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THE ELEMENTS OF

BACTERIOLOGICAL
TECHNIQUE

A LABORATORY GUIDE

FOR THE

MEDICAL, DENTAL, AND TECHNICAL STUDENT

J. W. H. EYRE, M.D., M.S., F.R.S. EDIN.

Bacteriologist to Guy's Hospital, London, and Lecturer on Bacteriology in the Medical

and Dental Schools ; formerly Lecturer on Bacteriology and Joint-Lecturer

on Practical Hygiene in the Charing Cross Hospital Medical

School, and Bacteriologist to Charing Cross Hospital, etc.

WITH 170 ILLUSTRATIONS

PHILADELPHIA AND LONDON

W. B. SAUNDERS & COMPANY
1903

LIBRARY

Registered at Stationers' Hall, London, England

TO THE MEMORY OF

JOHN WICHENFORD WASHBOURN, C.M.G., M.D., F.R.C.P.

Physician to Guy's Hospital and Lecturer on Bacteriology in the
Medical School, and Physician to the London Fever Hospital

MY TEACHER, FRIEND, AND CO-WORKER

225266

PREFACE.

IN the following pages I have endeavoured to arrange
briefly and concisely the various methods at present
in use for the study of bacteria, and the elucidation
of such points in their life-histories as are debatable
or still undetermined.

Of these methods, some are new, others are not;
but all are .reliable, only such having been included
as are capable of giving satisfactory results even in
the hands of beginners. In fact, the bulk of the
matter is simply an elaboration of the typewritten
notes distributed to some of my laboratory classes in
practical and applied bacteriology; consequently an
attempt" has been made to present the elements of
bacteriological technique in their logical sequence.

I make no apology for the space devoted to illus-
trations, nearly all of which have been prepared
especially for this volume; for a picture, if good,
possesses a higher educational value and conveys a
more accurate impression than a page of print; and
even sketches of apparatus serve a distinct purpose
in suggesting to the student those alterations and
modifications which may be rendered necessary or
advisable by the character of his laboratory equip-
ment.

The excellent and appropriate terminology intro-
duced by Chester in his recent work on " Determina-
tive Bacteriology" I have adopted in its entirety,
for I consider that it only needs to be used to convince

one of its extreme utility, whilst its inclusion in an

1 2 PREFACE.

elementary manual is calculated to induce in the
student habits of accurate observation and concise
description.

With the exception of Section XVII,— " Outlines
for the Study of Pathogenic Bacteria," — introduced
with the idea of completing the volume from the point
of view of the medical and dental student, the work
has been arranged to allow of its use as a laboratory
guide by the technical student generally, whether of
brewing, dairying, or agriculture.

So alive am I to its many imperfections that it
appears almost superfluous to state that the book is
in no sense intended as a rival to the many and ex-
cellent manuals of bacteriology at present in use,
but aims only at supplementing the usually scanty
details of technique, and at instructing the student
how to fit up and adapt apparatus for his daily work,
and how to carry out thoroughly and systematically
the various bacterioscopical analyses that are daily
demanded of the bacteriologist by the hygienist.

Finally, it is with much pleasure that I acknowledge
the valuable assistance received from my late assistant,
Mr. J. B. Gall, A.I.C., in the preparation of the section
dealing with the chemical products of bacterial life,
and which has been based upon the work of Lehmann,

JOHN W. H. EYRE.

GUY'S HOSPITAL, S. E.,
September, 1902.

CONTENTS.

PAGE.
I. GLASS APPARATUS IN COMMON USE 17

Cleaning of Glass Apparatus, 24 — Plugging Test-tubes and
Flasks, 29.

II. METHODS OF STERILISATION 32

Sterilising Agents, 32 — Methods of Application, 33.

III. THE MICROSCOPE 51

Methods of Micrometry, 60.

IV. MICROSCOPICAL EXAMINATION OF BACTERIA AND OTHER MI-

CRO-FUNGI 65

Apparatus and Reagents Used in Ordinary Microscopical
Examination, 65 — Methods of Examination, 69.

V. STAINING METHODS 81

Bacteria Stains, 81 — Contrast Stains, 83 — Tissue Stains, 84
— Methods of Demonstrating Structure of Bacteria, 86 — Dif-
ferential Methods of Staining, 93.

VI. METHODS OF DEMONSTRATING BACTERIA IN TISSUES .... 98
Freezing Method, 99 — Paraffin Method, 100 — Special Stain-
ing Methods for Sections, 104.

VII. CLASSIFICATION OF FUNGI 107

Morphology of the Hyphomycetes, 107 — Morphology of the
Blastomycetes, 109.

VIII. SCHIZOMYCETES Ill

Anatomy, 113 — Physiology, Ii6 — Biochemistry, 123.

IX. NUTRIENT MEDIA 125

Meat Extract, 127— Standardisation of Media, 132 — The
Filtration of Media, 136 — Tubing Nutrient Mecfi&j-r^S.

X. STOCK CULTURE MEDIA 141

XI. INCUBATORS . < 174

XII. METHODS OF CULTIVATION 177

Aerobic, 177 — Anaerobic Cultivations, 186.

XIII. METHODS OF ISOLATION 196

)( XIV. METHODS OF INDENTIFICATION 205

Scheme of Study, 205 — Macroscopical Examination of
Cultivations, 207 — Microscopical Methods, 218 — Chemical
Methods, 221 — Physical Methods, 238.

XV. EXPERIMENTAL INOCULATION OF ANIMALS 262

Methods of Inoculation, 274.

XVI. POST-MORTEM EXAMINATION OF EXPERIMENTAL ANIMALS . . 287

14 CONTENTS.

PAGE.
XVII. OUTLINES FOR THE STUDY OF THE PATHOGENIC BACTERIA . . 294

XVIII. BACTERIOLOGICAL ANALYSES ........ ..... 316

Bacteriological Examination of Water, 316 — Examination of
Sewage and Sewage Effluents, 334 — Examination of Air, 335
— Examination of Soil, 338 — Examination of Milk, 344 —
Ice Cream, 354 — Examination of Cream and Butter, 354 —
Examination of Unsound Meats, 356 — Examination of Fil-
ters, 358 — Examination of Disinfectants, 359«

INDEX .... 363

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BACTERIOLOGICAL
TECHNIQUE.

L GLASS APPARATUS IN COMMON
USE.

THE equipment of the bacteriological laboratory, so
far as the glass apparatus is concerned, differs but
little from that of a chemical laboratory, and the clean-
liness of the apparatus is equally important. The
glassware comprised in the following list, in addition
to being clean, must be stored in a sterile or germ-
free condition.

Test=tubes. — It is convenient to keep several sizes
of test-tubes in stock, to meet special requirements,
viz. :

1. 1 8 by 1.5 cm., to contain media for ordinary
tube cultivations.

2. 1 8 by 1.3 cm., to contain media used for pouring
plate cultivations, and also for holding sterile " swabs."

3. 1 8 by 2 cm., to contain wedges of potato, beet-
root, or other vegetable media.

4. 13 by 1.5 cm., to contain inspissated blood-serum.
The tubes should be made from the best German

potash glass, " blue-lined," stout and heavy, with the
edge of the mouth of the tube slightly turned over,
but not to such an extent as to form a definite rim.
(Cost about $4.25, or 9 shillings per gross.) Such
tubes are sufficiently stout to resist rough handling,
do not usually break if accidentally allowed to drop,

can be cleaned, sterilised, and used over and over again,
2 17

1 8 GIvASS APPARATUS IN COMMON USE.

and by their length of life fully justify their initial
expense.

A point to be noted is that the manufacturers rarely
turn out such tubes as these absolutely uniform in
calibre, and a batch of 1 8 by 1.5 cm. tubes usually con-
tains such extreme sizes as 1 8 by 2 cm. and 18 by 1.3
cm. Consequently, if a set of standard tubes is kept
for comparison, each new supply of 1 8 by 1.5 cm. tubes
may be easily sorted out into these three sizes, and so
simplify ordering.

5. 5 by 0.9 cm., for use in the inverted position
inside the tubes containing carbohydrate media, as
gas-collecting tubes.

6. 5 by 0.5 cm., for sedimentation reactions, etc.

Fig. I. — Bohemian flask. Fig. 2. — Pear-shaped Fig. 3. — Erlenmeyer flask

flask. (narrow neck).

These tubes may be of common thin glass, "un-
rimmed," as less than two per cent, are fit for use a
second time.

Bohemian Flasks (Fig. i). — These are the ordinary
flasks of the chemical laboratory. A good variety,
ranging in capacity from 250 to 3000 c.c., should be
kept on hand. A modified form, known as the " pear-
shaped" (Fig. 2), is preferable f6r the smaller sizes—
i. e., 250 and 500 c.c.

Erlenmeyer's Flasks (Fig. 3). — Erlenmeyer's flasks
of 75, loo, and 250 c.c. capacity are extremely useful.
For use as culture flasks care should be taken to select

PETRl'S DISHES OR "PIRATES."

only such as have a narrow neck of about 2 cm. in
length.

Kolle's Culture Flasks (Fig. 4).— These thin, flat
flasks (to contain agar or gelatine, which is allowed to
solidify in a layer on one side) are extremely useful
on account of the large nutrient surface available for
growth. A surface cultivation in one of these will
yield as much growth as ten or twelve " oblique" tube
cultures. The wide mouth, however, is a disadvantage,
and for many purposes thin, flat culture bottles (Fig. 5)
are to be preferred.

Filter Flasks or Kitasato's Serum Flasks (Fig. 6).—
Various sizes, from 250 to 2000 c.c. capacity. These

Fig. 4.— Kolle flask. Fig. 5.— Culture bottles. Fig. 6.— Filter flask.

must be of stout glass, to resist the pressure to which
they are subjected, and must be thoroughly well an-
nealed, in order to withstand the temperature neces-
sary for sterilisation.

Petri's Dishes or "Plates" (Fig. 7, a).— These have
now completely replaced the rectangular sheets of glass
introduced by Koch for the plate method of cultiva-
tion. Each "plate" consists of a pair of circular discs
of glass with sharply upturned edges, thus forming
shallow dishes, one of slightly greater diameter than
the other, and so, when inverted, forming a cover or

GLASS APPARATUS IN COMMON USE.

cap for the smaller. Plates having an outside diam-
eter of 10 cm. and a height of 1.5 cm. are the most
generally useful. Such plates are sterilised and stored
in batches of eighteen in cylindrical copper boxes with
a "pull-off" lid, 30 cm. high by 12 cm. diameter. In-
side the box is a copper stirrup with a circular bottom,
upon which the plates rest, and by means of which
each can be raised in turn to the mouth of the box
(Fig. 8).

Capsules (Fig. 7, b and c). — These are Petri's dishes
of smaller diameter but greater depth than those termed

Fig. 7. — Petri dish and capsules.

Fig. 8.-— Plate box with
stirrup.

plates. Two sizes will be found useful — viz., 4 cm.
diameter by 2 cm. high, capacity about 14 c.c.; and 5
cm. diameter by 2 cm. high, capacity about 25 c.c.
These are stored in copper cylinders of similar con-
struction to those used for plates, but measuring 20
by 6 cm. and 20 by 7 cm., respectively.

Graduated Pipettes. — Several varieties of these are
required, viz. :

1. Pipettes of i c.c. capacity graduated in o.i c.c.

2. Pipettes of i c.c. capacity graduated in o.oi c.c.
(Fig. 9, a).

CAPILLARY PIPETTES OR PASTEUR'S PIPETTES. 21

3. Pipettes of 10 c.c. capacity graduated in o.i c.c.

(Fig. 9,*).

These should be about 30 cm. in length (i and 2
of fairly narrow bore), graduated to the extreme point,
and having at least a 10 cm. length of clear space be-
tween the first graduation and the upper end. Each
variety should be stored in a separate cylindrical copper
case some 36 by 6 cm., with " pull-off" lid, upon which
is stamped, in plain figures, the capacity of the con-
tained pipettes.

V U

a b

Fig. 9. — Measuring pipettes, a and b.

a b c

Fig. 10. — Pasteur's pipettes, a, b, c.

Capillary Pipettes or Pasteur's Pipettes (Fig. 10, a). —
These little instruments are invaluable, and a goodly
supply should be kept on hand. They are prepared
from soft glass tubing of various-sized calibre in the
following manner: Hold a 14 cm. length of glass tube
by each end, and whilst rotating it heat the central
portion in the Bunsen flame until the glass is red hot
and soft. Now remove it from the flame and steadily
pull the ends apart, so drawing the heated portion
out into a capillary tube; break the capillary portion

GLASS APPARATUS IN COMMON USE.

at its centre, seal the broken ends in the flame, and
round off the edges of the open end of each pipette.
A loose plug of cotton-wool in the open mouth com-
pletes the capillary pipette. After a number have been
prepared, they are sterilised and stored in batches,
either in metal cases similar to those used for the gradu-
ated pipettes or in large-sized test-tubes — sealed ends
downwards and plugged ends towards
the mouth of the case.

A modification of this pipette, in
which a constriction or short length
of capillary tube is introduced just
below the plugged mouth (Fig. 10, b),
will also be found extremely useful in
the collection and storage of morbid
exudations.

A third form, where the capillary
portion is about 4 or 5 cm. long and
only forms a small fraction of the
entire length of the pipette (Fig. 10,
c), will also be found useful.

"Blood" Pipettes (Fig. n).— Spe-
cial pipettes for the collection of fairly
large quantities of blood (as suggested
by Pakes) should also be prepared.
These are made from soft glass tub-
ing of i cm. bore in a similar manner
to the Pasteur pipettes, except that
a blowpipe flame must be used in order to obtain the
sharp shoulder at either end of the central bulb. The
terminal tubes must retain a diameter of at least i
mm., in order to avoid capillary action during the col-
lection of the fluid.

For sterilisation and storage each pipette is placed
inside a test-tube, resting on a wad of cotton-wool,
and the tube plugged in the ordinary manner. As
these tubes are used almost exclusively for blood work,

Fig. II. — Blood
pipettes and hare-lip
pin in a test-tube.

FERMENTATION TUBES.

it is usual to place a lance-headed hare-lip pin inside
the tube so that the entire outfit may be sterilised at
one time.

Graduated Capillary Pipettes (Fig. 12). — These should
also be made in the laboratory, — from manometer
tubing, — of simple, convenient shape,
and graduated by the aid of a i c.c.
pipette (in hundredths) to contain
such quantities as 10, 50, and 90
c.mm., and carefully marked with a
writing diamond. These, previously
sterilised in large test-tubes, will be
found extremely useful in preparing
accurate percentage solutions, when
only minute quantities of fluid are
available.

Sedimentation Tubes (Fig. 13).—
These are prepared from 10 cm.
lengths of narrow glass tubing by
sealing one extremity, blowing a
small bulb at the centre, and plugging the open end
with cotton- wool; after sterilisation the open end is
provided with a short piece of rubber tubing and a glass
mouthpiece. When it is necessary to observe sedi-
mentation reactions in very small quantities of fluid,

Fig. 12. — Capillary
graduated pipettes.

Fig. 13. — Sedimentation tube.

these tubes will be found much more convenient than
the 5 by 0.5 cm. test-tubes previously mentioned.

Fermentation Tubes (Fig. 14). — These are used for
the collection and analysis of the gases liberated from
the media during the growth of some varieties of bac-

GLASS APPARATUS IN COMMON USE.

teria and may be either plain (a) or graduated (6).
A simple form (Fig. 14, c) may be made from 14
cm. lengths of soft glass tubing of 1.5 cm. diameter.
The Bunsen flame is applied to a spot some 5 cm. from
one end of such a piece of tubing and the tube slightly
drawn out to form a constriction, the constricted part

Fig. 14. — Fermentation tubes.

is bent in the bat's-wing flame, to an acute angle, and
the open extremity of the long arm sealed off in the
blowpipe flame. The open end of the short arm is
rounded off and then plugged with cotton- wool, and
the tube is ready for sterilisation.

CLEANING OF GLASS APPARATUS.

All glassware used in the bacteriological laboratory
must be thoroughly cleaned before use, and this rule
applies as forcibly to new as to old apparatus, although
the methods employed may vary slightly.

To Clean New Test=tubes.—

1. Place the tubes in a bucket or other convenient
receptacle, fill with water and add a handful of " sapon"
or other soap powder. See that the tubes are full and
submerged.

2. Fix the bucket over a large Bunsen flame and boil
for thirty minutes.

3. Cleanse the interior of the tubes with the aid of
test-tube brushes, and rinse thoroughly in cold water.

INFECTED TEST-TUBES. 25

4. Invert the tubes and allow them to drain com-
pletely.

5. Dry the tubes and polish the glass inside and out
with a soft cloth, such as selvyt.

New flasks, plates, and capsules must be cleaned in
a similar manner.

To Clean New Graduated Pipettes.—

1. Place the pipettes in a convenient receptacle,
filled with water to which soap powder has been added.

2. Boil the water vigorously for twenty minutes over
a Bunsen flame.

3. Rinse the pipettes in running water and drain.

4. Run distilled water through the pipettes and
drain.

5. Run rectified spirits through the pipette and drain
as completely as possible.

6. Place the pipettes in the hot-air oven (vide page
35), close the door, open the ventilating slide, and
run the temperature slowly up to about 80° C. Turn
off the gas and allow the oven to cool.

Or 6a. Attach each pipette in turn to the rubber
tube of the foot bellows, or blowpipe air-blast, and
blow air through the pipette until the interior is dry.

Glassware that has already been used is regarded as
infected, and is treated in a slightly different manner.

Infected Test=tubes.—

1. Pack the tubes in the wire basket of the auto-
clave (having previously removed the cotton-wool
plugs, caps, etc.), in the vertical position, and before
replacing the basket see that there is a sufficiency of
water in the bottom of the boiler. Now attach a
piece of rubber tubing to the nearest water tap, and
by means of this fill each tube with water.

2. Disinfect completely by exposing the tubes, etc.,
to a temperature of 120° C. for twenty minutes (vide
page 42).

(If an autoclave is not available, the tubes must be

26 GLASS APPARATUS IN COMMON USE.

placed in a digester, or even a large pan or pail with a
tightly fitting cover, and boiled vigourously for some
thirty to forty-five minutes to ensure disinfection.)

3. Whilst still hot, empty each tube in turn and
roughly clean its interior with a stiff test-tube brush.

4. Place the tubes in a bucket or other convenient
receptacle, fill with water and add a handful of sapon
or other soap powder. See that the tubes are full and
submerged.

