the steam for one hour. Afterwards they may be
steamed for 15 to 20 minutes on 3 successive days.

(c) Kanthack and Stephen’s serum agar mixture.
In making this culture material it is first necessary to
determine the amount of 10% caustic soda (or potash)
that will be needed to transform the albumins of the
serum into alkali albumins, so that no coagulation
occurs on boiling. 2 cc. is about the usual amount re-
quired per 100 cc, of serum.

To 100 cc. of the serum (or serous exudate) add 2 cc.
of 10 % caustic soda and 1 .5 grammes of prepared agar-
agar fibre. Boil up together and add 4 cc. glycerine
and .5 gramme of grape sugar. Again boil and filter,
using the warming funnel.

This culture material can be treated as agar. It
possesses all the advantages of both serum and agar,
except the clearness of the latter material.

Peptone solution of Dunham. To 100 cc. of dis-
tilled water add 1 gramme Witte’s or Fairchild’s pep-
tone and .5 gramme of common salt. Boil together for
15 minutes, neutralize if necessary with sodium carbon-
ate and again bring to a boil. Filter into flasks or

58 BACTERIOLOGICAL ANALYSIS.

tubes, and sterilize these for 20 minutes in the steam
sterilizer, on 3 successive days,

Milk. Separated milk (readily obtained from cream-
eries) should be used, as otherwise the cream is trouble-
some. If the milk is fresh it may be at once sterilized,
but if acid, first neutralize with sodium carbonate solu-
tion. Sterilization is best effected by steaming for 20
minutes on 3 successive days.

Litmus Milk is made by adding sufficient litmus
solution to neutralized milk, to give a distinct blue
tinge. The milk is then sterilized in the usual manner.

Potatoes. Select fair sized smooth potatoes and
wash thoroughly. Slice off the ends, and with a cork
borer, slightly smaller than the test tubes to be used,
cut out cylinders from 14 to 2 inches long. Cut these
cylinders diagonally across and allow them to soak in a
.5% solution of sodium carbonate for one hour. Now
place in the bottom of the test tubes to be used, a small
plug of moistened absorbent cotton and then put in each,
one of the potato plugs. Sterilize in the autoclave for
45 minutes at 120°C.

Preparation of Utensils.

Test Tubes. If the tubes are new, rinse several
times in water and then swab out with strong nitric
acid. Thoroughly rinse through water and allow to
dry on a draining rack. When dry, rinse out with
methylated spirit and again permit to dry.

Plug these tubes with raw cotton and sterilize in the
dry air oven, at 150°C for 1 hour.

PREPARATION OF UTENSILS. 59

Tubes which contain old culture media or growths
should, after removal of the cotton stoppers, be placed
in 5% carbolic acid for 24 hours. The tubes should
then be boiled in a 5% solution of washing soda for 4
hour. This softens and allows the ready removal of
any remaining material in the tubes. The tubes are
then rinsed in water and treated like new tubes.

Slides. New slides need no particular treatment be-
fore use. Slides used, shauld be placed in turpentine
for 24 hours, so as to free the cover glasses. On removal
from the turpentine rinse through “ waste” alcohol and
then in water. Boil for a few minutes in 5% solution
of washing soda, and again rinse in water, and dry.
Slides may then be kept in alcohol.

Glassware. Thorough rinsing in water suffices for
the cleaning of all glassware not contaminated with
culture media, or like material. When so contaminat-
ed boil in 5% solution of washing soda, and then rinse
in water. Flasks should be rinsed in methylated spirit
and allowed to dry, before sterilization. Sterilization is
effected in the hot air sterilizer at 150° to 300°C, for one
hour.

Instruments. Knives, scissors, forceps and hypo-
dermic needles are best sterilized by boiling from 5 to
10 minutes, after protecting any cutting edges by wrap-
ping absorbent cotton about them. Sterilization may
also be effected in 5% carbolic acid solution or in
1% formalin, exposing the instruments to their action
for at least 4 hour. Sterilization is readily and rapidly
effected, by placing the instruments in the flame, but of -

60 BACTERIOLOGICAL ANALYSIS.

course this rapidly destroys the instrument. Boiling is
certainly the most efficacious method for daily use.

Dressings. Gauze, gauze sponges, towels and ap-
rons are best sterilized by exposing them to the action
of steam in the steam sterilizer for $ to 1 hour. They
should be first loosely wrapped in towels. If necessary,
they may be dried in the hot air sterilizer at 120°C for
15 minutes.

WATER ANALYSIS. 61

WATER ANALYSIS.