5. Fix the bucket over a large Bunsen flame and
boil for thirty minutes.

6. Cleanse the interior of the tubes with the aid of
test-tube brushes, and rinse thoroughly in cold water.

7. Drain off the water and immerse tubes in a large
jar containing water acidulated with 2 to 5 per cent,
hydrochloric acid. Allow them to remain there for
about fifteen minutes.

8. Remove from the acid jar, drain, rinse thoroughly
in running water, then with distilled water.

9. Invert the tubes and allow them to drain com-
pletely.

Dry the tubes and polish the glass inside and out
with a soft cloth, such as selvyt,

Infected flasks, plates, and capsules must be treated
in a similar manner.

Flasks which have been used only in the preparation
of media must be cleaned immediately they are finished
with. Fill each flask with water to which some soap
powder and a few crystals of potassium permanganate
have been added, and let boil over the naked flame.
The interior of the flask can then usually be perfectly
cleaned with the aid of a flask brush, but in some cases
water acidulated with 5 per cent, nitric acid, or a large
wad of wet cotton- wool previously rolled in silver sand,
must be shaken around the interior of the flask, after
which rinse thoroughly with clean water, dry, and
polish.

COVER-SUPS. 27

Infected Pipettes.—

1. Plunge infected pipettes immediately after use
into tall glass cylinders containing a 2 per cent, solu-
tion of lysol, and allow them to remain therein for
some days.

2. Remove from the jar and drain. Boil in water
to which a little soap has been added, for thirty min-
utes.

3. Rinse thoroughly in cold water.

4. Immerse in 5 per cent, nitric acid for an hour or
two.

5. Rinse again in running water to remove all traces
of acid.

6. Complete the cleaning as described under "new
pipettes."

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w t

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«T

Fig. 15. — Slides and cover-slips, actual size.

Slides and cover=slips (Fig. 15), when first purchased,
have " greasy" surfaces, upon which water gathers in
minute drops and effectually prevents the spreading
of thin, even films.

Microscopical Slides. — The slides in general use are
those known as "three by one" slips (measuring 3
inches by i inch, or 76 by 26 mm.), and should be
of good white crown glass, with ground edges.

New slides should be allowed to remain in alcohol
acidulated with 5 per cent, hydrochloric acid for some
hours, rinsed in running water, roughly drained on a
towel, dried, and finally polished with a selvyt cloth.

Cover=slips. — The most useful sizes are the 19 mm.

28 GLASS APPARATUS IN COMMON USE.

squares for ordinary cover-glass film preparations, and
38 by 19 mm. rectangles for blood films and serial sec-
tions; both varieties must be of "No. i" thickness,
which varies between 0.15 and 0.22 mm., that they
may be available for use with the high-power immer-
sion lenses.

Cover-slips should be cleaned in the following manner:

1 . Drop the cover-slips one by one into an enamelled
iron pot or tall glass beaker, containing a 10 per cent,
solution of chromic acid.

2. Heat over a Bunsen flame and allow the acid
to boil gently for twenty minutes.

NOTE. — A few pieces of pipe-clay or pumice may be
placed in the beaker to prevent the "spurting" of the
chromic acid.

3. Turn the cover- slips out into a flat glass dish and
wash in running water under the tap until all trace
of yellow colour has disappeared. During the wash-
ing keep the cover-slips in motion by imparting a
rotatory movement to the dish.

4. Wash in distilled water in a similar manner.

5. Wash in rectified spirit.

6. Transfer the cover-slips, by means of a pair of
clean forceps, previously heated in the Bunsen flame
to destroy any trace of grease, to a small beaker of
absolute alcohol.

Drain off the alcohol and transfer the cover-slips,
by means of the forceps, to a wide-mouthed glass pot,
containing absolute alcohol, in which they are to be
stored, and stopper tightly.

NOTE. — After once being placed in the chromic acid,
the cover-slips must on no account be touched by the
fingers.

Used Slides and Cover=slips. — Used slides with the
mounted cover-slip preparations, and cover-slips used
for hanging-drop mounts, should, when discarded, be

PLUGGING TEST-TUBES AND FLASKS. 29

thrown into a pot containing a 2 per cent, solution of
lysol.

After immersion therein for a week or so, even the
cover-slips mounted with Canada balsam can be readily
detached from their slides.

Slides. —

1 . Wash the slides thoroughly in running water.

2. Boil the slides in water to which "sapon" has
been added, for half an hour.

3. Rinse thoroughly in cold water.

4. Dry and polish with a dry cloth.
Cover-slips.—

1 . Wash the cover-slips thoroughly in running water.

2. Boil the cover- slips in 10 per cent, solution of
chromic acid, as for new cover-slips.

3. Wash thoroughly in running water.

4. Pick out those cover-slips which show much ad-
herent dirty matter, and rub them between thumb
and forefinger under the water tap. The dirt usually
rubs off easily, as it has become friable from contact
with the chromic acid.

5. Return all the cover-slips to the beaker, fill in
fresh chromic acid solution, and treat as new cover-
slips.

NOTE. — Test-tubes, plates, capsules, etc., which, from
long use, have become scratched and hazy, or which
cannot be cleaned in any other way, may be dealt
with by immersing them in an enamelled iron bath,
containing water acidulated with hydrofluoric acid i
per cent., for ten minutes, rinsing thoroughly in water,
drying, and polishing.

PLUGGING TEST-TUBES AND FLASKS.

Before sterilisation all test-tubes and flasks must
be carefully plugged with cotton- wool, and for this
purpose best absorbent cotton-wool (preferably that
put up in cylindrical one-pound packets and inter-

3O GIvASS APPARATUS IN COMMON USE.

leaved with tissue paper — known as surgeons' wool)
should be employed.

1. For a test-tube or a small flask, tear a strip of
cotton- wool some 10 cm. long by 2 cm. wide from the
roll.

2. Turn in the ends neatly and roll the strip of wool
lightly between the thumb and fingers of both hands
to form a long cylinder.

3. Double this at the centre and introduce the now
rounded end into the open mouth of the tube or flask.

Fig. 16. — Plugging test-tubes : a, Cylinder of wool being rolled; b, cylinder
of wool being doubled ; r, cylinder of wool being inserted in tube.

4. Now, whilst supporting the wool between the
thumb and fingers of the right hand, rotate the test-
tube between those of the left, and gradually screw
the plug of wool into its mouth for a distance of about
2.5 cm., leaving about the same length of wool project-
ing.

The plug must be firm and fit the tube or flask fairly
tightly, sufficiently tightly in fact to bear the weight
of the glass plus the amount of medium the vessel is
intended to contain, but not so tightly as to prevent it
from being easily removed by a screwing motion when

PLUGGING TEST-TUBES AND FLASKS. 31

grasped between the fourth, or third and fourth fingers,
and the palm of the hand.

For a large flask a similar but larger strip of wool
must be taken; the method of making and inserting
the plug is identical

IL METHODS OF STERILISATION.

STERILISING AGENTS.

STERILISATION — i. e., the removal or the destruction
of germ life — may be effected by the aid of various
agents. As applied to the requirements of the bac-
teriological laboratory, many of these agents, such as
electricity, sunlight, etc., are practically useless ; whilst
others are limited in their applications, or are so well
suited to particular purposes as to be almost entirely
restricted to such.

The sterilising agents in common use are:

Chemical Reagents. — Disinfectants (for the disinfec-
tion of glass and metal apparatus and of morbid tis-
sues).

Heat.— (a) Dry Heat:

1. Naked flame (for the sterilisation of platinum
needles, etc.).

2. Muffle furnace (for the sterilisation of filter can-
dles, and for the destruction of morbid tissues).

3. Hot air (for the sterilisation of all glassware and
of metallic substances).

(b) Moist Heat:

1. Water at 56° C. (for the sterilisation of certain
albuminous fluids).

2. Water at 100° C. (for the sterilisation of surgical
instruments, rubber tubing, and stoppers, etc.).

3. Streaming steam at 100° C. (for the sterilisation
of media).

4. Superheated steam at 115° C. or 120° C. (for the
disinfection of contaminated articles and old culti-
vations of bacteria).

METHODS OF APPLICATION. 33

Filters.-

1 . Cotton- wool filters (for the sterilisation of air and
gases).

2. Porcelain filters (for the sterilisation of various
liquids).

METHODS OF APPLICATION.

Chemical Reagents, such as belong to the class known
as antiseptics (i. e., substances which inhibit the growth
of, but do not destroy, bacterial life), are obviously
useless. Disinfectants or germicides (i. e., substances
which destroy bacterial life), on the other hand, are of
value in the disinfection of morbid material, and also
of various pieces of apparatus, such as pipettes, pend-
ing their cleansing and complete sterilisation by other
processes. To this class belong:

1. Lysol, 2 per cent, solution;

2. Perchloride of mercury, o.i per cent, solution;

3. Carbolic acid, 5 per cent, solution;

4. Absolute alcohol;
5- Ether;

6. Chloroform;

7. Volatile oils, such as oil of mustard, oil of garlic,
arranged in order of general utility. Formaldehyde is
perhaps a more powerful germicide than any of the
above, but its penetrating vapor restricts its use. These
disinfectants are but little used in the final sterilisa-
tion of apparatus, chiefly on account of the difficulty
of completely removing such substances, for even
traces of these chemicals are sufficient to so inhibit
or alter the growth of bacteria brought into contact
with them, as to vitiate subsequent experiments con-
ducted by the aid of apparatus sterilised in this manner.

NOTE. — Tubes, flasks, filter flasks, pipettes, glass
tubing, etc., may be rapidly sterilised, in case of emer-
gency, by washing, in turn, with distilled water, per-
chloride of mercury solution, alcohol, and ether, drain-
3

METHODS OF STERILISATION.

ing, and finally gently heating over a gas flame to com-
pletely drive off the ether vapor. Chloroform or vola-
tile oils may be added to various fluids in order to
effect the destruction of contained bacteria, and when
this has been done, may be completely driven off from
the fluid by the application of gentle heat.

Dry Heat. — The naked flame of the Bunsen burner
is used for sterilising the platinum needles, the points
of forceps, or other small instruments, cover-glasses,
etc., a very short exposure to this heat being sufficient
to ensure sterilisation.

Muffle Furnace (Fig. 17). — This form of heat is

chiefly used for the destruc-
tion of the dead bodies of
small infected animals, mor-
bid tissues, etc., but is also
employed for the sterilisation
of porcelain filter candles.

Filter candles are disin-
fected immediately after use
by boiling in a beaker of
water for some fifteen or
twenty minutes. This treat-
ment, however, leaves the
dead bodies of the bacteria

upon the surface and blocking the interstices of the
filter.

To destroy the organic matter and prepare the filter
candle for further use proceed as follows :

1. Roll each bougie up in a piece of asbestos cloth,
secure the ends with a few turns of copper wire, and
place inside the muffle (a small muffle 76 by 88 by
163 mm. will hold perhaps four small Berkfeld candles).

2. Light the gas and raise the contents of the muffle
to a white heat; maintain this temperature for five
minutes.

3. Extinguish the gas, and when the muffle and its

Fig. 17. — Muffle furnace.

HOT AIR.

contents have become quite cold, remove the filter
candles from the interior of the muffle, and store with-
out removing the asbestos wrappings, in sterile metal
boxes.

Hot Air. — Hot air at 150° C. destroys all bacteria,
spores, etc., in about thirty minutes, whilst a few
minutes' exposure to a temperature of 170° to 180°
C. will effect the same result. This method is only
applicable to glass and metallic substances, and
the small bulk of cot-
ton-wool comprised in
the test-tube plugs,
etc. Large masses of
fabric are not effectu-
ally sterilised by dry
heat, as its power of
penetration is not
great.

Sterilisation by hot
air is effected in the
hot-air oven (Fig. 18).
This is a rectangular,
double -walled metal
box, mounted on a
stand and heated from

Fig. 1 8. — Hot-air oven.

below by a large Bun-
sen burner. One of

the sides is hinged to form a door. The central portion
of the metal bottom, on which the Bunsen flame would
play, is cut away, and replaced by firebrick plates, which
slide in metal grooves and are easily replaced when
broken or worn out. The top of the oven is provided
with a perforated ventilator slide and two tubulures,
the one for the reception of a centigrade thermometer
graduated to 200° or 250° C., the other for a thermo-
regulator. The thermo-regulator is by no means a
necessity, and it is more convenient to replace it by a

36 METHODS OF STERILISATION.

large bore thermometer with a sliding platinum point,
connected with an electric bell, which can be easily
adjusted to ring at any given temperature. The in-
terior of the oven is provided with loose shelves upon
which the articles to be sterilised are arranged, either
singly or packed in square wire baskets or crates, kept
specially for this purpose.

To prepare crates for the reception of test-tubes, etc.,
cover the bottom with a layer of thick asbestos cloth ; or
take some asbestos fibre, moisten it with a little water and
knead it into a paste ; plaster the paste over the bottom of
each crate, working it into meshes and smoothing its surface
by means of a pestle. When several crates have been thus
treated, place them inside the hot-air oven, close the door,
open the ventilating slide, light the gas, and run the temper-
ature of the interior up to 160° C. After an interval of
about ten minutes extinguish the gas, open the oven door,
and allow the contents to cool. The asbestos now forms
a smooth, dry, spongy layer over the bottom, which will
last many months before needing renewal, and will con-
siderably diminish the loss of tubes from breakage.

Copper cylinders and large test-tubes intended for the
reception of pipettes are prepared in a similar manner, to
protect the points of these articles.

To USE THE HOT-AIR OVEN. —

1 . Place the crates of test-tubes, plates and pipettes
in their metal cases, loose apparatus, etc., inside the
oven, taking particular care that none of' the cotton-
wool plugs are in contact with the walls of the oven,
otherwise the heat transmitted by the metal will char
or even flame them.

2. Close the oven door, set the electric alarm to ring
at 100° C., light the gas below, and open the ventilating
slide, in order that any moisture left in the tubes, etc.,
may escape.

3. When the temperature of the oven has reached
100° C., close the ventilating slide; reset the alarm to
ring at 175° C.

4. Run the temperature up to 175° C,

MOIST HEAT. 37

5. Extinguish the gas at once, and allow the appa-
ratus to cool.

6. When the temperature of the interior, as recorded
by the thermometer, has fallen to 60° C., — but not
before, — the door may be opened and the sterile articles
removed and stored away.

N. B. — Neglect of this precautionary cooling of the
oven to 60° C. will result in numerous cracked and
broken tubes.

On removal from the oven, the cotton- wool plugs will
probably be slightly brown in colour.

Metal instruments, such as knives, scissors, and
forceps, may be sterilised in the hot-air oven as de-
scribed above, but exposure to 175° C. is likely to
seriously affect the temper of the steel and certainly
blunts the cutting edges. If, however, it is desired
to sterilise surgical instruments by hot air, they should
be packed in a metal box, or boxes, and heated to
130° C. and retained at that temperature for about
thirty minutes.

Moist Heat.— Water at 56° C.— This temperature, if
maintained for thirty minutes, is sufficient to destroy
the vegetative forms of bacteria, but has practically
no effect on spores. Its use is limited to the sterilisa-
tion of such albuminous "fluid" media as would co-
agulate at a higher temperature.

METHOD.—

1. Fit up a water-bath, heated by a Bunsen flame
which is controlled by a thermo-regulator, so that the
temperature of the water remains at 56° C.

2. Immerse the tubes or flasks containing the albu-
minous fluid in the water-bath so that the upper level
of such fluid is at least 2 cm. below the level of the
water. (The temperature of the bath will now fall
somewhat, but after a few minutes will again rise to
56° C.)

38 METHODS OF STERILISATION.

3. After thirty minutes' exposure to 56° C., ex-
tinguish the gas, remove the tubes or flasks from the
bath, and subject them to the action of running water
so that their contents are rapidly cooled.

4. The vegetative forms of bacteria present in the
liquid being killed, stand it for twenty-four hours in
a cool, dark place; at the end of that time some at least
of the spores will have germinated and assumed the
vegetative form.

5. Destroy these new vegetative forms by a similar
exposure to 56° C. on the second day, whilst others,
of slower germination, may be caught on the third day,
and so on.

6. In order to ensure thorough sterilisation, repeat
the process on each of six successive days.

This method of exposing liquids to a temperature
of 56° C. in a water-bath for half an hour on each of
six successive days is termed fractional sterilisation.

Water at 100° C. destroys the vegetative forms of
bacteria almost instantaneously, and spores in from
five to fifteen minutes. This method of sterilisation
is applicable to the metal instruments, such as knives,
forceps, etc., used in animal experiments, syringes,
rubber corks, rubber and glass tubing, and other small
apparatus, and is effected in what is usually spoken of
as the "water steriliser."

This is a rectangular copper box, 26 cm. long, 18 cm,
wide, and 12 cm. deep, mounted on legs, heated from
below by a Bunsen or radial gas burner, and containing
a movable copper wire tray, 2 cm. smaller in every
dimension than the steriliser itself, and which is pro-
vided with handles. The top of the steriliser is hinged
to form a lid.

METHOD. —

i. Place the instruments, etc., to be sterilised inside
the copper basket, and replace the basket in the ster-
iliser.

STREAMING STEAM.

2. Pour a sufficient quantity of water into the ster-
iliser, shut down the lid, and light the gas below.

3. After the water has boiled and steam has been
issuing from beneath the lid for ten minutes, extinguish
the gas, open the lid, and lift out the wire basket by its
handles; the contained instruments, etc., are now
sterile and ready for use.

4. After use, or when accidentally contaminated,
replace the instruments in the basket and return that

Fig. 19. — Koch's steriliser.

Fig. 20. — Arnold's steriliser.

to the steriliser ; completely disinfect by a further boil-
ing for fifteen minutes.

5. After disinfection, and whilst still hot, take out
the instruments, carefully dry them at once, and return
them to their store cases.

Streaming steam — i. e., steam at 100° C. — destroys
the vegetative forms of bacteria in from fifteen to
twenty minutes, and the sporing forms in from one
to two hours. This method is chiefly used for the
sterilisation of the various nutrient media intended for

40 METHODS OF STERILISATION.

the cultivation of bacteria, and is carried out in a
steam kettle of special construction, known as Koch's
steam steriliser (Fig. 19) or in one of its many modifi-
cations, the most efficient of which is Arnold's (Fig. 20).

The steam steriliser in its simplest form consists of
a tall tinned-iron or copper cylindrical vessel, divided
into two unequal parts by a movable perforated metal
diaphragm, the lower, smaller portion serving for a
water reservoir, and the upper for the reception of
wire baskets containing the articles needing sterilisa-
tion. The vessel is closed by a loose conical lid, pro-
vided with handles, and perforated at its apex by a
tubulure; it is mounted on a tripod stand and heated
from below by a Bunsen burner. The more elaborate
steriliser is cased with felt or asbestos board, and pro-
vided with a water gauge, also a tap for emptying the
water compartment.

To USE THE STEAM STERILISER. —

1. Fill the water compartment to the level of the
perforated diaphragm, place the lid in position, and
light the Bunsen burner.

2. After the water has boiled, allow sufficient time
to elapse for steam to replace the air in the sterilising
compartment, as shown by the steam issuing in a
steady, continuous stream from the tubulure in the lid.

3. Remove the lid, quickly lower the wire basket
containing media tubes, etc., into the sterilising com-
partment until it rests on the diaphragm, and replace
the lid.

4. After an interval of twenty minutes in the case
of fluid media, or thirty minutes in the case of solid
media, take off the lid and remove the basket with its
contents. , •

5. Now, but not before, extinguish the gas.

NOTE. — After removing tubes, flasks, etc., from the
steam steriliser, they should be at once separated
freely in order to prevent moisture condensing upon

SUPERHEATED STEAM. 41

the cotton-wool plugs and soaking through into the
interior of the tubes.

This treatment will destroy any vegetative forms of
bacteria ; during the hours of cooling any spores present
will germinate, and the young organism will be de-
stroyed by repeating the process twenty-four hours
later; a third sterilisation after a similar interval makes
assurance doubly sure.

The method of sterilising by exposure to streaming
steam at 100° C. for twenty minutes on each of three
consecutive days is termed discontinuous or intermittent
sterilisation.

Continuous sterilisation, or exposure to steam at
100° C. for a period of one or two hours, is not to be
recommended.

Superheated steam — i. e., steam under pressure in
sealed vessels at a temperature of 115° C. — will destroy
both the vegetative and the sporing forms of bacteria
within fifteen minutes; if the pressure is increased,
and the temperature raised to 120° C., the same end
is attained in ten minutes. This method was formerly
employed for the sterilisation of media, but when it
was realised that hydrolytic changes occurred in media
subjected to this high temperature, which rendered
them unfit for the cultivation of the more delicate micro-
organisms, its use was restricted almost entirely to
the disinfection of such contaminated articles, old cul-
tivations, etc., as could not be dealt with by dry heat
or the actual furnace. Sterilisation by means of super-
heated steam is carried out in a special boiler — Cham-
berland's autoclave (Fig. 21). The autoclave consists
of a stout copper cylinder, provided with a copper or
gun-metal lid, which is secured in place by means of
bolts and thumbscrews, the joint between the cylinder
and its lid being hermetically sealed by the interposi-
tion of a rubber washer. The cover is perforated for
a branched tube carrying a vent cock, a manometer, and

METHODS OF STERILISATION.

a safety valve. The copper boiler is mounted in the
upper half of a cylindrical sheet-iron case — two con-
centric circular rows of Bunsen burners, each circle
having an independent gas-supply, occupying the
lower half. In the interior of the boiler is a large mov-
able wire basket, mounted
on legs, for the reception
of the articles to be ster-
ilised.

To USE THE AUTO-
CLAVE.—

1. Pack the articles to
be sterilised in the wire
basket.

2. Run water into the
boiler to the level of the
bottom of the basket ; also
fill the contained flasks
and tubes with water.

3. See that the rubber
washer is in position, then
replace the cover and
fasten it tightly on to the
autoclave by means of the
thumbscrews.

4. Open the vent cock and light both rings of burners.

5. When steam is issuing in a steady, continuous
stream from the vent tube, shut off the vent cock and
extinguish the outer ring of gas burners.

6. Wait until the index of the manometer records a
temperature of 120° C., then regulate the gas and the
spring safety valve in such a manner that this tem-
perature is just maintained, and leave it thus for
twenty minutes.

7. Extinguish the gas and allow the manometer
index to fall to zero.

8. Now open the vent cock slowly, and allow the

Fig. 21. — Autoclave.

FILTERS. 43

internal pressure to adjust itself to that of the atmos-
phere.

9. Remove the cover and take out the sterilised con-
tents.

Filters. — (a) Cotton-wool. — Practically the only
method in use in the laboratory for the sterilisation
of air or of a gas is by filtration through dry cotton-
wool or glass-wool, the fibres of which entangle the
micro-organisms and prevent their passage.

Perhaps the best example of such a filter is the cotton-
wool plug which closes the mouth of a culture tube.
Not only does ordinary diffusion take place through it,
but if a tube plugged in the usual manner with cotton-
wool is removed from the hot incubator, the tempera-
ture of the contained air rapidly falls to that of the

Fig. 22. — Air filter.

laboratory, and a partial vacuum is formed; air passes
into the tube, through the cotton- wool plug, to restore
the equilibrium, and, so long as the plug remains dry,
in a germ-free condition. If, however, the plug be-
comes moist, either by absorption from the atmosphere,
or from liquids coming into contact with it, micro-
organisms (especially the mould fungi) commence to
multiply, and the long thread forms rapidly penetrate
the substance of the plug, gain access to and contami-
nate the interior of the tube.

If it is desired to sterilise gases before admission to
a vessel containing a pure cultivation of a micro-
organism, as, for instance, when forcing a current of
oxygen over or through a broth cultivation of the

METHODS OF STERILISATION.

diphtheria bacillus, this can be readily effected as
follows :

1. Take a length of glass tubing of, say, 1.5 cm.
diameter, in the centre of which a bulb has been blown,
fill the bulb with dry cotton- wool, wrap a layer of
cotton- wool around each end of the tube, and secure
in position with a turn of thread or string; then ster-
ilise the piece of apparatus in the hot-air oven.

2. Prepare the cultivation in a Ruffer or Woodhead
flask (Fig. 23) the inlet tube of which has a layer
of cotton-wool wrapped round it and secured by

Fig. 23.— Ruffer' s flask.

thread, whilst the exit tube is plugged in the usual
manner.

3. Sterilise a short length of rubber tubing by boil-
ing. Transfer it from the boiling water to a beaker
of absolute alcohol.

4. Remove the rubber tube from the alcohol by
means of a pair of forceps, drain it thoroughly, and
pass through the flame of a Bunsen burner to burn off
the last traces of alcohol.

5. Remove the cotton- wool wraps from the entry
tube of the flask and from one end of the filter tube
and rapidly couple them up by means of the sterile
rubber tubing.

APPARATUS REQUIRED.

6. Connect the other end of the bulb tube with the
delivery tube from the gas reservoir.

The gas in its passage through the dry sterile cotton-
wool in the bulb of the filter tube will be freed from
any contained micro-organisms and will enter the
flask in a sterile condition.

(b) Porcelain. — The sterilisation of liquids by filtra-
tion is effected by passing them through a cylindrical
vessel, closed at one end like a test-tube, and made
either of porous "biscuit" porcelain, hard-burnt and
unglazed (Chamberland system), or of Kieselguhr, a
fine diatomaceous earth (Berkfeld
system), and termed a "bougie" or
"candle" (Fig. 24).

In this method the bacteria are re-
tained in the pores of the filter while
the liquid passes through in a germ-
free condition.

It is obvious that to be effective
the pores of the filter must be ex-
tremely minute, and therefore the
rate of filtration will usually be slow.
To overcome this defect, aspiration or
pressure, or a combination of the two,
may be employed to hasten the process.

T-, . j Fig. 24. — Filter

Apparatus Required. — & candle.

1. Separatory funnel containing the
unfiltered fluid.

2. Sterile filter candle, the open end fitted with a rubber
stopper perforated to receive the delivery tube of the sep-
aratory funnel, and its neck passed through a large rubber
washer (a) which fits the mouth of the filter flask.

3. Sterile filter flask of suitable size, for the reception of
the filtered fluid, its mouth closed by a cotton-wool plug.

4. Water injector pump, or Geryk's pump (an air pump
on the hydraulic principle, sealed by means of low vapor-
tension oil, Fig. 25).

If this latter is employed, a Wulff's bottle, fitted as a
wash-bottle and containing sulphuric acid, must be inter-
posed between the filter flask and the pump, in order to
prevent moist air reaching the oil in the pump.

METHODS OF STERILISATION.

5. Air filter (vide page 43) sterilised.

6. Pressure tubing.

7. Screw clamps (Fig. 26).

To filter flask

Fig. 25. — Geryk air pump.

METHOD. —

i. Couple the exhaust pipe of the suction pump
with the lateral tube of the filter flask (first removing the

cotton-wool plug from this
latter), by means of pressure
tubing, interposing, if neces-
sary, the wash-bottle of acid.
2. Remove the cotton- wool
plug and adjust the porce-
Fig. 26.— Screw clamps. lain candle in the neck of the

filter flask.

3. Attach the nozzle of the separatory funnel to the
filter candle by means of the perforated rubber stopper
(Fig. 27).

TO FILTER. 47

4. Open the tap of the funnel, and exhaust the air
from the filter flask and wash-bottle; maintain the
vacuum until the filtration is complete.

5. Adjust a screw clamp to the pressure tubing
attached to the lateral branch of the filter flask;
screw it up tightly, and disconnect the acid wash-
bottle.

6. Attach the air filter to the open end of the pressure
tubing; open the screw clamp gradually, and allow

To exhaust
pump

Fig. 27. — Apparatus arranged for filtering — aspiration.

filtered air to enter the flask, to destroy the negative
pressure.

7. Detach the rubber tubing from the lateral branch
of the flask, flame the end of the branch in the Bunsen,
and plug its orifice with sterile cotton-wool.

8. Remove the filter candle from the mouth of the
flask, flame the mouth, and plug with sterile cotton-
wool.

METHODS OF STERILISATION.

9. Disinfect the filter candle and separatory funnel
by boiling.

If it is found necessary to employ pressure in addi-
tion to or in place of suction, insert a perforated rubber
stopper into the mouth of the separatory funnel and
secure in position with copper wire; next fit a piece of
glass tubing through the stopper, and connect the
external orifice with an air-pressure pump of some
kind (an ordinary foot pump such as is employed for
inflating bicycle tires is one of the most generally use-

Fig. 28. — Apparatus arranged for filtering.

ful, for this purpose) or with a cylinder of compressed
air or other gas.

Some forms of filter candle are made with the open
end contracted into a delivery nozzle, which is glazed.
In this case the apparatus is fitted up in a slightly
different manner; the fluid to be filtered is contained
in an open cylinder into which the candle is plunged,
while its delivery nozzle is connected with the filter
flask by means of a piece of flexible pressure tubing
(previously sterilised by boiling), as in figure 28.

TO STERIUSE. 49

In order to filter a large bulk of fluid very rapidly it
is necessary to use a higher pressure than glass would
stand, and in these cases the metal receptacle designed
by Pakes, to hold the filter candle itself as well as the
fluid to be filtered, should be employed. (A vacuum
should also be maintained in the filter flask, by means
of an exhaust pump, during the entire process.)

This piece of apparatus consists of a brass cylinder,
capacity 2500 c.c., with two shoulders; and an opening
in the neck at each end, provided with screw threads.

A nut carrying a pressure gauge fits into the top
screw; and into the bottom is fitted a brass cylinder
carrying the filter candle and prolonged downwards
into a delivery tube. Leakage is prevented by means
of rubber washers.

Into the top shoulder a tube is inserted, bent at
right angles and provided with a tap. All the brass-
work is tinned inside (Fig. 29, a). In use the reservoir
is generally mounted on a tripod stand.

To Sterilise.—

1 . Insert the filter candle into its cylinder and screw
this loosely on.

2. Wrap a layer of cotton- wool around the delivery
tube and fasten in position.

3. Remove the nut carrying the pressure gauge and
plug the neck with cotton- wool.

4. Heat the whole apparatus in the autoclave at
120° C. for twenty minutes.

METHOD.—

1. Remove the apparatus from the autoclave, and.
allow it to cool.

2. Screw home the box carrying the bougie.

3. Set the apparatus up in position, with its delivery
tube (from which the cotton-wool wrapping has been
removed) passing through a perforated rubber stopper
in the neck of a filter flask.

4. Fill the fluid to be filtered into the cylinder and

50 METHODS OF STERILISATION.

screw on the nut carrying the pressure gauge. (This
nut should be immersed in boiling water for a few
minutes previous to screwing on, in order to sterilise it.)

5. Connect the horizontal arm of the entry tube
with a cylinder of compressed oxygen (or carbon di-
oxide, Fig. 29, b), by means of pressure tubing.

6. Connect the lateral arm of the filter flask with

LJL.J

Fig. 29 — Fakes' filtering reservoir— pressure and aspiration.

the exhaust pump (Fig. 29, c) and start the latter work-
ing.

7. Open the tap of the gas cylinder; then open the
tap on the entry tube of the filter cylinder and raise
the pressure in its interior until the desired point
is recorded on the manometer. Maintain this pressure
until filtration is completed, by regulating the tap on
the entry tube.

m. THE MICROSCOPE.

THE essentials of a microscope for bacteriological
work may be briefly summed up as follows:

The instrument of the monocular type must be of

Fig. 30. — Microscope complete.
51

52 THE MICROSCOPE.

good workmanship and well finished, rigid, firm, and
free from vibration, not only when upright, but also
when inclined to an angle or in the horizontal position.
The various joints and movements must work smoothly
and precisely, equally free from the defects of "loss
of time" and "slipping." All screws, etc., should con-
form to the Royal Microscopical Society's standard.
It must also be provided with good lenses and a suffi-
ciently large stage. The details of its component parts,
to which attention must be specially directed, are as
follows :

1. The Base or Foot (Fig. 30, a). — Two elementary
forms — the tripod (Fig. 3 1 , a) and the vertical column

Fig. 31. — Foot, three types.

set into a plate (Fig. 31, b) — serve as the patterns for
countless modifications in shape and size of this portion
of the stand. The chief desiderata — stability and ease
of manipulation — are attained in the first by means of
the "spread" of the three feet, which are usually shod
with cork; in the second, by the dead weight of the
foot-plate. The tripod is mechanically the more
correct form, and for practical use is much to be pre-
ferred. Its chief rival, the Jackson foot (Fig. 31, c), is
based upon the same principle, and on the score of
appearance has much to recommend it.

2. The body tube (Fig. 30, b) maybe either that known
as the "long" or "English" (length 250 mm.), or the
"short" or "Continental" (length 160 mm.). Neither

COARSE ADJUSTMENT.

length appears to possess any material advantage over
the other, but it is absolutely necessary to secure objec-
tives which have been manufactured for the particular
tube length chosen. In the high-class microscope of
the present day the body tube is usually shorter than
the Continental, but is provided with a draw tube
which, when fully extended, gives a tube length greater

Fig. 32. — Coarse adjustment.

Fig. 33. — Fine adjustment.

than the English, thus permitting the use of either
form of objective.

(Optical tube length = distance from the back lens of
the objective to the field glass of the ocular.

Mechanical tube length — distance from the end of the
nosepiece to the eyeglass of the ocular.)

3. The coarse adjustment (Fig. 30, c) should be a
rack-and-pinion movement, steadiness and smoothness
of action being secured by means of deeply bevelled
edges and careful countersinking (Fig. 32).

THE MICROSCOPE.

4. The fine adjustment (Fig. 30, d) should on no ac-
count depend upon the action of springs, but should
be of the lever pattern, preferably the Nelson (Fig. 33).
In this form the unequal length of the arms of the lever
secures very delicate movement, and, moreover, only
a small portion of the weight of the body tube is
transmitted to the thread of the vertical screw actuat-
ing the movement.

5. The stage (Fig. 30, e) should be square in shape
and large in area, — at least 12 cm., — flat and rigid, in

Fig. 34. — Mechanical stage.

order to afford a safe support for the Petri dish used for
plate cultivations; and should be supplied with spring
clips (removable at will) to secure the 3 by i glass slides.
A mechanical stage must be classed as a necessity
rather than a luxury so far as the bacteriologist is con-
cerned, as when working with high powers, and espe-
cially when examining hanging-drop specimens, it is
almost impossible to execute sufficiently delicate move-
ments with the fingers. In selecting a mechanical
stage, preference should be given to one which forms

SUBSTAGE CONDENSER. 55

an integral part of the ordinary stage (Fig. 34) rather
than one which needs to be clamped on every time it
is required. The mechanical stage should be fitted
with three (removable) screw studs, so that if necessary
a Vernier finder (Fig. 34, D), such as is usually fitted
to this class of stage, or a Maltwood finder, may be
used.

6. Diaphragm. — Separate single diaphragms must
be avoided; a revolving plate pierced with different-
sized apertures and secured below the stage is prefer-
able, but undoubtedly the best form is the "iris"
diaphragm (Fig. 35).

ig- 35- — Diaphragm iris.

7. The substage condenser is a necessary part of the
optical outfit. Its purpose is to collect the rays of
light reflected by the mirror, by virtue of a short focus
system of lenses, into a cone of large aperture (redu-
cible at will by means of an iris diaphragm mounted
as a part of the condenser), which can be accurately
focussed on the plane of the object. This focussing
must be performed anew for each object, on account
of the variation in the thickness of the slides.

The form in most general use is that known as the
Abbe (Fig. 36) and consists of a plano-convex lens
mounted above a biconvex lens. This combination is
carried in a screw-centering collar below the stage of

THE MICROSCOPE.

Fig- 36.— Optical part of Abbe
illuminator.

the microscope (Fig. 30, /), and must be accurately
adjusted so that its optical axis coincides with that of

the objective. Vertical move-
ment of the entire substage
apparatus effected by means
of a rack and pinion is a de-
cided advantage, and some
means should be provided for
temporarily removing the
condenser from the optical
axis of the microscope.

8. Mirrors. — Below the substage condenser is at-
tached a reversible circular frame bearing a plane
mirror on one side and a concave mirror on the other
(Fig. 30, g) . The plane mirror is that usually employed,
but occasionally, as for example when using low powers
and with the condenser racked down and thrown out
of the optical axis, the concave mirror is used.

9. Oculars, or Eyepieces. — Those known as the
Huyghenian oculars (Fig. 37) will be sufficient for all
ordinary work without resorting

to the more expensive ' ' compensa-
tion" oculars. Two or three, mag-
nifying the "real" image (formed
by the objective) four, six, or eight
times respectively, form a useful
equipment.

10. Objectives. — Three objec-
tives are necessary: one for low-
power work — e. g., i inch, f inch,
or \ inch; one for high-power
work — e. g., ^ inch oil immersion
lens; and an intermediate lens —
e. g.,\ inch or J inch (dry). These
lenses must be carefully selected,

especial attention being paid to the following points:
(a) Correction of Spherical Aberration. — Spherical

Fig. 37. — Huyghenian
eyepiece.