For public health purposes a bacteriological exami-
nation of water calls for an answer to the questions, (a)
are any pathogenic bacteria present; (b) has the water
been polluted with sewage ; (c) are there many bacteria
present apart from sewage forms, and if so whence are
they derived. Practically the first and second questions
have to be considered together, for water-borne diseases
are primarily sewage-borne diseases. The third question
seldom assumes much importance from a purely public
health view point. Many bacteria are present in water,
laden with vegetable organic matter, and this class of
water would afford the basis for most examinations in
answer to question (c).

Care should be exercised in the collection of the
sample, so as to obtain a specimen which will give
the general biological characters of a water. For
this reason the water should be collected in cleansed
sterilized jars, bottles or flasks, stoppered with a
closely fitting cork. The samples should be sent
at once for analysis, as even several hours in warm
weather may change, to a considerable extent, the bac-
terial content of the water. If there is any delay before
an analysis can be made, the sample should be carefully
packed in ice.

Methods of Examination.

For measuring exact quantities of water a pipette
graduated to .1 cc. is necessary. This should be prev-
iously sterilized, a small cotton plug being pushed in at

62 BACTERIOLOGICAL ANALYSIS.

the upper end. Push over large end some rubber tub-
ing with a clip. -To keep this pipette sterile during the
manipulations, place it in a flask of sterile water, kept
boiling. Before use, it is, of course, allowed to cool.

1. (a) Add .1 cc. of the water to each of three lique-
fied gelatine and two agar tubes (kept at 42°C).

Pour into plates, and incubate the former at 20°C,
and the latter at 37°C.

(b) Add .1 cc. of the water to each of three liquefied
gelatine tubes and two agar tubes.

Pour into plates, place these in Novy’s or Buchner’s
jars for incubation, anaerobically.

As soon as colonies appear on the agar and gelatine
plates, remove and examine.

Note the number of colonies, counting the colonies
by dividing the plate surface into 16 equal segments,
and counting one or more segments.

Contrast the growths aerobically and anaerobically,
both as to the number and character of the colonies.
Nearly all sewage and pathogenic bacteria are faculta-
tive anaerobes, while the ordinary water bacteria (saph-
rophytes) are aerobic. Hence a large growth on the
anaerobic plate is always suspicious. Good potable
water, as a rule, does not contain over 100 bacteria per
lec., but this may run up to 1,000 and yet water be
fairly good. Large numbers are always suspicious of
some form of organic pollution.

Note the character of the colonies. Look for colon-
ies suspicious of Bacillus coli communis, and also for
liquefying forms. ,

WATER ANALYSIS. 63

Make subcultures of the varieties present on the
various media, particularly any colonies which in naked
eye and microscopic appearances, resemble the Colon
bacillus, Typhoid bacillus or the Proteus varieties.

In sewage contamination the Colon bacillus is con-
stantly present, usually with one or more of the putre-
factive species. These, with a chemical examination
indicating excess of chlorides, nitrates or albuminoid
ammonia, make sewage contamination a certainty.

Tests for the Colon bacillus and putrefactive species
are carried out more completely, by some of the pro-
cesses later described.

2. To fermentation tubes containing sterile grape
sugar broth, add 1 cc. of water. Incubate at 37°C for 24
hours. If gas is formed, Bacillus coli communis is
probably present.

Agar and gelatine plates should be made in such a
case, and the gas forming bacterium separated and
identified.

3. (a) Add .1 cc of the water to each of three lique-
fied gelatine tubes, containing respectively .1%, .12%
and .15% of carbolic acid. Pour plates and incubate at
20°C.

The majority of bacteria are checked in growth in
this medium, while the Colon and Typhoid bacilli
flourish well. Any colonies which appear can be fur-
ther examined by subculture, to aid identification.

(b) Add .1 ce of the water to each of three liquefied
tubes of Elsner’s potato gelatine medium. (Elsner uses
the water in which potatoes have been boiled, in place

64 BACTERIOLOGICAL ANALYSIS.

of broth, to make gelatine. To it he adds 1% of po-
tassium iodide). Pour plates in the usual manner.
This medium has the same effect on bacterial growth as
the carbolic gelatine.

4. Add .1 cc. of the water to 6 or more peptone
tubes, and incubate for 24 hours at 37°C. Strips of fil-
ter paper, moistened with a solution of acetate of lead
may be fixed to the stoppers. Putrefactive bacteria de-
velop sulphuretted hydrogen during growth, and if this
is formed, the lead paper is blackened. The indol test
may also be applied, with and without the addition of
nitrites. Plates may also be made from the surface of
the fluid, and the bacteria present, separated and identi-

fied.

Peptone solution is used extensively when examin-
ing for the Spirillum of Asiatic Cholera. The method
used is either to use the “concentrate,” as described in
(5), or to add to 50 cc. flasks of peptone, from 10 to 25
cc. of the water.