NUMERICAL APERTURE. 57

aberration gives rise to a distorted image, due to the
central and peripheral rays focussing at different points.
(6) Correction of Chromatic Aberration. — Chromatic
aberration gives rise to a coloured fringe around the
edge of the field, which is due to the fact that the
different-coloured rays of the spectrum possess vary-
ing refrangibilities and that a simple lens acts towards
them as a prism.

(c) Flatness of Field. — The visual field should be
large and, above all, flat; in other words, objects at the
periphery of the field should be as distinctly " in focus"
as those in the centre. Failing this, the lens should
possess a large central "flat" area and the entire per-
ipheral ring should come into focus at the same moment
and with the least possible movement of the fine ad-
justment.

(d) Good Definition. — Actual magnification is, within
limits, of course, of less value than clear definition and
high resolving power, for it is upon these properties
we depend for our knowledge of the detailed structure
of the objects examined.

(e) Numerical Aperture (N . A.). — The numerical
aperture may be defined, in general terms, as the ratio
of the effective diameter of the back lens of the objective
to its equivalent focal length. The determination of
this point is a process requiring considerable technical
skill and mathematical ability, and is completely
beyond the powers of the average microscopist.1

Although with the increase in power it is increasingly
difficult to combine all these corrections in one objec-
tive, they are brought to a high pitch of excellence
in the present-day " achromatic" objectives, and so

1 Its importance will be realised, however, when it is stated in the words of
Professor Abbe : " The numerical aperture of a lens determines all its
essential qualities ; the brightness of the image increases with a given magni-
fication and other things being equal, as the square of the aperture ; the resolv-
ing and defining powers are directly related to it, the focal depth of differentia-
tion of depths varies inversely as the aperture, and so forth."

58 THE MICROSCOPE.

remove the necessity for the use of the higher priced
and less durable apochromatic lenses.

In selecting objectives the best "test" objects to
employ are:

1. A thin (one cell layer), even " blood film," stained
with eosin and counterstained with methylene-blue.

2. A thin cover-slip preparation of a young culti-
vation of the B. diphtheria (showing segmentation)
stained with methylene-blue.

Accessories. — Nosepiece. — The first and most useful
accessory is a nosepiece to carry two of the objectives
(Fig. 38), or, better still, all three (Fig. 39). This
nosepiece, preferably constructed of aluminium, is
of the covered-in type, consisting of a curved plate

Fig. 38. — Double nosepiece. Fig. 39. — Triple nosepiece.

attached to the lower end of the body tube — a circular
aperture being cut to correspond to the lumen of that
tube. To the under surface of this plate is pivoted a
similarly curved plate, fitted with three tubulures,
each of which carries an objective. By rotating the
lower plate each of the objectives can be brought suc-
cessively in to the optical axis of the microscope.

Warm Stage (Fig. 40). — This is a flat metal case
through the interior of which water of any required
temperature can be circulated. It is made to clamp
on to the stage of the microscope, and is perforated
with a large hole coinciding with the optical axis of the
microscope; and by raising the temperature of hang-
ing-drop preparations, etc., placed upon it, above that
of the surrounding atmosphere, is extremely useful

MICROMETER.

for observations on spore germination, hanging- drop
cultivations, etc.

Eye Shade (Fig. 41). — This piece of apparatus con-
sists of a pear-shaped piece of blackened metal, hinged

Fig. 40. — Warm stage.

to a collar which rotates on the upper part of the
body tube of the microscope. It can be used to shut
out the image of surrounding objects from the un-
occupied eye, and when carrying out prolonged obser-
vations will be found of real service.

Fig. 41. — Eye screen.

Micrometer. — Some form of micrometer for the pur-
pose of measuring bacteria and other objects is also
essential. Details of those in general use will be found
in the following pages.

60 THE MICROSCOPE.

METHODS OF MICROMETRY.

The unit of length as applied to the measurement
of microscopical objects is the one-thousandth part
of a millimetre (o.ooi mm.), denominated a micron
(sometimes, and erroneously, referred to as a micro-
millimetre), and indicated in writing by the Greek
letter //. Of the many methods in use for the measure-
ment of bacteria, three only will be here described,
viz.:

By means of the stage micrometer.

Fig. 42. — Camera lucida, Abbe pattern.

By means of the ocular or eyepiece micrometer.

By means of the filar micrometer (Ramsden's mi-
crometer eyepiece).

(a) By means of the stage micrometer.

The stage micrometer is a 3 by i inch glass slip having
engraved on it a scale divided to hundredths of a milli-
metre (o.oi mm.), every tenth line being made longer
than the intervening ones, to facilitate counting. A
cover-glass is cemented over the scale to protect it
from injury.

EYEPIECE MICROMETER.

1 . Attach a camera lucida (of the Wollaston, Beale,
or Abbe pattern) to the eyepiece of the microscope.

2. Adjust the micrometer on the stage of the micro-
scope and accurately focus the divisions.

3. Project the scale of the stage micrometer on to
a piece of paper and with pen or pencil sketch in the
magnified image, each division of which corresponds
to 10 ii. Mark on the paper the optical combination
(ocular objective and tube length) employed to pro-
duce this particular magnification.

4. Repeat this procedure for each of the possible
combinations of oculars and objectives fitted to the
microscope supplied, and carefully preserve the scales
thus obtained.

Fig. 43. — Eyepiece micrometer,
ordinary.

Fig. 44. — Eyepiece micrometer, net.

To measure an object by this method simply project
the image on to the scale corresponding to the par-
ticular optical combination in use at the moment.
Read off the number of divisions it occupies and ex-
press them as micra.

In place of preserving a scale for each optical com-
bination, the object to be measured and the micrometer
scale may be projected and sketched, in turn, on the
same piece of paper.

(6) By means of the eyepiece micrometer.

The eyepiece micrometer is a circular glass disc having
engraved on it a scale divided to tenths of a millimetre
(o.i mm.) (Fig. 43), or the entire surface ruled in o.i

62 THE MICROSCOPE.

mm. squares (the net micrometer) (Fig. 44). It can be
fitted inside the mount of any ocular just above the
aperture of the diaphragm and must be adjusted
exactly in the focus of the eyeglass.

Some makers mount the glass disc together with a
circular cover-glass in such a way that when placed
in position in any Huyghenian eyepiece of their own
manufacture, the scale is exactly in focus.

The value of one division of the micrometer scale
must first be ascertained for each optical combination
by the aid of the stage micrometer, thus :

1. Insert the eyepiece micrometer inside the ocular
and adjust the stage micrometer on the stage of the
microscope.

2. Focus the scale of the stage micrometer accurately;
the lines will appear to be immediately below those of
the eyepiece micrometer. Make the lines on the two
micrometers parallel by rotating the ocular.

3. Make two of the lines on the ocular micrometer
coincide with those bounding one division of the stage
micrometer; this is effected by increasing or diminish-
ing the tube length; and note the number of included
divisions.

4. Calculate the value of each division of the eye-
piece micrometer in terms of /*, by means of the fol-
lowing formula:

x = 10 y.
Where x = the number of included divisions of the

eyepiece micrometer.

y = the number of included divisions of the
stage micrometer.

5. Note the optical combination employed in this
experiment and record it with the calculated microm-
eter value.

Repeat this process for each of the other combina-
tions. Carefully record the results.

To measure an object by this method read off the

FILAR MICROMETER. 63

number of divisions of the eyepiece micrometer it
occupies and express the result in micra by a refer-
ence to the standard value for the particular optical
combination employed.

Zeiss prepares a compensating eyepiece micrometer
for use with his apochromatic objectives, the divisions
of which are so computed that (with a tube length of
1 60 mm.) the value of each is equivalent to as many
micra as there are millimetres in the focal length of the
objective employed.

The filar or cobweb micrometer (Ramsden's microm-

Fig. 45. — Ramsden's micrometer.

Fig. 46. — Ramsden's
micrometer field.

eter eyepiece (Fig. 45) consists of an ocular having a
fine " fixed" wire stretching horizontally across the
field (Fig. 46), a vertical reference wire — fixed — ad-
justed at right angles to the first; and a fine wire, paral-
lel to the reference wire, which can be moved across
the field by the action of a micrometer screw; the
trap head is divided into one hundred parts, which
successively pass a fixed index as the head is turned.
In the field is also fixed a comb with the intervals be-
tween its teeth corresponding to one complete revolu-
tion of this screw-head.

64 THE MICROSCOPE.

As in the previous method, the value of each division
of the micrometer scale (i. e., the comb) must first be
determined for each optical combination. This is
effected as follows:

1 . Place the filar micrometer and the stage microm-
eter in their respective positions.

2. Rotate the screw of the filar micrometer until the
movable wire coincides with the fixed one, and the
index marks zero on the screw-head.

3. Focus the scale of each micrometer accurately,
and make the lines on them parallel.

4. Rotate the head of the micrometer screw until
the movable line has traversed one division of the
stage micrometer. Note the number of complete revo-
lutions (by means of the recording comb) and the frac-
tions of a revolution (by means of scale on the head
of the micrometer screw), which are required to meas-
ure the o.o i mm.

5. Make several such estimations and average the
results.

6. Note the optical combination employed in this
experiment and record it carefully, together with the
micrometer value in terms of //.

7. Repeat this process for each of the different
optical combinations and record the results.

To measure an object by this method, simply note
the number of revolutions and fractions of a revolu-
tion of the screw-head required to traverse such object
from edge to edge, and express the result as micra
by reference to the recorded values for that particular
optical combination.

IV. MICROSCOPICAL EXAMINATION OF
BACTERIA AND OTHER MICRO-FUNGL

APPARATUS AND REAGENTS USED IN ORDINARY
MICROSCOPICAL EXAMINATION.

EACH student is provided with a set consisting of
the following pieces of apparatus and reagents.

1. India-rubber "change-mat" upon which cover-
glasses may be rested during the process of staining.

2. Squares of blotting paper about 10 cm., for dry-
ing cover-slips and slides.

(The filter paper known as "German lined" — a
highly absorbent, closely woven paper, having an even
surface and no loose " fluff" to adhere to the specimens
— is the most useful for this purpose.)

3. Glass jar filled with 2 per cent, lysol solution for
the reception of infected cover-glasses and useless
slides.

4. Bunsen burner provided with by-pass.

5. Porcelain trough holding five or six hanging-drop
slides (Fig. 47).

A hanging-drop slide is prepared by cementing a
circular cell of tin, 13 to 15 mm. diameter, and i to 2
mm. in height, to the centre of a 3 by i slip by means
of Canada balsam. It is often extremely convenient
to have two of these cells cemented close together on
one slide.

Another form of hanging-drop slide is made in which
a circular or oval cell is ground out of the centre of a,
3 by i slip. These are more expensive, less convenient
to work with, and are more easily contaminated by
drops of material under examination, and should be
carefully avoided.

5 65

MICROSCOPICAL EXAMINATION.

6. Three aluminium rods (Fig. 48), each about 25
cm. long and carrying a piece of 0.015 gauge platino-
iridium wire 7.5 cm. in length. The end of one of the
wires is bent round to form an oval loop, of about i

Fig. 47. — Hanging-drop slides : a, Double cell seen from above ; b, single
cell seen from the side.

mm. in its short diameter, and is termed a loop or an
ose; the terminal 3 or 4 mm. of another wire is flat-
tened out by hammering it on a smooth iron surface to
form a "spatula"; the third is left untouched or is
pointed by the aid of a file. These instruments are

Fig. 48. — Ends of rods.

used for inoculating culture tubes and preparing speci-
mens for microscopical examination.

The method of mounting these wires may be de-
scribed as follows:

Take a piece of aluminium wire 25 cm. long and

APPARATUS AND REAGENTS. 67

about 0.25 cm. in diameter, and drill a fine hole com-
pletely through the wire about a centimetre from one
end. Sink a straight narrow channel along one side
of the wire, in its long axis, from the hole to the nearest
end, shallow at first, but gradually becoming deeper.

On the opposite side of the wire make a short cut,
2 mm. in length, leading from the hole in the same
direction.

Now pass one end of the platinum wire through the
hole, turn up about 2 mm. at right angles and press
the short piece into the short cut. Turn the long end
of the wire sharply, also at right angles, and sink it
into the long channel so that it emerges from about

Fig. 49. — Platinum rod in aluminium handle — method of mounting.

the centre of the cut end of the aluminium wire (Fig.
49). A few sharp taps with a small hammer will
now close in the side of the two channels over the wire
and hold it securely.

The platinum wire may be fused into the end of a
piece of glass rod, but such a handle is vastly inferior
to aluminium and is not to be recommended.

7. Two pairs of sharp-pointed spring forceps (10
cm. long), one of which must be kept perfectly clean
and reserved for handling clean cover-slips, the other
being for use during staining operations.

8. A box of clean 3 by i glass slips.

9. A glass capsule with tightly fitting (ground on)

MICROSCOPICAL EXAMINATION.

glass lid, containing clean cover-slips in absolute
alcohol.

10. One of Faber's " grease pencils" (yellow, red, or
blue) for writing on glass.

11. A wooden rack fitted with ten drop- bottles
(Fig. 50) each 60 c.c. capacity, containing

Aniline water.

Gentian violet, saturated alcoholic solution.

Gram's iodine.

Absolute alcohol.

Fig. 50. — Drop-bottle.

Fig. 51. — Canada balsam pot.

i per cent, aqueous solution,
saturated alcoholic , solution.

Eosin, yellowish,

Methylene-blue,

Fuchsin, basic,

Carbolic acid, 5 per cent, aqueous solution.

Acetic acid, i per cent, solution.

Sulphuric acid, 25 per cent, solution.
And two pots with air-tight glass caps (Fig. 51), filled
respectively with Canada balsam dissolved in xylol, and
sterile vaseline, and each provided with a piece of glass
rod.

METHODS OF EXAMINATION. 69

METHODS OF EXAMINATION.
Bacteria, etc., are examined microscopically, both

1. In the living state, unstained, or stained.

2. After having been fixed, killed, and stained by
suitable methods.

The preparation of a specimen from a tube cultiva-
tion for examination by these methods may be de-
scribed as follows:

1. Living, Unstained. — (a) "Fresh" Preparation. — •
i. Clean and dry a 3 by i glass slip and place it on
one of the squares of filter paper. Deposit a drop of
water (preferably distilled) or a drop of i per cent,
solution of caustic potash, on the centre of the slip,
by means of the platinum loop.

Fig. 52. — Holding tubes for removing cultivation, as seen from the front.

ft 2. Take the tube cultivation in the left hand

and ignite the cotton-wool plug by holding it
to the flame of the Bunsen burner. Extinguish
the flame by blowing on the plug, whilst rotating
the tube on its long axis, its mouth directed
vertically upwards, between the thumb and fin-
gers. (This operation is termed "flaming the
plug," and is intended to destroy any micro-
organisms that may have become entangled in
^ the loose fibres of the cotton- wool, and which,
if not thus destroyed, might fall into the tube

o 5

70 MICROSCOPICAL EXAMINATION.

when the plug is removed and so accidentally
contaminate the cultivation.)

3. Hold the tube at or near its centre between
the ends of the thumb and first two fingers of
the left hand, and allow the sealed end to rest
upon the back of the hand between the thumb
and forefinger, the plug pointing to the right.
Keep the tube as nearly in the horizontal posi-
tion as is consistent with safety, to diminish
the risk of the accidental entry of organisms

g. 52).

§ 4. Take the handle of the loop between the
thumb and forefinger of the right hand, holding
the instrument in a position similar to that
occupied by a pen or a paint-brush, and sterilise
o the platinum portion by holding it in the flame
55 of a Bunsen burner until it is red hot. Sterilise
the adjacent portion of the aluminium handle
by passing it rapidly twice or thrice through
the flame. After sterilising it, the loop must not
be allowed to leave the hand or to touch against
anything but the material it is intended to
examine, until it is finished with and has been
again sterilised.

5. Grasp the cotton- wool plug of the test-
tube between the little finger and the palm of
the right hand (whilst still holding the loop as
directed in step 4), and remove it from the
mouth of the tube by a "screwing" motion of
the right hand.

6. Introduce the platinum loop into the tube
and hold it in this position until satisfied that
it is quite cool. (The cooling may be hastened
by touching the loop on one of the drops of
moisture which are usually to be found con-
densed on the interior of the glass tube, or by
dipping it into the condensation water at the

o
g

K
O

METHODS OF EXAMINATION. 71

bottom; at the same time care must be taken
in the case of cultures of solid media to avoid
touching either the medium or the growth.)

7. Remove a small portion of the growth by
taking up a drop of liquid, in the case of a fluid
culture, in the loop or by touching it on the
surface of the growth when the culture is on
solid medium ; and withdraw the loop from the
tube without again touching the medium or
the glass sides of the tube.

8. Replace the cotton- wool plug in the mouth
of the tube.

9. Mix the contents of the loop thoroughly with
the drop of water on the 3 by i slide.

10. Again sterilise the loop as directed in step 4,
and replace it in its stand.

11. Replace the tube cultivation in its rack or jar.

12. Remove a cover-slip from the glass capsule by
means of the cover-slip forceps, rest it for a moment
on its edge, on a piece of filter paper to remove the
excess of alcohol, then pass it through the flame of the
Bunsen burner. This burns off the remainder of the
alcohol, and the cover-slip so "flamed" is now clean,
dry, and sterile.

13. I/ower the cover-slip, still held in the forceps,
on to the surface of the drop of fluid on the 3 by i
slip, carefully and gently, to avoid the inclusion of air
bubbles.

14. Examine microscopically (vide infra).
During the microscopical examination, stains and

other reagents may be run in under the cover-slips by
the simple method of placing a drop of the reagent
in contact with one edge of the cover-glass and apply-
ing the torn edge of a piece of blotting paper to the
opposite side. The reagent may then be observed
to flow across the field and come into contact with
such of the micro-organisms as lie in its path.

72 MICROSCOPICAL EXAMINATION.

(b) Hanging-drop Preparation. —

1. Smear a layer of sterile vaseline on the upper
surface of the ring cell of a hanging-drop slide by means
of the glass rod provided with the vaseline bottle, and
place the slide on a piece of filter paper.

2. "Flame" a cover- slip and place it on the filter
paper by the side of the hanging-drop slide.