After 16 to 18 hours, the surface layers of the fluid
are examined microscopically for vibrios. If these are
present apply indol tests, and make subcultures on gela-
tine and agar plates, to separate and identify the
species.

5. Filter through:a Berkfeld or Chamberland candle,
into a sterile flask, 500 cc. of the water to be examined.
Place about 20 cc. of the filtered water, (which is
“ sterile”), into a sterile beaker, and with a sterile brush
scrape the filter candle surface into the water. This
“concentrates” the bacteria present, in the water. The

MILK ANALYSIS. 65

concentrate can then be added to carbolic gelatine,
glucose broth fermentation tubes, and peptone tubes, as
before described,

This method is particularly applicable when testing
for the Typhoid and Colon bacilli, and the Cholera
spirillum.

Milk Analysis.

The general principles of milk analysis are those al-
ready given under water analysis. As milk is such an
excellent medium for bacterial growth, it is usually
necessary to dilute the sample at least 10 times with
‘sterile water, before making plates.

In milk analysis stress must be laid on the presence
or absence of the Tubercle bacillus, it being practically
the only bacterium transmitted directly from animals to
man via milk. We have two tests for Tubercle bacilli
in milk. (a) Microscopic examination of the sediment,
after sedimentation or centrifugation, at best an unreli-
able method. (6) The inoculation of guinea pigs. 5 to
10 cc. of the milk is thrown into the peritoneal cavity
of each of three or four guinea pigs. Three weeks
after, the animals are examined for developing tuber-
cular lesions.

From a practical standpoint, the proper application
of the tuberculin test is a more ready and certain
method of detecting the presence, and danger from
Tuberculosis in cattle.

PART III.
CLINICAL MICROSCOPY AND DIAGNOSIS.

SECTION I.
Urine.

The sample should be taken from the entire quan-
tity passed in 24 hours. Otherwise, select a sample
passed some hours after a meal. Do not use the morn-
ing’s urine for routine examination, as in this urine,
least evidence of disturbance of the renal functions
will be found. Putrefaction soon occurs when urine is
allowed to stand, before examination. A crystal or two
of thymol tends to prevent this, and does not destroy the
efficacy of any of the coarser chemical or microscopical
tests.

Methods of Examination.

1. Physical examination,

(a) Note the quantity passed in 24 hours. This is
important in calculating the actual daily excretion of
any normal or abnormal urinary constituents, as well as
determining whether oliguria or polyuria exists.

(b) Note the color and such general characters as
turbidity. Various changes in color and clearness are
found both in normal and abnormal urines. These
changes in normal urine depend upon the amount of

68 CLINICAL MICROSCOPY AND DIAGNOSIS.

the solid constituents, putrefactive changes, and the
like. The urine is variously colored by the presence of
such constituents as phosphates, urates, pus, blood and
bile.

(c) Take the specific gravity. By this we get (par-
ticularly in a 24 hours specimen), a good indication of
the amount of solids in the urine. The usual formula
given is to multiply the last two figures by 2, and so
obtain the number of grammes of solids per litre.

2. Chemical tests.

(a) Take the reaction with litmus paper. The re-
action is important in carrying out certain albumin
tests. It may also indicate the nature of a sediment.

(b) Examine the sample for albumin.

If the urine be at all turbid, filter through several
sheets of filter paper, before the application of the tests.

1. Use the heat and nitric acid test.

2. Apply the nitric acid test. Simon advises an
excellent modification of the usual application of this
method. 20 cc. of urine are placed in a conical urine
glass, and 6 to 10 cc. of nitric acid are added through a
pipette, passed to bottom of glass. At point of junction
of the two fluids we have the formation of the usual
contact ring in the presence of albumin. This ring is
usually colored at bottom, rose to brick red, from the
normal urinary pigment. If indican be present in ex-
cess, we have a violet ring. In biliary urine we have
the usual contact play of colors (acid must contain nit-
rous acid as well). On standing 5 to 10 minutes, high

URINE EXAMINATION. 69

up in the urine a distinct white band appears, very
faint normally but distinct with slight excess of uric
acid. If urea be present in larger amount than 25
grammes per litre, it crystallizes out, on side of urine
glass.

3. Apply the picric acid test, with Esbach’s albumi-

nometer. This is decidedly the best, day to day, clinical
quantitative test.

(c) Apply the tests for sugar.

In the presence of albumin the urine should first be
boiled and then filtered.

1. Use Fehling’s test, the cupric sulphate and alka-
line solutions being kept in separate bottles, equal parts
being mixed for use. 10 cc. of this solution are reduced
by .05 gm. sugar.

To apply this test quantitatively place 10 cc. of Feh-
ling’s solution, diluted with 40 cc. of water, in a porce-
lain capsule and bring to a boil. Add from a burette,
drop by drop, the urine diluted 10 times, till all blue
color is completely discharged, keeping the solution at
the boiling point. Amount of diluted urine required,
will contain .05 gm. of sugar.