3. Place a drop of water on the centre of the cover-
slip by means of the platinum loop.

4. Obtain a small quantity of the material it is de-
sired to examine, in the manner detailed above (steps
2 to ii must be followed in their entirety and with
the strictest exactitude whenever tube contents are
being handled), and mix it with the drop of water on
the cover-slip.

5. Raise the cover-slip in the points of the forceps
and rapidly invert it on to the ring cell of the hanging-
drop slide, so that the drop of fluid occupies the centre
of the ring. (Carefully avoid contact between the
drop of fluid and either the ring cell or the layer of
vaseline. Should this happen, the now infected hanging-
drop slide and its cover-slip must be dropped into the
pot of lysol and a new preparation made.)

6. Press the cover-slip firmly down into the vaseline
on to the top of the ring cell. (This spreads out the
vaseline into a thin layer, and besides ensuring the
adhesion of the cover-slip, seals the cells and so retards
evaporation.)

7. Examine microscopically (vide infra).
Microscopical Examination of the Unstained Speci=

mens.—

1 . Place the body tube of the microscope in the ver-
tical position.

2. Arrange the hanging-drop slide on the micro-
scope stage so that the drop of fluid is in the optical
axis of the instrument, and secure it in the position
by means of the spring clips.

EXAMINATION OF UNSTAINED SPECIMENS. 73

3. Use the J-inch objective, rack down the body
tube until the front lens of the objective is almost in
contact with the cover-slip — that is, well within its
focal distance. This is best done whilst bending down
the head to one side of the microscope, so that the
eyes are on a level with the stage.

4. Apply the eye to the ocular and adjust the plane
mirror to the position which secures the best illumina-
tion.

5. Rack the condenser down slightly and cut down
the aperture of the iris diaphragm so that the light,
although even, is dim.

6. Rack up the body tube by means of the coarse
adjustment until the bacteria come into view; then
focus exactly by means of the fine adjustment.

Some difficulty is often experienced at first in finding
the hanging drop, and if the first attempt is unsuccess-
ful, the student must not on any account, whilst still
applying his eye to the ocular, rack the body tube
down (for by so doing there is every likelihood of the
front lens of the objective being forced through the
cover-glass, and not only spoiling the specimen, but
also contaminating the objective) ; but, on the con-
trary, withdraw his eye, rack the tube up, and com-
mence again from step 2.

The examination of a "fresh" specimen or a "hang-
ing-drop" preparation is directed to the determination
of the following data:

1. The nature of the bacteria present — e. g., cocci,
bacilli, etc.

2. The purity of the cultivation; this can only be
determined when gross morphological differences exist
between the organisms present.

3. The presence or absence of spores, which show
their typical refrangibility exceedingly well when
examined by this method.

4. The presence or absence of mobility. In a hang-

74 MICROSCOPICAL EXAMINATION.

ing-drop specimen some form of movement can prac-
tically always be observed, and its character must be
carefully determined by noting the relative positions
of adjacent micro-organisms.

(a) Brownian or molecular movement. Minute par-
ticles of solid matter (including bacteria), when sus-
pended in a fluid, will always show a vibratory move-
ment affecting the entire field, but never altering the
relative positions of the bacteria. (Cocci exhibit this
movement, but with the exception of the Micrococcus
agilis, the cocci are non-motile.)

(6) Streaming movement. This is due to currents
set up in the hanging drop as a result of jarring of the
specimen or of evaporation, and although the relative
position of the bacteria may vary, still the flowing
movement of large numbers of organisms in some one
direction will usually be sufficient to demonstrate the
nature of this motion.

(c) Locomotive movement, or true motility, is deter-
mined by observing some one particular bacillus chang-
ing its position in the field independently of, and in a
direction contrary to, other organisms present.

When the examination is completed and the specimen
finished with, the " fresh specimen" — i. e., the slide with
the cover-slip attached — must be dropped into the
lysol pot. In the hanging-drop specimen, however,
the cover-slip only is infected, and this may be raised
from the ring cell by means of forceps and dropped
into the disinfectant.

Permanent Staining of the Hanging-drop Specimen.
— Occasionally it is necessary to fix and stain a hanging-
drop preparation. This may be done as follows:

1. Remove the cover- slip from the cell by the aid of
the forceps.

2. If the drop is small, fix it by dropping it face
downwards, whilst still wet, on to the surface of some
Gulland's solution or corrosive sublimate solution (vide

KILLED, STAINED. 75

page 76) in a watch-glass. If the drop is large, place
it face upwards on the rubber mat, cover it with an
inverted watch-glass, and allow it to dry. Then fix
it in the alcohol and ether solution (vide infra) .

3. Dip the cover-glass into a beaker containing hot
water in order to remove some of the vaseline adher-
ing to it.

4. Wash successively in alcohol, xylol, ether, and
alcohol, to remove the last traces of grease.

5. Wash in water.

6. Stain, wash, dry, and mount as for an ordinary
cover-slip film preparation (vide page 78).

2. Killed, Stained. — In this method three distinct
processes are necessary:

1. " Preparing" and " fixing" the film.

2. Staining.

3. Mounting.

i a. Preparing the Film. —

1 . Proceed as in making a hanging-drop preparation,
steps 2 to 4.

2. Spread the drop of emulsion evenly over the
cover-slip in the form of a square film to within i mm.
of each edge of the cover-slip.

3. Allow it to dry completely in the air.

i b. Fixing. — Fix by passing the cover-slip, held in
the fingers, three times through the flame of a Bunsen
burner. In preparing films for staining, it is sometimes
necessary (as in the case of those intended for micro-
metric observations) to fix by exposure to a uniform
temperature of 115° C., for twenty minutes. This is
best done in a carefully regulated hot-air oven. Fixa-
tion may also be effected by immersing in some fixative
fluid, such as one of the following:

1. Absolute alcohol.

A , , i -L i f equal parts, for five to thirty

2. Absolute alcohol, . J

< minutes (e. g., for blood or

I milk) .

76 MICROSCOPICAL EXAMINATION.

3. Osmic acid, i per cent, aqueous solution, for .thirty
seconds.

4. Corrosive sublimate, saturated aqueous solution,
for five minutes.

5. Corrosive sublimate (I^ang), for five minutes.
This solution is prepared by dissolving:

Sodium chloride 0.75 gramme

Hydrarg. perchlor 12.00 grammes

Acetic acid 5.00 "

In distilled water loo.oo c.c.

Filter.

6. Gulland's solution, for five minutes. This solu-
tion is prepared by mixing :

Absolute alcohol 25.0 c.c.

Ether 25.0 "

Corrosive sublimate, 20 per cent, alcoholic solution . 0.4 "

Either of these methods of fixation coagulates the
albuminous material and ensures perfect adhesion of
the film to the cover-slip.

Wash the cover-slip thoroughly in running water and
proceed with the staining.

If the film has been prepared from broth, liquefied
gelatine, or pus or other morbid exudations, saturate
the film after fixation with acetic acid 2 per cent, and
allow it to act for two minutes.

Wash with alcohol, then let the alcohol remain on
the cover-slip for two minutes. (This will "clear"
the groundwork and give a much sharper and cleaner
film than would otherwise be obtained.)

If the film has been prepared from blood or blood-
stained fluid, treat with acetic acid 2 per cent, for two
minutes after fixation. Wash with water, dry, and
proceed with the staining. (This will remove the
haemoglobin and facilitate examination.)

2. Staining. —

i. Rest the cover-slip, film side uppermost, on the
rubber mat.

STAINING. 77

2. By means of a drop-bottle, cover the film side of
the cover-slip with the selected stain, allow it to act
for a few minutes, then wash off the excess in running
water.

The penetrating power of stains is increased by (a)
physical means — e. g., heating the stain; (b) chemical
means — e. g., by the addition of carbolic acid, 5 per
cent, aqueous solution; caustic alkalies, 2 per cent,
aqueous solution; water saturated with aniline oil;
borax, 0.5 per cent, aqueous solution.

The most commonly used dyes for cover-slip film
preparations are the aniline dyes.

(A) Basic:

(a) Methylene-blue.

(b) Gentian violet.

These dyes are kept in saturated alcoholic (90 per
cent.) solutions so that decomposition may be re-
tarded.

Two or three drops of alcoholic solution of these
dyes to, say, 4 c.c. water, usually makes a sufficiently
strong staining fluid for cover-slip film preparations.

Carbolic methylene-blue (C.M.B.) and carbol f uchsin
(C.F.) are prepared by covering the cover-slip with 5
per cent, solution of carbolic acid and adding a few
drops of the saturated alcoholic solution of methylene-
blue or f uchsin respectively to it. For aniline gentian
violet (A.G.V.) the stain is added to a saturated solu-
tion of aniline oil in water.

(d) Thionin blue.

(e) Bismarck brown.

(B) Acid:

(a) Eosin, aqueous yellowish.

(b) Safranine.

These dyes are kept in i per cent, aqueous solution
to which is added 5 per cent, of alcohol, as a preserva-
tive. They are generally used in this form.

78 MICROSCOPICAL EXAMINATION.

A few nuclear stains (carmine, haemal oxylin) are
occasionally used more especially in " section" work.

2a. Decolonisation. — After overstaining, films may
be decolourised by washing for a longer or shorter time
in one of the following reagents arranged in ascending
order of power

1. Water.

2. Chloroform.

3. Acetic acid, i per cent.

4. Alcohol.

5. Alcohol absolute, |
Acetic acid, i per cent., /

Hydrochloric, i per cent, aqueous

solution.
Hydrochloric, i per cent, alco-

6. Mineral acids

holic (90 per cent.) solution.

Sulphuric, 25 per cent, aqueous

solution.

Nitric, 33 per cent, aqueous solu-
tion.

2b. Counter staining. — Use colours which will con-
trast with the first stain; e. g.,

^^ ' ( for films stained by methylene-blue or
Eosin, V ~ , • , •
_^ . ( Gram s method.
Fuchsm, )

Methylene-blue, ") r £1

~ . t > for films stained by fuchsm.

Gentian violet, j

3. Mounting.—

1. Wash the film carefully in running water.

2. Blot off the superfluous water with the filter
paper, or dry more completely between two folds of
blotting paper.

3. Complete the drying in the air, or by holding the
cover-slip in the fingers at a safe distance above the
flame of the Bunsen burner.

4. Place a drop of xylol balsam on the centre of a
clean 3 by i glass slide and invert the cover-slip over

IMPRESSION FILMS. 79

the balsam, and lower it carefully to avoid the inclusion
of air bubbles.

NOTE. — Xylol is used in preference to chloroform to
dissolve Canada balsam, as it does not decolourise
the specimen.

Impression films (Klatschprdparat) are prepared
from isolated colonies of bacteria in order that their
characteristic formation may be examined by higher
powers than can be brought to bear on the living culti-
vation. They are prepared from plate cultivations
(vide page 181), in the following manner.

1. Remove a clean cover-slip from the alcohol pot
with sterile forceps and burn off the spirit.

2. Open the plate and rest one edge of the cover-
slip on the surface of the medium a little to one side
of the selected colony. I/ower it cautiously over the
colony until horizontal. Avoid any lateral movement
or the inclusion of bubbles of air.

3. Make gentle vertical pressure on the centre of the
cover-slip with the points of the forceps to ensure
perfect contact with the colony.

4. Steady one edge of the cover-slip with the forceps
and pass the point of a mounted needle just under
the opposite edge and raise the cover-slip carefully;
the colony will be adherent to it. When nearly verti-
cal, grasp the cover-slip with the forceps and remove
it from the plate. Re-cover the plate.

5. Place the cover-slip, film uppermost, on the rubber
mat, and cover it with an inverted watch-glass until
dry.

6. Fix by immersing in one of the fixing fluids pre-
viously mentioned (vide page 75).

7. Clear with acetic acid and alcohol.

8. Stain and mount as an ordinary cover-slip film
preparation, being careful to perform all washing op-
erations with extreme gentleness.

80 MICROSCOPICAL EXAMINATION.

Microscopical Examination of the Stained Specimen.

— (The body tube of the microscope may be vertical
or inclined to an angle.)

1 . Secure the slide on the stage of the microscope by
means of the spring clips.

2. Place a drop of cedarwood oil on the centre of the
cover-slip.

3. Use the y^-inch oil immersion lens of the micro-
scope. Rack down the body tube till the front lens
of the objective is in contact with the oil and nearly
touching the cover-slip.

4. Rack up the condenser until it is in contact with
the under surface of the slide.

5. Apply the eye to the ocular and arrange the plane
mirror so as to obtain the greatest possible amount of
light.

6. Rack up the body tube until the stained film
comes into view.

7. Focus the condenser accurately on the film.

8. Focus the film accurately by means of the fine
adjustment.

V. STAINING METHODS.

BACTERIA STAINS.

IN the following pages are collected the various
"stock" stains in everyday use in the bacteriological
laboratory, together with a selection of the most con-
venient and generally useful staining methods for
demonstrating particular structures or differentiating
groups of bacteria. The stains employed should either
be those prepared by Griibler, of Leipzig, or Merck, of
Darmstadt. The methods printed in ordinary type
are those which a long experience has shown to be the
most reliable, and to give the best results

Methylene=blue. —

1. Saturated Aqueous Solution.
Weigh out

Methylene-blue 1.5 grammes

Place in a stoppered bottle having a capacity of
from 150 to 200 c.c. and add

Distilled water 100.0 c.c.

Allow the water to remain in contact with the dye
for two weeks, shaking the contents of the bottle vig-
ourously for a few moments every day. Filter.

2. Saturated Alcoholic Solution.
Weigh out

Methylene-blue . . 1.5 grammes

Place in a stoppered bottle of 150 c.c. capacity and
add

Alcohol, 90 per cent 100.0 c.c.

Allow the alcohol to remain in contact with the dye
for two hours, shaking vigourously every few minutes.
Filter.

6 81

82 STAINING METHODS.

3. Carbolic Methylene-blue (Kiihne).
Weigh out

Methylene-blue 1.5 grammes

Carbolic acid . . . 5.0 "

and dissolve in

Distilled water 100.0 c.c.

and add

Absolute alcohol 10.0 "

Filter.

4. Alkaline Methylene-blue (Loffler).
Measure out and mix

Methylene-blue, saturated alcoholic solution . . 30.0 c.c.
Caustic potash, o. I per cent, aqueous solution . 100.0 "

Filter.

Fuchsin (Basic). —

5. Saturated Aqueous Solution.
Weigh out

Basic fuchsin 1.5 grammes

and proceed as in preparing the corresponding solution
of methylene-blue (q. v.).

6. Saturated Alcoholic Solution.
Weigh out

Basic fuchsin 3.5 grammes

and proceed as in preparing the corresponding solu-
tion of methylene-blue.

7. Carbolic Fuchsin (Ziehl).
Weigh out

Basic fuchsin i.o gramme

Carbolic acid 5.0 grammes

dissolve in

Distilled water 100.0 c.c.

and add

Absolute alcohol 10.0 "

Filter.

CONTRAST STAINS. 83

Gentian Violet. —

8. Saturated Aqueous Solution.
Weigh out

Gentian violet ; 2.25 grammes

and proceed as in preparing the corresponding solution
of methylene-blue.

9. Saturated Alcoholic Solution.
Weigh out

Gentian violet 5.0 grammes

and proceed as in preparing the corresponding solu-
tion of methylene-blue.

10. Carbolic Gentian Violet (Nicolle).
Measure out and mix

Gentian violet, saturated alcoholic solution . . 10.0 c.c.
Carbolic acid, I per cent, aqueous solution . . 100.0 "

Filter.

Thionine Blue (or Lauth's Violet).—

11. Carbolic Thionine Blue (Nicolle).
Weigh out

Thionine blue . i.o gramme

Carbolic acid 2.5 grammes

and dissolve in

Distilled water 100.0 c.c.

Filter.

Before use dilute with equal quantity of distilled
water and again filter.

CONTRAST STAINS.

Eosin. — There are several commercial varieties of
eosin, which, from the bacteriological point of view,
possess very different values. Griibler lists four varie-
ties, of which two only are useful for bacteriological
work:

Eosin, aqueous yellowish.

Eosin, aqueous bluish.

84 STAINING METHODS.

12. Eosin Aqueous Solution (Yellowish or Bluish
Shade), i per cent.

Weigh out

Eosin, aqueous i.o gramme

dissolve in

Distilled water loo.o c.c.

and add

Absolute alcohol 5.0 "

Filter.

13. Eosin Alcoholic Solution, 0.5 per cent.
Weigh out

Eosin, alcoholic 0.5 gramme

and dissolve in

Alcohol (70 percent.) 100.0 c.c.

Filter.

Vesuvin (or Bismarck Brown). —

14. Saturated A queous Solution.
Weigh out

Vesuvin 0.5 gramme

and dissolve in

Distilled water . 100.0 c.c.

Filter.
Safranine. —

Weigh out

Safranine 0.5 gramme

and dissolve in

Distilled water 100.0 c.c.

Filter.

TISSUE STAINS.
Lithium Carmine (Orth). —
Weigh out

Carmine 2.5 grammes

and dissolve in

Lithium carbonate, cold saturated solution . . . loo.o c.c.

Filter.
Picrocarmine. —

Weigh out

Picrocarmine 2.0 grammes

and dissolve in

Distilled water loo.o c.c.

TISSUE STAINS. 85

Hsematoxylin. —

1 . Weigh out

Hsematoxylin 2.0 grammes

and dissolve in

Absolute alcohol loo.o c.c.

2. Weigh out

Ammonium alum 2.0 grammes

and dissolve in

Distilled water 100.0 c.c.

3. Mix i and 2, allow the mixture to stand forty-
eight hours, then filter.

4. Add

Glycerine 85.0 c.c.

Acetic acid, glacial 10.0 "

5. Allow the stain to stand for one month; then
filter ready for use.

Aniline Gentian Violet (For Weigert's Fibrin Stain). —
Weigh out

Gentian violet i.o gramme

and dissolve in

Absolute alcohol 15.0 c.c.

Distilled water 80.0 "

then add

Aniline oil 3.0 "

Shake well and filter before use.
Alum Carmine (Meyer). —
Weigh out

Alum 2.5 grammes

Carmine I.o gramme

and place in a glass beaker.
Measure out in a measuring cylinder,

Distilled water 100.0 c.c.

Place the beaker on a sand-bath, add the water in
successive small quantities, and keep the mixture'boil-
ing for twenty minutes. Measure the solution and
make up to 100 c.c. by the addition of distilled water.
Filter.

86 STAINING METHODS.

METHODS OF DEMONSTRATING STRUCTURE OF
BACTERIA.