2. If in doubt as to the accuracy of the preceding
test, place urine to which a small amount of yeast
(Fleischmann’s) has been added, in a fermentation
tube and keep at 37°C (98.6°F) for 24 hours, for gas
formation.

3. Phenyl-hydrazin test. 10 cc. of the urine are
boiled in a water bath for 30 minutes with 3 or 4
grammes of acetate of soda, and 2 to .5 gramme of

10 CLINICAL MICROSCOPY AND DIAGNOSIS.

phenyl-hydrazin. The tube is cooled by plunging into
cold water, and a microscopic examination made for
phenyl-glucosazone crystals. This is a delicate test.

(d) Estimate the percentage of urea using Doremus’
or larger ureometers.

These tests will be all that are usually called for in
routine clinical work. Quantitative estimates may be
required at times of uric acid, phosphates, chlorides,
or sulphates. For these, a work on urine analysis
should be consulted.

3. Microscopic examination.

Urine which is turbid, may be examined at once but
it is better to wait till a sediment has formed. This is
best effected by placing the urine in a conical glass and
allowing to settle from 3 to 24 hours. The supernatant
fluid is then decanted, and the sediment is placed with
a pipette upon a slide and examined under the low
power of the microscope. Care should be taken to
withdraw the lowest sediment with the pipette. A cen-
trifuge may be used for rapid sedimentation.

Much can be learned of the nature of a sediment by
noting its color, general characters, the reaction of the
urine, and whether in urine at the time of passing from
bladder. The elements examined for microscopically
and which appear as a rule as sediments may be divided
into three classes, (1) chemical, (2) anatomical, (3) bac-
terial.

1. The chemical sediments consist of various crystal-
line and amorphous chemical bodies which may exist
normally or be present abnormally in urine. We may

URINE EXAMINATION. val

sub-divide them according to the reaction of urine in ;
which found, color, and whether crystalline or amor-
phous. ;

(a) Sediments in acid urine:

Urate of soda, amorphous, _ brick red.
Uric acid, crystalline, bright red.
Oxalate of lime, crystalline, white.

Monocalcium phosphate, crystalline (rare).

Ammonio-magnesic phosphate, crystalline, white.
(found in urine becoming ammoniacal),

(6) Sediments in alkaline urine:

1. Alkalinity due to fixed alkalies;

Basic phosphates of Ca. and Mg. amorphous, white.

Calcium carbonate, crystalline.
Dicalcic phosphate, crystalline.

2, Alkalinity due to ammonia (usually fermentative):
Ammonium urate, crystalline, yellow.

Ammonio-magnesic phosphate.
Calcium carbonate.
Tricalic phosphate.

Besides these sediments we have at times the rarer
forms of chemical sediments, such as those of leucin
and tyrosin, lime and magnesia soaps, cystin, xanthin
and hippuric acid. It must be remembered that the
presence of many of these chemical sediments, does not
of necessity mean an increase in their amount in the
urine. The degree of concentration, the reaction and
putrefactive changes determine the presence of many
of them, so that in considering their import, these points
must be taken into consideration.

72 CLINICAL MICROSCOPY AND DIAGNOSIS.

2. The anatomical sediments consist of cellular ele-
ments derived from the urinary passages or from the
blood. They consist of various forms of epithelium,
tube casts, pus cells and blood. In all normal urines a
few epithelial cells, (usually derived from the bladder)
are found. Asarule, too, a few leucecytes (polymor-
phonuclear) can be seen. Apart from these constitu-
ents, the presence of other elements means some lesion
of the urinary tract.

(a) Epithelium may be derived from,
Kidney;
Renal pelvis and ureter;

Bladder and urethra;
(Vulva and vagina.)

There are no positive differential features between
the epithelium from these parts, but as a rule the cells
are fairly characteristic. A diagnosis cannot be based
on the presence of epithelium alone, such features as
albuminuria or pyuria having always to be considered.

(b) Tube casts are characteristic of lesions of the
renal parenchyma. We find various forms of these casts
depending on the intensity and nature of the lesion.

Tube casts may be epithelial, granular, fatty, hyaline,
waxy, uratic, or consist of blood cells.

(c) Blood may be derived from any part of the urin-
ary tract. Note must be taken of the presence of clots,
the nature of the admixture with urine and the presence
of ureteral or true blood casts, in determining the
source of the haemorrhage.

URINE EXAMINATION. 13

(d) Pus cells (leucocytes) are found in all irritative
or inflammatory lesions of the urinary tract. Only
rarely are they found alone, (rupture of abscess into
passages). As a rule we find, and must consider the
presence of albumin, epithelium or casts, as well as the
chemical characters of the urine, in determining the
site of lesion.