To Demonstrate Capsules.
1. MacConkey. —

Stain. —
Weigh out

Dahlia 0.5 gramme

Methyl green (oo crystals) 1.5 grammes

rub up in a mortar with

Distilled water 100.0 c.c.

Add

Fuchsin, saturated alcoholic solution 10.0 "

and make up to 200 c.c. by the addition of

Distilled water 90.0 c.c.

Filter.

Allow the stain to stand for two weeks before use;
keep in a dark place or in an amber glass bottle. Owing
to the unstable character of the methyl green, this
stain deteriorates after about six months.

METHOD. — ,

1 . Prepare and fix film in the usual manner.

2. Flood the cover-slip with the stain and allow it
to act for five to ten minutes.

3. Wash very thoroughly in water; if necessary,
direct a powerful stream of water on the film from a
wash-bottle.

4. Dry and mount.

2. Welch's Method.—

1. Prepare and fix film in the usual manner.

2. Flood the slide with acetic acid 2 per cent.; allow the
acid to remain in contact with the film for two minutes.
This swells up and fixes the capsule and enables it to take
the §tain.

3. Blow off the acetic acid by the aid of a pipette.

4. Immerse in aniline gentian violet, five to thirty seconds.

5. Wash in water.

6. Dry and mount.

TO DEMONSTRATE FLAGEU/A. 87

3. Ribbert's Method.—

Stain. —

Measure out and mix

Acetic acid, glacial 12.5 c.c.

Alcohol, absolute 50.0 "

Distilled water loo.o "

Warm to 36° C. (e. g.y in the "hot" incubator) and satu-
rate with dahlia. Filter.
METHOD.—

1. Prepare and fix films in the usual manner.

2. Cover the film with the stain and allow it to act for one
or two seconds only.

3. Wash thoroughly in water.

4. Dry and mount.

To Demonstrate Flagella.

1. Muir's Modified Pitfield.— This is the best method
and gives the most reliable results, for not only is the
percentage of successful preparations higher than with
any other, but the bacilli and flagella retain their rela-
tive proportions.

(a) Mordant. —

Tannic acid, 10 per cent, aqueous solution . . . 10 c.c.

Corrosive sublimate, saturated aqueous solution . 5 "

Alum, saturated aqueous solution 5 '«

Carbolic fuchsin (Ziehl) 5 "

Mix thoroughly.

A precipitate forms which must be allowed to settle
for a few hours.

Decant off the clear fluid into tubes and centrif ugalise
thoroughly.

This solution keeps for about a couple of weeks,
but must be re-centrifugalised each time, before use.

(b) Stain.—

Alum, saturated aqueous solution 25 c.c.

Gentian violet, saturated alcoholic solution ... 5 "

Filter.

This stain must be freshly prepared.
METHOD. — The cultivations employed should be
smear agar cultures, twelve to eighteen hours old if

88 STAINING METHODS.

incubated at 37° C., twenty-four to thirty hours if
incubated at 22° C.

1. Remove a very small quantity of the growth by
means of the platinum spatula.

2. Emulsify it with a few cubic centimetres of dis-
tilled water in a watch-glass, by gently moving the
spatula to and fro in the water. Do not rub up the
growth on the side of the watch-glass.

3. Spread a thin film of the emulsion on a newly
flamed cover-slip, using no force, but rather leading
the drop over the cover-slip with the platinum loop.

4. Allow the film to dry in the air, properly protected
from falling dust.

5. Fix by passing thrice through the Bunsen flame,
holding the cover-slip whilst doing so by one corner
between the finger and thumb.

6. Pour on the film as much of the mordant as the
cover-glass will hold. Grasp the cover-slip with the
forceps and hold it, high above the flame, until steam
rises. Allow the steaming mordant to remain in con-
tact with the film two minutes.

7. Wash well in water and dry carefully.

8. Pour on the film as much of the stain as the cover-
glass will hold. Steam over the flame as before for
two minutes.

9. Wash well in water.

10. Dry and mount.

2. •« Pitfield " Original Method.^-

(a) Mordant. —

Tannic acid I gramme

Water 10 c.c.

(6) Stain.—

Saturated aqueous solution of alum ...... 10 c.c.

Saturated alcoholic solution of gentian violet . . I "
Distilled water 5 "

Mix equal parts of a and b before using.

i. Prepare and fix the film in the manner described above.

VAN ERMENGEM'S METHOD. 89

2. Boil the mixture and immerse the cover-slip in it,
whilst still hot, for one minute.

3. Wash in water.

4. Examine in water; if satisfactory, dry and mount in
Canada balsam.

3. MacCrorrie's Method. —
Mordant-Stain. —
Measure out and mix

Night blue, saturated alcoholic solution .... IO c.c.
Potash alum, saturated aqueous solution .... IO "
Tannin, 10 per cent, aqueous solution 10 "

NOTE. — The addition of gallic acid, o.i to 0.2 gramme,
may improve the solution, but is not necessary.
METHOD. —

1. Prepare and fix the films as above.

2. Pour some of the mordant-stain on the film and warm
gently, high above the flame, for two minutes (or place in
the "hot" incubator for a like period).

3. Wash thoroughly in water.

4. Dry and mount.

4. Van Ermengem's Method. — This method, being
merely a precipitation of a silver salt on the micro-organ-
isms and not a true stain, is unsatisfactory; the relative pro-
portions of bacteria and flagella are almost invariably de-
stroyed. '

(a) Fixing Fluid. —

Osmic acid, 2 per cent, aqueous solution . , . . 10 c.c.
Tannic acid, 20 per cent, aqueous solution ... 20 "
Acetic acid, glacial i "

The fixing fluid should be prepared some days before use
and filtered as required. In colour it should be distinctly
violet.

(6) Sensitising Solution. —

Silver nitrate, o. 5 per cent, aqueous solution.

This solution must be kept in a dark blue glass bottle or
in a dark cupboard.

Filter immediately before use.
(c) Reducing Solution. —
Weigh out

Gallic acid 5 grammes

Tannic acid 3 "

Potassium acetate, fused 10 "

and dissolve in

Distilled water 350 c.c.

Filter.

90 STAINING METHODS.

This solution will keep active for several days, but fresh
solution must be used for each preparation.
METHOD. —

1. Prepare emulsion, make and fix films therefrom as de-
scribed in the preceding method, steps i to 4.

2. Pour on the film as much of the fixing solution as the
cover-glass will hold, heat carefully over the flame till steam
rises, and allow the steaming fixing fluid to act for five
minutes.

3. Wash well in water.

4. Wash in absolute alcohol.

5. Wash in distilled water.

6. Pour some of the sensitising solution on the film and
allow it to act for from thirty seconds to one minute; blot
off the excess of fluid with filter paper.

7. Without washing, transfer the film to a watch-glass
containing the reducing solution and allow it to remain
therein for from thirty seconds to one minute; blot off the
excess of fluid with filter paper.

8. Without washing, again treat the film with the sensi-
tising solution, this time until the film commences to turn
black.

9. Wash in distilled water.

10. Dry and mount.

5. Loffler's Method.—
(a) Mordant. —

Tannic acid, 20 per cent, aqueous solution . . . 10 c.c.

Ferrous sulphate, saturated aqueous solution . . 5 "

Hsematoxylin solution (prepared by boiling I
gramme logwood with 8 c.c. distilled water,
filtering and replacing the loss from evapora-
tion) ................. 3 "

Carbolic acid, I per cent, aqueous solution ... 4 "

This solution must be freshly prepared.
Alternative Mordant. —

Tannic acid, 20 per cent, aqueous solution . . .
Ferrous sulphate, saturated aqueous solution . .
Fuchsin, saturated alcoholic solution .....

(6) Stain.—

Weigh out

Methylene-blue .............. \

Or methylene-violet ............ 1 4 grammes

Or fuchsin ................ J

and dissolve in

Aniline water, freshly saturated and filtered . . 100 c.c.

TO STAIN SPORES. 91

METHOD. —

1. Prepare and fix films as above.

2. Pour the mordant on to the film and warm cautiously
Over the flame till steam rises; keep the mordant gently
steaming for one minute.

3. Wash well in distilled water till no more colour is dis-
charged; if necessary, wash carefully with absolute alcohol.

4. Filter a few drops of the stain on to the film, warm
as before, and allow the steaming stain to act for one minute.

5. Wash well in distilled water.

6. Dry and mount.

NOTE. — The flagella of some organisms can be demon-
strated better by means of an alkaline stain or an acid stain
— a point to be determined for each. According to require-
ments, therefore, LofBer recommends the addition of sodium
hydrate, i per cent, aqueous solution, i c.c. ; or an equal
quantity of an exactly comparable solution of sulphuric
acid.

To Stain Spores.
1. Single Stain.—

1. Prepare cover-slip film in the usual way.

2. In fixing, pass the cover-slip film fifteen or thirty
times through the flame instead of only three. This
destroys the resisting power of the spore membrane
and allows the stain to reach the interior.

3. Stain in the usual way with methylene-blue or
fuchsin.

4. Wash in water.

5. Dry and mount
2. Double Stain.—

1. Prepare and fix film in the usual way — i. e., pass
three times through flame to fix.

2. Cover the film with hot carbol-fuchsin and hold
in the forceps above a small flame until the fluid begins
to steam. Set the cover-slip down and allow it to
cool. Repeat the process when the stain ceases to
steam and continue to repeat until the stain has been
in contact with the film for twenty minutes. (This
stains both spores and bacteria.)

3. Wash in water.

92 STAINING METHODS.

4. Decolourise in alcohol, 2 parts; acetic acid, i per
cent., i part. (This removes the stain from everything
but the spores.)

5. Wash in water.

6. Mount the cover-slip in water and examine micro-
scopically with the ^-inch objective. (Spores should
be red, and the rest of the film colourless or a very
light pink.) If satisfactory, pass on to section 7; if
unsatisfactory, repeat steps 2 to 5.

7. Counterstain in weak methylene-blue. (Now
spores red, bacilli blue.)

8. Wash in water.

9. Dry and mount.

The spores of different bacilli differ greatly in their
resistance to decolourising reagents; even the spores
of the same species of organisms vary according to
their age. Young spores are more easily decolourised
than those more mature.

Sulphuric acid, i per cent, aqueous solution, and
hydrochloric acid, 0.5 per cent, alcoholic (90 per cent.)
solution, are useful decolourising reagents.

3. Muller's Method.—

1. Prepare and fix films in the usual manner.

2. Immerse in absolute alcohol for two minutes, then in
chloroform for two minutes ; wash in water. This dissolves
out any fat or crystals that might otherwise retain the
" spore" stain.

3. Immerse in chromic acid, 5 per cent, aqueous solution,
for one minute; wash in water.

4. Pour Ziehl's carbolic fuchsin on the film, warm as in
previous methods, and allow it to act for ten minutes.

5. Wash in water.

6. Decolourise in sulphuric acid, 5 per cent, aqueous solu-
tion, for five seconds.

7. Wash in water.

8. Counterstain with Kuhne's carbolic methylene-blue for
one or two minutes.

9. Wash in water.

10. Dry and mount.
(Spores red, bacilli blue.)

DIFFERENTIAL METHODS OF STAINING. 93

4. Abbott's Method.—

1. Prepare and fix films in the usual manner.

2. Pour I/jffler's alkaline methylene-blue on the film;
warm cautiously over the flame till steam rises and allow
the hot steam to act for one to five minutes.

3. Wash thoroughly in water.

4. Decolourise in nitric acid, 2 per cent, alcoholic (80 per
cent.) solution.

5. Wash thoroughly in water.

6. Counterstain in eosin, i per cent, aqueous solution.

7. Wash.

8. Dry and mount.
(Spores blue, bacilli red.)

DIFFERENTIAL METHODS OF STAINING.

Gram's Method. — This method depends upon the
fact that the protoplasm of some bacteria permits
aniline gentian violet and Lugol's iodine solution,
when applied consecutively, to enter into a chemical
combination which results in the formation of a new
blue-black pigment, only very sparingly soluble in
alcohol. Such organisms are said to " stain by Gram."

1. Prepare a cover-slip film and fix in the usual way.

2. Stain in aniline gentian violet three to five minutes.

To prepare aniline water, pour 4 or 5 c.c. aniline oil into
a stoppered bottle and add distilled water, 100 c.c. Shake
vigourously and filter immediately before use. The excess of
oil sinks to the bottom of the bottle and may be used again.

Filter as much aniline water on to the cover-slip as
it will hold; then add the smallest quantity of alcoholic
solution of gentian violet which suffices to saturate the
aniline water and form a "bronze scum" upon its
surface.

3. Wash in water.

4. Treat with Lugol's iodine solution until the film
is black or dark brown.

To do this treat with iodine solution for a few seconds,
wash in water, and examine the film over a piece of
white filter paper. Note the colour. Repeat this

94 STAINING METHODS.

process until the film ceases to darken with the fresh
application of iodine solution.

I/ugol's solution is prepared by dissolving

Iodine I gramme

Iodine of potassium 3 grammes

In distilled water 300 c.c.

5. Wash in water.

6. Wash with alcohol until no more colour is dis-
charged and the alcohol runs away clear and colourless.

7. Wash in water.

8. Counterstain very lightly with dilute eosin, dilute
fuchsin, or vesuvin.

NOTE. — This section may be omitted when dealing
with films prepared from pure cultivations.

9. Wash in water.

10. Dry and mount.

Qram=Weigert Method. — (Also extremely useful for
sections.)

1-5. Proceed as for the corresponding sections of
Gram's method (quod vide).

6. Dry in the air.

7. Wash in aniline oil, i part, xylol, 2 parts, until
no more colour is discharged.

8. Wash in xylol.

9. Mount in xylol balsam.

(Then fibrin and hyaline tissue are stained deep blue,
whilst bacteria which " stain Gram" appear of a deep
blue- violet colour.)

Modified Qram=Weigert Method. — (Employed to
demonstrate trichophyta in hair.)

1 . Soak the hairs in ether for ten minutes to remove
the fat.

2. Stain ten to sixty minutes in a tar-like solution
of aniline gentian violet (prepared by adding 15 drops
of the alcoholic solution of gentian violet to 3 drops
of aniline water).

TO DIFFERENTIATE THE DIPHTHERIA BACIIJvUS. 95

3. Dry the hairs between pieces of blotting paper.

4. Treat with perfectly fresh iodine solution.

5. Again dry between blotting paper.

6. Treat with aniline oil. (If necessary, add a drop
or two of nitric acid to the oil.)

7. Again treat with aniline oil.

8. Treat with aniline oil and xylol, equal parts.

9. Clear with xylol.

10. Mount in xylol balsam.

To Differentiate the Tubercle Bacillus and Other Acid=
fast Bacilli (Ziehl-Neelsen's Method). -

1. Smear a thin, even film of the specimen on the
cover-slip by means of the platinum loop. (In the
case of sputum, if it is a very watery specimen, allow
the film to dry, then spread a second and even a third
layer over the first.)

2. Fix by passing three times through the flame.

3. Stain in hot carbol-fuchsin (as in staining for
spores) for five to ten minutes. (This stains every-
thing on the film.)

4. Decolourise by dipping in sulphuric acid, 25 per
cent. (This removes stain from everything but acid-
fast bacilli; e. g., tubercle, leprosy, and smegma bacilli.)

5. Wash in water.

6. Wash in alcohol till no more colour is discharged.
(This usually, but not invariably, removes the stain
from acid-fast bacilli other than tubercle; e. g., smegma
bacillus.)

7. Wash in water.

8. Counterstain in weak methylene-blue. (Stains
non-acid-fast bacilli, leucocytes, epithelial cells, etc.)

9. Wash in water, dry, and mount.

To Differentiate the Diphtheria Bacillus (Neisser's
Method). -
Stain I. —
Weigh out

Methylene-blue I gramme

96 STAINING METHODS.

and dissolve in

95 per cent, alcohol 20 c.c.

Distilled water 950 "

then add

Acetic acid (glacial) 50 "

Filter.
Stain II. —
Weigh out

Vesuvin 2 grammes

and dissolve in

Distilled water, boiling 1000 c.c.

Cool and filter.
METHOD.—

1. Prepare and fix films in the usual way.

2. Pour stain I on the film and allow it to act for
thirty seconds.

3. Wash thoroughly in water.

4. Pour stain II on to the film and allow it to act for
thirty seconds.

5. Wash thoroughly in water.

6. Dry and mount.

NOTE. — The cultivation from which the films are pre-
pared must be upon blood-serum which has been in-
cubated at 37° C. for from nine to eighteen hours.

The bacilli are stained a light brown by the vesuvin,
which contrasts well with the two or three black spots,
situated at the poles and occasionally one in the centre
representing protoplasmic aggregations (? meta-chro-
matic granules) stained by the acid methylene-blue.

To Demonstrate the (?) Syphilis Bacillus (Lustgarten's
Method).—

1. Prepare and fix the film in the usual manner.

2. Stain in aniline gentian violet or aniline fuchsin, twenty-
four hours.

3. Wash in water.

4. Place in 1.5 per cent, solution of permanganate of

TO DEMONSTRATE THE SYPHILIS BACILLUS. 97

potash (a brown precipitate of oxide of manganese forms),
three to four seconds.

5. Wash in watery solution of pure SO2. If not decolour-
ised, repeat sections 4 and 5 until satisfactory.

6. Wash in water, dry, and mount.
Or—

1. Prepare and fix the film in the usual manner.

2. Stain in hot carbol-fuchsin.

3. Wash in water to which has been added 2 or 3 drops
of chloride of lime.

4. Decolourise in concentrated solution of chloride of iron.

5. Wash thoroughly in water.

6. Dry and mount.
Syphilis bacilli remain red.

NOTE. — These methods are equally applicable to tissue
sections.

VL METHODS OF DEMONSTRATING
BACTERIA IN TISSUES.

FOR bacteriological purposes, sections of tissues are
most conveniently prepared by either the freezing
method or the paraffin method.

The latter is decidedly preferable, but as it is of
greater importance to demonstrate the bacteria, if such
are present, than to preserve the tissue elements un-
altered, the "frozen" sections are often of value.

Whichever method is selected, it is necessary to
take small pieces of the tissue for sectioning, — 0.5 c.c.
cubes when possible, — not exceeding half a centimetre
in thickness. Post-mortem material should be secured
as soon after the death of the animal as possible.

The tissue is prepared for cutting by—

(a) Fixation; that is, by causing the death of the
cellular elements in such a manner that they retain
their characteristic shape and form.

The fixing fluids in general use are : Absolute alcohol ;
corrosive sublimate, saturated aqueous solution; cor-
rosive sublimate, Lang's solution (vide page 76);
formaldehyde, i per cent, aqueous solution. (Of
these, Lang's corrosive sublimate solution is decidedly
the best all-round " fixative.")