3. Bacteria in the urine may be derived from the

urinary passages or may enter the urine after its pass-
age from the body (putrefaction).

The bacteria which may be found in affections of
the urinary tract are as follows:

(a) The Tubercle bacillus is found in Tuberculosis
of any part of the urinary tract. It is examined for as
described at page 32.

(6) Occasionally certain bacteria which maf be ex-
creted through the kidney, multiply in the passages ex-
citing a bacteriuria. This is seen occasionally after
Typhoid fever and in some infections with the Bacillus
coli communis and more rarely with other microbes
during the course of a septicaemia or pyaemia.

(c) Bacteria may make their way to the bladder and
upper urinary passages by being introduced through
urethra on catheters, etc. This usually excites the
condition of ammoniacal cystitis and by extension gives
rise to a form of “ surgical” kidney.

These three forms must be distinguished from the
bacteria which enter and cause putrefaction of the urine
after its passage from the bladder.

14 CLINICAL MICROSCOPY AND DIAGNOSIS.

SECTION II.
Blood.

Blood examinations require the use of special instru-
ments and reagents. The instruments which will be
needed besides a microscope, are a haemoglobinometer
(Fleischl’s), a haemacytometer (Thoma’s) and a spear
headed needle for obtaining blood. A haematocrit and
centrifuge are very useful when much blood work has
to be undertaken.

The following reagents will be required:

Toison’s dilution fluid,

Glycerine 3 ‘ : F : 30 parts
Sodium sulphate ‘ ‘ ‘ o 380%
Sodium chloride ; ‘ : : 1
Methy] violet : : ; : . 025
Water . ; ‘ ; : : 160 “

Benzol and chloroform, and a mixture of these two
having a specific gravity of 1055.
Ehrlich’s triple stain,
Saturated aqueous solution acid fuchsin 24 parts
Distilled water : ‘ . : ® dies
Saturated aqueous solution orange G. 6
% ‘“* methylene green 64 “
Add the last slowly, drop by drop, constantly stir-
ring.
Absolute alcohol : : ‘ ‘ 5 parts
Distilled water i : : ; . 10 “

BLOOD EXAMINATION. 15

If the methylene green precipitates add more acid
fuchsin.

Eosin .56% solution in 70% alcohol.

Loefiler’s methylene blue.

Ehrlich’s eosin-haematoxylin stain,

Haematoxylin . ‘ 4-5 parts
Alcohol, glycerine, and water . 100 “ each
Glacial acetic acid : : 20 “

Alum F ; ‘ ' to saturation.

Allow to ripen in sun 5 or 6 weeks, and add 1% of
eosin.

In blood examination the points to be ascertained
are the specific gravity, the amount of haemoglobin and
the number and characteristics of the red and white
blood cells. Examination bacteriologically by cover
glass preparations and cultures may also be required.

The blood may be obtained from the lobe of the ear,
root of the finger nail or the ball of the finger, the first
being undoubtedly the best situation. The part should
be washed with soap and water, then with a mixture of
alcohol and ether before puncturing. The first drop of
blood which exudes should be wiped away with sterile
gauze. No pressure should be made to obtain the
blood.

Specific Gravity. Allow a drop of blood to fall into
a cylindrical vessel containing the benzol-chloroform
mixture. If it floats add more benzol, if it sinks add
chloroform, till the drop remains stationery about the
middle of the fluid. Take the specific gravity of the
mixture and this will be the reading for the blood.

716 CLINICAL MICROSCOPY AND DIAGNOSIS.

Estimation of Haemoglobin. After arranging the
apparatus (Fleischl’s) cover the bottom of one of the
cells with distilled water. Fill the capillary pipette
with blood, taking care not to soil its exterior, and to
have the tube just full. Rinse out the pipette in the
water of. the cell, and by dropping on more distilled
water. Fill the cell almost full and with the handle of
the pipette thoroughly mix the contents of the cell.
Both cells are now carefully filled with water till both
present a convex surface. Using an oil or gas lamp,
(not daylight) compare the tint of the blood cell with
that of the standard bar under the water cell, moving
the bar till the two tints are the same. Read off the
percentage of normal on the bar index.

Estimation of the Red Corpuscles. Carefully fill
the pipette of the haemacytometer up to 1 with blood.
At once draw in dilution fluid till mark on bulb, 101, is
reached. Mix the blood and the fluid by twirling the
pipette between the fingers fora minute. After blow-
ing out the fluid that fills the tube of the pipette, place
a drop of the mixture in the bulb, on the centre of the
counting slide. Place over this a cover glass, taking
care not to allow the blood to. overflow the cell edges.
Examine under the microscope and finding the ruled
squares, count the red corpuscles on at least 40 squares.
This should be repeated several times and an average
taken. As each ruled square is z)55 c. mm. we can
readily calculate the number of corpuscles per c.
mm., by the following formula:

BLOOD EXAMINATION. TT

Number of corpuscles per 1 c. mm. =

No. of corpuscles x 4,000 x 100 (dilution)
No. of squares.

Normally the red blood cells number between 5,000,-
000 and 5,500,000 per c. mm.

By this method of examination, information can also
be obtained of the size and shape of the red corpuscles,

Estimation of White Corpuscles. The white cor-
puscles may be estimated at the same time as the red,
by counting at least 200 squares.

It is better to estimate them separately, using the
large pipette of the haemacytometer. Blood is drawn
into this pipette up to 1 and then the bulb is
filled up to 11 with a .4% solution of acetic acid. The
acid destroys the red corpuscles leaving the white cells.
Examination is then made in the same manner as for
the red corpuscles. Normally there are on an average
10,000 white corpuscles per c. mm.

The Haematocrit may also be used to determine
the numbers of red and white cells. The blood is drawn
into the graduated pipette and at once centrifuged, at
10,000 revolutions per minute, for 3 minutes. Normally
the red corpuscles will stand at 50 (ona scale of 100) so
that 1% —=100,000 corpuscles. To calculate the number
of red corpuscles one simply adds five ciphers to the
percentage reading. The leucocytes are found on the
surface of the red discs, as a narrow white band. Any
increase in their numbers will be detected by an in-
crease of this band.

18 CLINICAL MICROSCOPY AND DIAGNOSIS.

Microscopic Examination of Blood Films,

(a) Direct examination of moist films is called for,
in determining poikilocytosis, leucocytosis, and in ex-
amining for the malarial plasmodium.

Moist preparations are made by touching the blood
drop with a thoroughly clean cover glass, and then in-
verting this upon a slide. Only a small drop must be
taken on the cover glass so that it will spread out in a
single layer on the slide. To prevent drying, the cover
_ glass may be ringed with vaseline.

(6) Examination of films dried and stained, is of
much wider application, being employed to determine
the same characters as the moist films, and in addition
giving the forms and relative proportions of the white
cells and the staining characters of the red corpuscles,

The blood films are prepared by touching one cover
glass to the blood, so as to obtain a small drop, then
dropping on this cover glass a second, and sliding the
two apart. The films are dried in the air and are then
ready for fixation. This may be effected by heat or in
certain fixing fluids. Heat fixation gives the best
results. The cover glasses may be placed in an oven
for 2 hours at a temperature of 110°C. As this
method cannot always be carried out, a cheap and effec-
tive substitute, is made by the use of a long triangle of
brass, about .5 cm. in thickness. A gas or spirit lamp
flame is applied to the apex and after 5 or 10 minutes
tests are made for the boiling point on the triangle.
The cover glasses are placed 2 to 3 cm. in front of this

BLOOD EXAMINATION. 719

point, where the temperature averages between 110° and
120°C, and are allowed to remain for 2 hours.

A more rapid method of fixation is to place the films for
1 minute in a solution made by taking 1 part of forma-
lin, 9 parts water and 90 parts of alcohol. This method
produces almost as satisfactory results as are obtained
by heat fixation.

Staining of the Films. (a) For general purposes
Ehrlich’s triple stain is perhaps the best. It stains the
red corpuscles orange, the eosinophile granules red,
the neutrophile granules violet, nuclei of red corpuscles
(when present) greenish black, nuclei of leucocytes
greenish blue, the nuclei of the lymphocytes being, as
a rule, stained less deeply than the others. In using
this stain care must be taken not to shake it up. The
stain is best withdrawn on a glass rod and the films
covered for 6 to8 minutes, then carefully washed in
water, dried and mounted in Canada balsam.

(b) Eosin and Loeffler’s methylene blue.

Place the films in eosin 2 minutes, then wash and
dry. Place in methylene blue for 4 to 1 minute, wash
dry and mount in Canada balsam.

The red corpuscles and eosinophile granules are
stained red, nuclei blue, malarial parasites blue.

(c) Ehrlich’s eosin-haematoxylin stein.

The films are placed in this stain for 24 hours, ex-
posed to light, and are then washed, dried and mounted.

The red corpuscles and eosinophile granules are
stained red, nuclei of red discs and lymphocytes, black,
nuclei of other white cells, bluish black.

80 CLINICAL MICROSCOPY AND DIAGNOSIS.

SECTION III.

Gastric Contents.