(6) Hardening; that is, by rendering the tissue of
sufficient consistency to admit of thin slices or " sec-
tions" being cut from it. This is effected by passing
the tissue successively through alcohols of gradually
increasing strength: 30 per cent, alcohol, 50 per cent,
alcohol, 75 per cent, alcohol, 90 per cent, alcohol, recti-
fied alcohol, absolute alcohol.

In both these processes a large excess of fluid should

always be used.

FREEZING METHOD.

1. Fixation. Place the pieces of tissue in a wide-
mouthed glass bottle and fill with absolute alcohol. Al-
low the tissues to remain therein for twenty-four hours.

2. Hardening. Remove the alcohol (no longer abso-
lute, as it has taken up water from the tissues) from
the bottle and replace

it with fresh absolute
alcohol. Allow the tis-
sues to remain therein
for twenty-four hours.

NOTE. — If not need-
ed for cutting imme-
diately, the hardened
tissues can be stored
in 50 per cent, alco-
hol.

3. Remove the alco-
hol from the tissues by
soaking in water from
one to two hours.
Remove the stopper
from the bottle; rest
a glass funnel in the
open mouth and place
under a tap of running
water. The water, of

course, overflows, but the tissues remain in the bottle.

4. Impregnate the tissues with mucilage for twelve
to twenty-four hours, according to size. Transfer the
pieces of tissue to a bottle containing sterilised gum
mixture.

Formula. —

Gum arable 5 grammes

Saccharose I gramme

Boric acid I "

Water . . 100 c.c.

Fig- 53' — Washing tissues.

100 TO DEMONSTRATE BACTERIA IN TISSUES.

5. Place the tissue on the plate of a freezing micro-
tome (Cathcart's is perhaps the best form), cover and
surround with fresh gum mixture; freeze, and cut
sections.

6. Float the sections off the knife into a glass dish
containing tepid water and allow them to remain
therein for about an hour to dissolve out the gum.

(If not required at once, store in 50 per cent, alcohol.)

7. Transfer to a glass capsule containing the selected
staining fluid, by means of a section lifter.

8. Transfer the sections in turn to a capsule con-
taining absolute alcohol (to dehydrate) and to one
containing xylol or oil of cloves (to clear).

Alternative Method. —

8a. Place the stained section in a dish of clean water
and introduce a glass slide obliquely beneath the sec-
tion; with a mounted needle draw the section on to
the slide and hold it there; gently remove the slide
from the water, taking care that any folds in the sec-
tion are floated out before the slide is finally removed
from the water.

86. Drain away as much water as possible from the
section. Drop absolute alcohol on to the section from
a drop bottle, to dehydrate it.

Sc. Double a piece of blotting paper and gently press
it on the section to dry it.

Sd. Drop on xylol to clear the section.

9. Place a large drop of xylol balsam on the section
and carefully lower a cover-glass on to the balsam.

PARAFFIN METHOD,

i. Fixation. Place the pieces of tissue, resting on
cotton- wool, in a wide-mouthed glass bottle. Pour on
a sufficient quantity of the corrosive sublimate fixing
fluid; allow the tissue to remain therein for twelve to
twenty-four hours according to size.

PARAFFIN METHOD.

101

2. Pour off the fixing fluid and wash thoroughly in
running water for twenty minutes to half an hour to
remove the excess of corrosive sublimate.

3. Hardening. Place the tissues in each of the fol-
lowing strengths of alcohol in turn for from twelve to
twenty-four hours: 50 per cent., 75 per cent., 90 per
cent., absolute.

4. Dehydrate by transferring to fresh absolute alco-
hol.

5. Clear by immersing in equal parts of absolute alco-
hol and xylol for six to eighteen hours ; then in pure
xylol for from fifteen minutes to six hours. The tissue
should not be allowed to remain in the xylol longer
than is necessary. When "cleared," the tissue be-
comes more or less transparent.

Fig. 54. — L- shaped brass moulds.

Fig. 55.— Paraffin kettle.

6. Transfer the tissues to a vessel containing melted
paraffin. Place this vessel in a paraffin water-bath
regulated for 2° C. above the melting-point of the
paraffin used and allow the tissues to soak for some
six to twelve hours to ensure complete impregnation.
The paraffin used should have a melting-point of not
more than 58° C. For all ordinary purposes 54° C.
will be found quite high enough.

7. Imbed in fresh paraffin in a metal ( or paper) mould,
(a) Arrange a pair of L-shaped pieces of metal on

a plate of glass to form a rectangular trough (Fig. 54).

102 TO DEMONSTRATE BACTERIA IN TISSUES.

(b) Pour fresh melted paraffin into the mould from a
special vessel (Fig. 55).

(d) Blow gently on the surface of the paraffin in the
mould, and as soon as a film of solid paraffin has formed,
carefully lift the glass plate on which the mould is set
and lower plate and mould together into a basin of
cold water.

(e) When the block is cold, break off the metal L's;
trim off the excess of paraffin from around the tissue
with a knife, taking care to retain the rectangular
shape, and store the block in a pill-box.

When several pieces of tissue have to be imbedded
at one time, shapes of copper, 10
cm., 5 cm., and 2.5 cm. square re-
spectively, and 0.75 cm. deep (Fig.
56) will be found extremely useful.
These placed upon plates of glass
replace the pair of L's in the above

Fig. 56.— Paraffin

' mould. process.

8. Cement the block on the

carrier of a "paraffin" microtome (the Minot, the
Jung, or the Cambridge Rocker) with a little melted
paraffin. Greater security is obtained if the paraffin
around the base of the block is melted by means of a
hot metal or glass rod.

9. Cut sections — thin, and if possible in ribbands.
Mounting Paraffin Sections. —

1. Place a large drop of 30 per cent, alcohol on
the centre of a slide (or cover-slip) and float the sec-
tion on to the surface of the drop, from a section
lifter.

2. Hold the slide in the fingers of one hand and warm
cautiously over the flame of a Bunsen burner, touch-
ing the under surface of the glass from time to time
on the back of the other hand. As soon as the slide

STAINING PARAFFIN SECTIONS.

I03

ig. 57. — Section rack.

feels distinctly warm to the skin, the paraffin section
will flatten out and all wrinkles disappear.

(The slide with the section floating on it may be
rested on the top of the paraffin bath for two or three
minutes, instead of warm-
ing over the flame as here
described.)

3. Cautiously tilt up
the slide and blot off the
excess of spirit with blot-
ting paper, leaving the
section attached to the
centre of the slide.

4. Place the slide in a
wire rack (Fig. 57), section
downwards, in the "hot"
incubator for twelve to
twenty-four hours. At

the end of this time the section is firmly adherent to
the glass, and is treated during the subsequent steps
as a "fixed" cover-glass film preparation.

NoTS. — If large, thick sections have to be manipulated,
or if time is of importance, it is often advisable to add a
trace of Mayer's albumin to the alcohol before floating out
the section. If this substance is employed, a sojourn of
twenty minutes to half an hour in the "hot" incubator will
be found ample to ensure firm adhesion of the section to the
slide. Mayer's albumin is prepared as follows:

Weigh out

Salicylate of soda I gramme

and dissolve in

Glycerine 50 c.c.

Add

White of egg 50 "

Mix thoroughly by means of an egg whisk.
Filter into a clean bottle.

Staining Paraffin Sections. —

i. Warm paraffin section over the Bunsen flame to

104 TO DEMONSTRATE BACTERIA IN TISSUES.

soften (but not to melt) the paraffin, then dissolve out
the wax with xylol.

2. Remove xylol by flushing the section with alcohol.

3. If the tissue was originally "fixed" in a corrosive
sublimate solution, the section must now be treated
with lyUgol's iodine solution for five minutes.

4. Stain deeply, if using a single stain, as the sub-
sequent processes decolourise.

5. Wash in water, decolourise if necessary.

6. Flood with several changes of absolute alcohol to
dehydrate the section.

7. Clear in xylol. (Oil of cloves is not usually em-
ployed, as it decolourises the section.)

8. Mount in xylol balsam.

SPECIAL STAINING METHODS FOR SECTIONS.

Double=staining Carmine and Gram=Weigert.—

1. Prepare the sec-
tion for staining as
above, sections i to 3.

2. Stain in lithium
carmine ( Orth's ) or
picrocarmine for ten
to thirty minutes in a
porcelain staining pot
(Fig. 58).

3. Wash in picric acid
solution until yellow.

At this stage cell nuclei are red, protoplasm is yellow,
and bacteria are colourless.

Picric acid solution is prepared by mixing

Picric acid, saturated aqueous solution . . . . 40 c.c.

Hydrochloric acid i "

Alcohol (90 per cent.) 160 "

4. Wash in water.

5. Wash in alcohol.

Fig. 58. — Staining pot.

TO DEMONSTRATE CAPSULES. 105

6. Stain in aniline gentian violet.

7. Wash in iodine solution till dark brown or black.

8. Wash in water.

9. Dip in absolute alcohol for a second.

10. Decolourise with aniline oil till no more colour
is discharged.

11. Wash with aniline oil, 2 parts, xylol, i part.

12. Clear with xylol.

13. Mount in xylol balsam.

Alternative Qram=Weigert Method for Sections. —

1 . Fix paraffin section on slide and prepare for stain-
ing in the usual manner.

2. Stain in alum carmine for about fifteen minutes.

3. Wash thoroughly in water.

4. Filter aniline gentian violet solution on to the
section on the slide and allow to stain about twenty-
five minutes.

5. Wash thoroughly in water.

6. Treat with Lugol's iodine until section ceases to
become any blacker.

7. Wash thoroughly in water.

8. Treat with a mixture of equal parts of aniline
oil and xylol until no more colour comes away.

9. Wash thoroughly with xylol.

10. Decolourise and dehydrate rapidly with abso-
lute alcohol until there remains only a very faint
bluish tint.

1 1 . Clear with xylol.

12. Mount in xylol balsam.
To Demonstrate Capsules.—

1. MacConkey's Method. — Stain precisely as for
cover-slip films.

2. Friedldnder's Method. —
Stain.—

Gentian violet, saturated alcoholic solution . . . 50 c.c.

Acetic acid, glacial 10 "

Distilled water , . 100 "

106 TO DEMONSTRATE BACTERIA IN TISSUES.

METHOD. —

1. Prepare the sections for staining, secundum artem.

2. Stain sections in the warm (e. g,, in the hot incubator)
for twenty-four hours.

3. Wash with water.

4. Decolourise lightly with acetic acid, i per cent.

5. Dehydrate rapidly with absolute alcohol.

6. Clear with xylol.

7. Mount in xylol balsam.

To Demonstrate Acid=fast Bacilli.—

1. Prepare the sections for staining in the usual
way.

2. Stain with haematin solution ten to twenty sec-
onds, to obtain a pure nuclear stain ; then wash in water.

3. Stain with carbolic fuchsin twenty to thirty
minutes at 47° C.; then wash in water.

4. Treat with aniline hydrochlorate, 2 per cent,
aqueous solution, for two to five seconds.

5. Decolourise in 75 per cent, alcohol till section
appears free from stain — fifteen to thirty minutes.

6. Dehydrate with absolute alcohol.

7. Clear very rapidly with xylol.

8. Mount in xylol balsam.

VH. CLASSIFICATION OF FUNGI.

FUNGI are divided into:

1. Hymenomycetes (including the mushrooms, etc.).

2. Hyphomycetes (moulds).

3. Blastomycetes (yeasts and torulse).

4. Schizomycetes (bacteria).

NOTE. — Formerly myxomycetes were included in
the fungi ; they are now recognised as belonging to the
animal kingdom, and are termed "mycetozoa."

MORPHOLOGY OF THE HYPHOMYCETES.

At the commencement of his studies, the attention
of the student is directed to the various non-pathogenic
moulds and yeasts, not only that he may gain the
necessary technique whilst handling cultivations of
harmless organisms, but also because these very species
are amongst the commonest of those that may acci-
dentally contaminate his future preparations.

The hyphomycetes are composed of a mycelium of
short jointed rods or "hyphae" springing from an axis
or germinal tube which develops from the spore. Hy-
phae are —

(a) Nutritive or submerged.

(6) Reproductive or aerial.

The protoplasm of these cells contains granules,
pigment, oil globules, and sometimes crystals of cal-
cium oxalate.

Reproduction. — Apical spore formation — asexual;

zoospores — sexual.

Mucorinse. — Mucor (Fig. 59). — Note the branching
filaments— "mycelium" (a), "hyphse" (6).

Note the asexual reproduction..

107

io8

CLASSIFICATION OF FUNGI.

1 . A filament grows upwards. At its apex a septum
forms, then a globular swelling appears — " sporagium"
(d). This possesses a definite membrane.

2. From the septum grows a club-shaped mass of
protoplasm — "columella" (c).

Fig- 59- — Mucor mucedo.

Fig. 60. — Aspergillus.

3. The rest of the contained protoplasm breaks up
into "swarm spores" (e).

Finally the membrane ruptures and spores escape.
Perisporaceae. — Aspergillus (Fig. 60). — Note the
branching filaments — "mycelium" (a).

Note the asexual
reproduction. -

1. A filament (b)
grows upwards, its
termination becomes
clubbed ; on the
clubbed extremity
flask-shaped cells ap-
pear — " sterigmata"

Fig. 61. — Penicillium. (c).

2. At free end of

each sterigma is formed an oval body — a spore or
"gonidium" (d), which, when ripe, is thrown off from
the sterigma.

MORPHOLOGY OF THE B^ASTOMYCETES. 109

Penicillium (Fig. 6 1 ) . — Note the branching filaments
— " mycelium" (a) (frequently containing globules).
Note the asexual reproduction.

1 . A filament grows upwards — ' ' goniodophore' ' ( b ) —
and its apex divides up into several branches —
"basidia"(c).

2. At the apex of each branch a flask-shaped cell,
"sterigma" (d), appears.

3. At the apex of each sterigma appears a row of
oval cells — "spores" or "conidia" (e). These, when
ripe, are cast off from the sterigmata.

Ascomycetse. — O'idium (Fig. 62). — (This family is
perhaps as nearly re-
lated to the blastomy-
cetes as it is to the hy-
phomycetes.)

Note the branching
filaments — "pseudo-
mycelium " ( a ) . Here
and there filaments are
broken up at their ends
into oval or rod-shaped Fig' 62'~ Oidium-

segments, "oi'dia," and behave as spores.

Note the asexual reproduction. From the pseudo-
mycelium arise true hyphae (b), each of which in turn
ends in a chain of spores (c).

MORPHOLOGY OF THE BLASTOMYCETES.

The blastomycetes are composed of spherical or oval
cells (8 to 9.5 /i in diameter), which, when rapidly
multiplying by budding, may form a spurious myce-
lium. A thin cell-wall encloses the granular proto-
plasm, in which vacuoles and sometimes a nucleus
may be noted. This latter is best seen when stained
with osmic acid or haematoxylin.

During their growth and multiplication the blasto-

CLASSIFICATION OF FUNGI.

mycetes split up solutions containing sugar into alcohol
and CO2.

Reproduction. — Budding, ascospores — asexual.

(Torulae, whilst resembling yeasts in almost every
other respect, never form spores.)

Saccharomyces (Fig. 63). — Note the round or oval
cells of granular protoplasm (a) containing solid par-
ticles and vacuoles (c), and surrounded by a definite
envelope.

Note the asexual reproduction.

i. "Gemmation" — that is, the budding out of
daughter cells (b) from various parts of the gradually
enlarging mother cell. These are eventually cast off

Fig. 63. — Saccharomyces
with ascospores.

Fig. 64. — Torula.

and in turn become mother cells and form fresh groups
of buds.

2. Spore formation — "ascospores" (e). These are
formed at definite temperatures and within well-de-
fined periods; e. g., Saccharomyces cerevisiae, thirty
hours at 25° to 37° C., or ten days at 12° C.

Torulae (Fig. 64). — Note the points in morphology,
etc., as above.

Note the absence of ascospore formation.

VIIL SCHIZOMYCETES.

Classification and Morphology. — Bacteria are often
classified, in general terms, according to their life
functions, into —

Saprogenic, or putrefactive bacteria;
Zymogenic, or fermentative bacteria;
Pathogenic, or disease-producing bacteria;
or according to their metabolic products into —

Chromogenic, or pigment-producing bacteria;
Photogenic, or light-producing bacteria;
Aerogenic, or gas-producing bacteria;
and so on.

Such broad groupings as these have, however, but
little practical value when applied to the systematic
study of the fission fungi.

On the other hand, no really scientific classification
of the schizomycetes has yet been drawn up, and the
varying morphological appearances of the members
of the family are still utilised as a basis for classifica-
tion, as under—

i. Micrococci, or Cocci (Fig. 65). — Rounded or oval
cells, subdivided according to the arrangement of the
individuals after fission, into —

Diplococci and Streptococci, where division takes
place in one plane only, and the individuals remain
attached (a) in pairs or (b) in chains.

Tetrads, Merismopedia, or Pediococci, where divi-
sion takes place alternately in two planes at right
angles to each other, and the individuals remain at-
tached in flat tablets of four, or its multiples.

Sarcinae, where division takes place in three planes
successively, and the individuals remain attached in
cubical packets of eight and its multiples.

112

SCHIZOMYCETES.

Staphylococci, where division takes place in three
planes, but with no definite sequence; consequently
the individuals remain attached in pairs, short chains,
plates of four, cubical packets of eight, and irregular
masses containing numerous cocci.

Fig. 65. — Types of bacteria — cocci : I, Diagram of sphere indicating planes
of fission; 2, diplococci ; 3, streptococci; 4, tetrads; 5, sarcinse ; 6, staphy-
lococci.

2. Bacilli (Fig. 66, i to 3). — Rod-shaped cells. A
bacillus, however short, can usually be distinguished
from a coccus in that two sides are parallel. Some
bacilli after fission retain a characteristic arrangement
and may be spoken of as diplobacilli or streptobacilli.

Fig. 66. — Types of bacteria — bacilli, etc. : I, Bacilli ; 2, diplobacilli ; 3,
streptobacilli ; 4, spirilla ; 5, vibrios ; 6, spirochaetae.

(Leptothrix is a term that in the past has been loosely
used to signify a long thread, but is now restricted to
such forms as belong to the leptothriciae (vide page

3. Spirilla (Fig. 66, 4 to 6). — Curved and twisted
filaments. Classified, according to shape, into —

ANATOMY. 113

Spirillum.

Vibrio (comma).