Examinations of the gastric contents are required to
obtain information of the condition and digestive power
of the normal gastric juices, and to determine the
presence of any abnormal constituents, such, for in-
stance, as the products of fermentation. Accurate in-
formation, upon which conclusions can be based, is
only to be obtained after several examinations and the
use of test meals on a food free stomach, the contents
being withdrawn at a set period.

The most commonly employed test meal is the test
breakfast of Ewald and consists of 35 to 40 grammes
white bread, and 300 cc. of water, or tea without milk
or sugar. This is withdrawn one hour afterward, if
possible, without the use of water. If water has to be
used, measured small quantities only should be em-
ployed. At the expiration of one hour, normally about
40 cc. of contents can be withdrawn. and in this we
can demonstrate the presence of free and combined hy-
drochloric acid, pepsin, rennet, peptone and glucose.

Boas advises a special test meal in cases of suspected
carcinoma of stomach. It consists of from 350 to 400
cc. of oatmeal gruel made with water and salt, and given
at night, after lavage of the mouth and stomach. Hight
or ten hours afterwards the contents are expressed.
The meal is given to prevent the introduction of lactic
acid in the food, and the tests are made for lactic acid
alone.

GASTRIC CONTENTS. 81

The examinations made of the gastric contents are
physical, chemical and microscopical.

1. Physical examination.

(a) Measure the amount of contents obtained. More
than 40 ce. indicates loss of motor and absorptive power
of the stomach wall.

‘(b) Note the color, which is normally light yellow or
brownish yellow. The presence of blood, bile or much
mucus, changes the color.

(c) Note the odor. If fatty acids as acetic and
butyric acids are present, they can readily be detected
in this manner.

(d) Note the amount of mucus. This is increased
in nearly all forms of gastric affections, but more par-
ticularly so in “chronic” gastritis. The presence of
food fragments, portions of mucous membrane and the
like, must also be noted.

2. Chemical examination.

In a chemical examination of the gastric contents
the following reagents will be required.

Congo red solution. A dilute aqueous solution of
Congo red powder of a brick red color.

Phloroglucin-vanillin solution of Gunzburg. Phloro-
glucin 2 grammes, vanillin 1 gramme, absolute alco-
hol 30 ce.

Dimethyl-amido-azo-benzol. .5% alcoholic solution.

Phenolphthalein. 1% alcoholic solution.

Alizarin. 1% aqueous solution.

82 CLINICAL MICROSCOPY AND DIAGNOSIS,

Uffelmann’s reagent. 8drops of saturated aqueous
solution of the perchloride of iron are mixed with 3
drops of pure carbolic acid, and water added till an
amethyst blue solution is obtained.

Decinormal sodium hydrate solution. Contains 4
grammes of Na OH in a litre of water. 1 cc. of the deci-
normal solution neutralizes .00,365 grammes of HCl.

The chemical tests are applied to the filtrate, one-
half of the contents being filtered.

(a) Determine the acidity with litmus paper.

Acidity may be due to HCl free or combined, to
acid salts, or to the presence of such fermentative acids
as lactic, acetic or butyric.

(b) If acid, determine the total acidity by placing
5 cc. of the filtered contents in a capsule, adding one or
two drops of phenolphthalein solution, and then care-
fully adding from a burette, decinormal sodic hydrate
till completely neutralized. This will be indicated
when no further intensification of the red color occurs,
on the addition of more sodic hydrate. Normally from
40 to 65 cc. of the decinormal sodic hydrate solution are
required to neutralize 100 cc. of the gastric contents.

(c) If acid, is the acidity due to free acids or acid
salts. To determine this add a drop of the gastric con-
tents to a drop of the Congo red solution. If free acids
are present an azure blue coloration appears.

(d) If free acid is present, determine if free HCl
exists.

(1) To a drop of the filtrate in a porcelain capsule
add a drop of Gunzburg’s phloroglucin-vanillin solu-

GASTRIC CONTENTS. 83

tion. Carefully evaporate high over the flame. If free
HCl is present a rose red ring develops at margin of
evaporating drop. This test will determine the presence
of .05 parts of free HCl per mille.

(2) To a drop of the filtrate on a porcelain capsule,
add a drop of the dimethyl-amido-azo-benzol solution.
This gives a red coloration, in the presence of free
HCl when present in .02 parts or over, per mille. It is
a more delicate test than Gunzburg’s and can be used
to determine the amount of free HCl present.

(e) While in practice it is found that in the presence
of free HCl, tests for lactic acid may be disregarded, yet
when free acid is present a test should be made for lac-
tic acid.

Place in a graduate or test tube about 20 cc. of
Uffelmann’s reagent. Add drop by drop from a pipette
the filtered stomach contents, a lemon yellow precipitate
falls in the presence of lactic acid.

This test, while all that need be applied for clinical
purposes, is not always accurate as it is interfered with
by excess of HCl, presence of acetic and butyric acids,
alcohol or much glucose. A more satisfactory method
is to shake up 10 ce. of the filtrate with 30 cc. of ether,
repeating twice, and removing the ether with a pipette.
The ether is then carefully evaporated, the residue
extracted with water and used to make the test.

(f) The fatty acids particularly acetic and butyric
acids are at times present as fermentative products.
The amount of these acids usually rises and falls with
the amount of lactic acid. Their odor is the best quali-
tative test.

84 CLINICAL MICROSCOPY AND DIAGNOSIS.

(g) When the relative proportions of the various
constituents, which produce acidity, are to be deter-
mined the following method gives quite accurate results.
10 cc. of the filtered contents are placed in a beaker and
decinormal sodic hydrate solution is added, drop by
drop, till a drop taken out with a small platinum loop,
no longer gives a reaction with the dimethyl-amido-azo-
benzol solution. The reading on the burette gives the
amount of solution required to neutralize the free HCl
(which has the strongest affinity for the alkaline solu-
tion).

Now add a loop of the mixture to a drop of Congo
red solution. If this still indicates acidity, add more of
the soda solution till the Congo red no longer indicates.
The reading on the burette gives the amount of alkaline
solution required to neutralize the free organic acids
present.

A loop of the contents is next added to a drop of
alizarin (indicates alkalinity), and if there is no violet
reaction, add the soda solution till this develops. The
reading on the burette gives the amount of the soda
required to neutralize the acid salts.

If contents are still acid to phenolphthalein, add
more of the soda solution, till the rose-red color com-
pletely develops. The reading on the burette gives the
amount of solution required to neutralize the combined
HCL.

If there is no free HCl, nor free acid, titration can
be commenced at the corresponding portion of the pro-
cess just described.

GASTRIC CONTENTS. 85

(h) Test for pepsin by determining the digestive
powers of the juice. In the absence of free HCl no
pepsin will be found and tests must be directed towards
its zymogen, pepsinogen.

Add a small, shaving 1 cm. square and 1 mm. thick,
of coagulated egg albumin to 10 cc. of the filtrate, and
keep at 37°C, one to two hours. If there.is no free HCl
present, add 3 or 4 drops of a 25% solution of HCl, so
as to transform any pepsinogen into pepsin. After two
hours the shaving ought to be digested.

Quantitative estimation of the pepsin is carried out
by making a series of dilutions and testing the digestive
powers of these dilutions. This procedure is rarely
called for in clinical practice.

(7) Tests for the milk curdling ferment, “rennet?
and its zymogen.

1. In testing for rennet add 3 to 5 drops of the fil-
trate to 10 cc. milk and keep at 37°C. If rennet is
present the milk clots in 10 to 15 minutes.

(2) If no coagulation occurs, test for the rennet
zymogen, by neutralizing some of the gastric contents
with lime water, and then adding 3 to 5 drops, to the
milk, when if the zymogen is present, coagulation
occurs in 10 to 15 minutes. Quantitative tests can be
carried out by a series of dilutions as is done for pepsin,
bit are rarely required clinically.

(j) Tests for the condition of the various food con-
stituents of the test meal, may also be carried out.
Practically, tests are required only for presence of pep-
tone, propeptones, and the stage of starch digestion.

86 CLINICAL MICROSCOPY AND DIAGNOSIS.

1. Test for propeptones (or albumoses), by adding
to 5 cc. of the filtrate, a saturated solution of sodium
chloride, when any propeptone is precipitated. The
more turbid the fluid becomes, the greater the quantity
of propeptone. If no precipitate falls, a drop of acetic
acid (10% solution) may be added when, if present,
propeptone falls. This addition will only be required
in the absence of free acid.

2. Test for peptones by adding to the filtrate, after
propeptones have been precipitated and filtered out, an
equal quantity of caustic soda solution and a few drops
of 1% solution of copper sulphate. A violet red or purp-
lish color (biuret) is produced, if peptone is present.

(9) Starch should be so far changed in the gastric
contents as not to give either the reaction for starch or
for erythro-dextrin. Test for these, by adding to sev-
eral drops of the filtrate, a drop of tincture of iodine.
Starch, if present, will give a blue reaction, erythro-
dextrin, a purplish red.

3. Microscopical examination.

Portions of the unfiltered contents are examined
under the microscope, with the low and high lenses and
with the oil immersion lens. By this means we deter-
mine the nature of any food fragments, (starch granules,
milk and fat globules), the presence of blood, pus,
epithelium, and bacteria, including sarcinae and yeasts.

Any fragments resembling portions of a new growth
may be sectioned and examined in the usual manner.

Cultures may be made on the various laboratory
media to separate out and identify any bacteria present.

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