Spirochaeta.

Higher forms of bacteria are also met with, which
possess the following characteristics : They are attached,
unbranched, filamentous forms, showing —

(a) Differentiation between base and apex;

(b) Growth apparently apical;

(d) "Pseudo-branching" from apposition of cells;
and are classified into —

1 . Beggiotoa. 1 Free swimming forms, which contain

2. Thiothrix. / sulphur granules.

3. Crenothrix. -\ ^* £ . . , -

These forms do not contain sulphur

4. Cladothnx. > ,

. . ( granules.

5. Leptothnx. )

6. Strep tothrix. A group which exhibits true
branching, and contains some pathogenic species.

The morphology of the same bacterium may vary
greatly under different conditions of growth as to —

1. The composition, reaction, etc., of the nutrient
medium.

2. The atmosphere in which it is cultivated.

3. The temperature at which it is incubated.

4. Exposure to or protection from light.

For example, under one set of conditions the exami-
nation of a pure cultivation of a bacillus may show a
short oval rod as the predominant form, whilst another
culture of the same bacillus, but grown under different
conditions, may consist almost entirely of long fila-
ments or threads, a condition known as ' ' pleomorphism. ' "

ANATOMY.

i. Capsule (Fig. 67, b). — A gelatinous envelope
(probably akin to mucin in composition) surrounding
each individual organism, and preventing absolute con-

SCHIZOMYCETES.

Fig. 67. — Structure of bacteria.

tact between any two. In some species the capsule is
well marked, but it cannot be demonstrated in all. In
very well marked cases of gelatinisation of the cell wall,

the individual cells are
cemented together in a
coherent mass, to which
the term "zoogloea" is
applied. In some species
colouring matter or ferric
oxide is stored in the
capsule.

2. Cell Watt (Fig. 6?, c).
— A protective differen-
tiation of the outer layer
of the cell protoplasm;
difficult to demonstrate,
but treatment with io-
dine or salt solution
sometimes causes shrinkage of the cell contents — " plas-
molysis" — and so renders the cell wall apparent (e. g.y
B. megatherium) in the manner shown in figure 68.
Stained bacilli, when examined with
polarising microscope, often show a
doubly refractile cell wall (e. g., B.
tuberculosis and B. anthracis).

In some of the higher bacteria the
cell wall exhibits this differentia-
tion to a marked degree and forms
a hard sheath within which the cell

Fig. 68. — Plasmolysis.

protoplasm is freely movable; and

during the process of reproduction the cell protoplasm

may be extruded, leaving the empty tube unaltered in

shape.

3. Cell Contents. — Protoplasm (mycoprotein) con-
tains a high percentage of nitrogen, but is said to differ
from proteid in that it is not precipitated by C2HfiO.
It is usually homogeneous in appearance — sometimes

1 1 5

granular — and may contain oil globules or sap vacuoles
(Fig. 67, d), chromatin granules, and even sulphur
granules. Sap vacuoles must be distinguished from
spores, on the one hand, and the vacuolated appear-
ance due to plasmolysis, on the other.

The cell contents may sometimes be differentiated
into a parietal layer, and a central body (e. g., beg-
giotoa) when stained by haematoxylin.

4. Nucleus. — This structure has not been conclu-
sively proved to exist, but in some bacteria denser
masses of protoplasm situated at the poles and ex-
hibiting a more marked affinity than the rest of the cell
protoplasm for aniline dyes have been observed. These
are termed polar granules or Polkorner (Fig. 67, e).
Occasionally these aggregations of protoplasm alter
the colour of the dye they take up. They are then
known as metachromatic bodies or Ernst' schen Korner.

5. Flagella (Organs of Locomotion, Fig. 67, a).—
These are gelatinous elongations of the cell protoplasm
(or more probably of the capsule), occurring either at
one pole, at both poles, or

scattered around the entire
periphery. Flagella are not
pseudopodia. The possession
of flagella was at one time
suggested as a basis for a sys-
tem of classification, when the
following types of ciliation Fig. 69._Types of ciliavtion.
were differentiated:

1. Polar: (a) Monotrichous (a single flagellum situ-
ated at one pole; e. g., B. pyocyaneus).

(b) Amphitrichous (a single flagellum at each pole;
e. g., Spirillum volutans).

2. Diffuse : Peritrichous (flagella scattered around the
entire periphery; e. g., B. typhosus).

1 1 6 SCHIZOMYCETES.

PHYSIOLOGY.

Reproduction. — Active Stage. — Vegetative, i. e., by
the division of cells, or "fission."

1. The cell becomes elongated and the protoplasm
aggregated at opposite poles.

2. A circular constriction of the organism takes
place midway between these aggregations, and a sep-
tum is formed in the interior of the cell at right angles
to its length.

3. The division deepens, the septum divides into two
lamellae, and finally two cells are formed.

4. The daughter cells may remain united by the
gelatinous envelope for a variable time. Eventually
they separate and themselves subdivide.

Fig. 70. — Fission of cocci. Fig. 71. — Fission of bacteria.

Cultures on artificial media, after growing in the
same medium for some time, — i. e., when the pabulum
is exhausted, — show "involution forms " (Fig. 72), well
exemplified in cultures of B. pestis on agar two days
old, B. diphtherias on potato four to six days old.

They are of two classes, viz.:

(a) Involution forms characterised by alterations of
shape. (Not necessarily dead.)

(b) Involution forms characterised by loss of staining
power. (Always dead.)

Resting Stage. — Spore Formation. — Conditions in-
fluencing spore formation: In an old culture nothing
may be left but spores. It used to be supposed that
spores were always formed when

(a) The supply of nutrient was exhausted.

PHYSIOLOGY. 117

(6) The medium became toxic from the accumula-
tion of metabolic products.

So that the species might not
become extinct. This is not
altogether correct; e. g., the
temperature at which spores are
best formed is constant for each
bacterium, but varies with dif-
ferent species; again, aerobes
require oxygen for sporulation,
but anaerobes will not spore in
its presence.

(A) Arthrogenous : Noted only
in the micrococci. One complete

element resulting from ordinary Fis- 72-— involution forms.

fission becomes differentiated for

the purpose, enlarges, and develops a dense cell wall.

One or more of the cells in a series may undergo this

alteration.

This process is probably not real spore formation,
but merely relative increase of resistance. These so-
called arthrospores have never been observed to ''ger-
minate," nor is their resistance very marked, as they
fail to initiate new cultures, after having been exposed
to a temperature of 80° C. for ten minutes.

(B) Endogenous: The cell protoplasm becomes dif-
ferentiated and condensed into a spherical or oval
mass (very rarely cylindrical). After further contrac-
tion the outer layers of the mass become still more
highly differentiated and form a distinct spore mem-
brane, and the spore itself is now highly refractile.
It has been suggested, and apparently on good grounds,
that the spore membrane consists of two layers, the
exosporium and the endosporium. Each cell forms
one spore only, usually in the middle, occasionally at

a b c d e

IW?

Il8 SCHIZOMYCETES.

one end (four exceptions, however, are recorded; e. g.,
B. inflatus). The shape of the parent cell may be un-
altered, as in the anthrax bacillus, or altered, as in the
tetanus bacillus, and these points serve as the basis
for a classification of spore-bearing bacilli, as follows :

(A) Cell body of the parent
bacillus unaltered in shape
(Fig. 73, a).

(B) Cell body of the parent
bacillus altered in shape.

Fig- 73-— Types of spore-bear- _. r

ing bacilli. i. tlostr^d^um (Fig. 73, b):

Rod swollen at the centre and

attenuated at the poles; spindle shape; e. g., B. buty-
ricus.

2. Cuneate (Fig. 73, c) : Rods swollen slightly at one
pole and more or less pointed at the other; wedge-
shaped.

3. Clavate (Fig. 73, d) : Rods swollen at one pole and
cylindrical (unaltered) at the other; keyhole-shaped;
e. g., B. chauvei.

4. Capitate (Fig. 73, e) : Rods with a spherical en-
largement at one pole; drumstick-shaped; e. g., B.
tetani.

The endospores remain within the parent cell for a
variable time (in one case it is stated that germination
of the spore occurs within the interior of the parent
cell — "endo-germination"), but are eventually set free,
as a result of the swelling up and solution of the cell
membrane of the parent bacillus in the surrounding
liquid, or of the rupture of that membrane. They
then present the following characteristics:

1 . Well-formed, dense cell membranes, which renders
them extremely difficult to stain, but when once stained
equally difficult to decolourise.

2. High refractility, which distinguished them from
vacuoles.

3. Higher resistance than the parent organism to

GERMINATION. 119

such lethal agents as heat, desiccation, starvation,
time, etc., this resistance being due to

(a) Low water contents of plasma .

/IN T i j ^ °i the spore

(b) Low heat-conducting power

; ; ' , M. membrane.

This resistance varies somewhat with the particular
species, — e. g., some spores may resist boiling for a few
minutes, — but practically all are killed if the boiling is
continued for ten minutes.

Germination. — When transplanted to suitable media
and placed under favourable conditions, the spores
germinate, usually within twenty-four to thirty-six
hours, and successively undergo the following changes :

1. Swell up slowly and enlarge, through the ab-
sorption of water.

2. Lose their refrangibility.

3. At this stage one of three processes (but the par-
ticular process is always constant for the same species)
may be observed:

(a) The spore grows out into the new bacillus without
discarding the spore membrane (which in this case
now becomes the cell membrane); e. g., B. lepto-
sporus.

(6) It loses its spore membrane by solution; e. g.t B.
anthracis.

In this process the rupture may be either polar, and
taking place at one pole only (e. g., B. butyricus) or
occurring at both poles (e. g., B. sessile), or equatorial;
e. g., B. subtilis.

In those cases where the spore membrane is discarded
the cell membrane of the new bacillus may either be
formed from —

(a) The inner layer of the spore membrane, which
has undergone a preliminary splitting into parietal
and visceral layers; e. g., B. butyricus.

(6) The outer layers of the cell protoplasm, which

I2O

SCHIZOMYCETES.

become differentiated for that purpose; e. g., B. mega-
therium.

The new bacillus now increases in size, elongates,

O G

Fig. 74-

Fig. 75-

Fig. 76.

•^^^

o 0®

Fig. 77.

Fig. 78.

Figs- 74, 75, 76, 77, 78.— Types of germination : Fig. 74, simple ; Fig. 75,
solution; Fig. 76, polar; Fig. 77, bipolar; Fig. 78, equatorial.

FOOD STUFFS. 121

and takes on a vegetative growth, — i. e., undergoes
fission, — the bacilli resulting from which may in their
turn give rise to spores.

Food Stuffs. — i. Organic Foods.—-

(a) The pure parasites (e. g., B. leprae) will not live
outside the living body.

(6) Both saprophytic and facultative parasitic bac-
teria agree in requiring non-concentrated food.

(c) The facultative parasites need highly organised
foods; e. g., proteids or other sources of nitrogen and
carbon, and salts.

(d) The saprophytic bacteria are more easily culti-
vated; e. g.,

1. Some bacteria will grow in almost pure distilled
water.

2. Some bacteria will grow in pure solutions of the
carbohydrates .

2. Water is absolutely essential to the growth of
bacteria.

Food of a definite reaction is needed for the growth
of bacteria. As a general rule growth is most active
in media which react slightly acid to phenolphthalein
— that is, neutral or faintly alkaline to litmus. Mould
growth, on the other hand, is most vigourous in media
that are strongly acid to phenolphthalein.

Environment. — The influence of physical agents
upon bacterial life and growth is strongly marked.

i. Atmosphere. — The presence of oxygen is necessary
for the growth of some bacteria, and death follows
when the supply is cut off. Such organisms are termed
obligate aerobes.

Some bacteria appear to thrive equally well whether
supplied with or deprived of oxygen. These are termed
facultative anaerobes.

A third class will only live and multiply when the
access of free oxygen is completely excluded. These
are termed obligate anaerobes.

122 SCHIZOMYCETES.

2. Temperature. — Practically no bacterial growth
occurs below 5° C., and very little above 40° C. 30° C.
to 37° C. is the most favorable for the large majority
of micro-organisms. *

The maximum and minimum temperatures at which
growth takes place, as well as the optimum, are fairly
constant for each bacterium.

Bacteria have been classified, according to their
optimum temperature, into—

MIN. OPT. MAX.

1. Psychrophilic bacteria (chiefly water

organisms) o° C 15° C. 30° C.

2. Mesophilic bacteria (includes patho-

genic bacteria) 15° C. 27° C. 45° C.

3. Thermophilic bacteria 45° C. 55° C. 70° C.

The thermal death-point of an organism is another
biological constant; and is that temperature which
causes the death of the vegetative forms when the
exposure is continued for a period of ten minutes.

3. Light. — Many organisms are indifferent to the
presence of light. On the other hand, light frequently
impedes growth, and alters to a greater or lesser ex-
tent the biochemical characters of the organisms —
e. g., chromogenicity or power of liquefaction. Patho-
genic bacteria undergo a progressive loss of virulence
when cultivated in the presence of light.

4. Movements. — Movements, if slight and simply of
a flowing character, do not appear to injuriously affect
the growth of bacteria; but violent agitation, such as
shaking, absolutely kills them.

A condition of perfect rest would seem to be that
most conducive to bacterial growth.

The Metabolic Products of Bacteria. — Pigment Pro-
duction.— Many micro-organisms produce one or more
vivid pigments — yellow, orange, red, violet, fluorescent,
etc. — during the course of their life and growth. The
colouring matter usually exists as an intercellular

THE METABOLIC PRODUCTS OF BACTERIA. 123

excrementitious substance. Occasionally, however, it
appears to be stored actually within the bodies of the
bacteria. The chromogenic bacteria are therefore
classified, in accordance with the final destination of
the colouring matter they elaborate, into —

Chromoparous Bacteria: in which the pigment is
diffused out upon and into the surrounding medium.

Chromophorous Bacteria: in which the pigment is
stored in the cell protoplasm of the organism.

Parachromophorous Bacteria : in* which the pigment
is stored in the cell wall of the organism.

Different species of chromogenic bacteria differ in
their requirements as to environment, for the produc-
tion of their characteristic pigments; e. g., some need
oxygen, light, or high temperature; others again favor
the converse of these conditions.

Light Production. — Some bacteria, and usually those
originally derived from water, whether fresh or salt,
exhibit marked phosphorescence when cultivated
under suitable conditions. These are classed as " pho-
togenic."

Enzyme Production. — Many bacteria produce soluble
ferments or enzymes during the course of their growth,
as evidenced by the liquefaction of gelatine, the clot-
ting of milk, etc. These ferments may belong to either
of the following well-recognised classes: proteolytic,
diastatic, invertin, rennet.

Toxin Production. — A large number, especially of
the pathogenic bacteria, elaborate or secrete poisonous
substances concerning which but little exact knowledge
is available, although many would appear to be en-
zymic in their action.

These toxins are usually differentiated into —

Extracellular (or Soluble) Toxins: those which are
diffused into, and held in solution by, the surrounding
medium.

Intracellular (or Insoluble) Toxins : those which are

124 SCHIZOMYCETKS.

so closely bound up with the cell protoplasm of the
bacteria elaborating them that up to the present time
no means has been devised for their separation or ex-
traction.

End-products of Metabolism. — Under this heading
are included —

Organic Acids (e. g., lactic, butyric, etc.).

Alkalies (e. g., ammonia).

Aromatic Compounds (e. g., indol, phenol).

Reducing Substances (e. g., those reducing nitrates
to nitrites).

Gases (e. g., sulphuretted hydrogen, carbon dioxide,
etc.).

And while the discussion of their formation, etc.,
is beyond the scope of a laboratory handbook, the
methods in use for their detection and separation
come into the ordinary routine work and will therefore
be described (vide page 221 et seq.).

IX. NUTRIENT MEDIA.

IN order that the life and growth of bacteria may
be accurately observed in the laboratory, it is neces-
sary—

1. To isolate individual members of the different
varieties of micro-organisms.

2. To cultivate isolated organisms apart from other
associated or contaminating bacteria — i. e., in pure
culture.

For the successful achievement of these objects it is
necessary to provide nutriment in a form suited to the
needs of the particular bacterium or bacteria under
observation, and in a general way it may be said that
the nutrient materials should approximate as closely
as possible, in composition and character, to the natural
soil of the organism.

The general requirements of bacteria as to their
food-supply have already been indicated (page 121)
and many combinations of proteids and of carbohy-
drates have been devised, from time to time, on these
lines. These, together with various vegetable tissues,
physiological or pathological fluid secretions, etc., are
collectively spoken of as nutrient media or culture media.

The greater number of these media are primarily
fluid, but, on account of the rapidity with which bac-
terial growth diffuses itself through a liquid, it is im-
possible to study therein the characteristics of indi-
vidual organisms. Many such media are, therefore,
subsequently rendered solid by the addition of sub-
stances like gelatine or agar, in varying proportions,
the proportions of such added material being generally
mentioned when referring to the media; e. g., 10 per
cent, gelatine, 2 per cent. agar. Gelatine is employed

125

126 NUTRIENT MEDIA.

for the solidification of those media it is intended to
use in the cultivation of bacteria at the room tem-
perature or in the "cold" incubator. In the percent-
ages usually employed, gelatine media become fluid at
25° C. ; higher percentages remain solid at somewhat
higher temperatures, but the difficulty of filtering
strong solutions of gelatine militates against their gen-
eral use.

Agar media, on the other hand (even in 2 per cent,
solutions), only become liquid when exposed to 90° C.
for a considerable period, and again solidify at 40° C.

When it becomes necessary to render these media
fluid, heat is applied, upon the withdrawal of which they
again assume their solid condition. Such media should
be referred to as liquefiable media; in point of fact,
however, they are usually grouped together with the
solid media.

NOTE. — It must here be stated that the designation
10 per cent, gelatine or 2 per cent, agar refers only to
the quantity of those substances actually added in
the process of manufacture, and not to the percentage
of gelatine or agar, as the case may be, present in
the finished medium; the explanation being that the
commercial products employed contain a large propor-
tion of insoluble material which is separated off by
filtration during the preparation of the liquefiable
media.

Other media, again, — e. g., potato, coagulated blood-
serum, etc., — cannot be again liquefied by physical
means, and these are spoken of as solid media.

The following pages detail the method of preparing
the various nutrient media, those in general use being
printed in bolder type than those occasionally required
for more highly specialised work. It must be premised
that scrupulous cleanliness is to be observed with
regard to all apparatus, vessels, funnels, etc., employed
in the preparation of media.

MEAT EXTRACT. 12 J

MEAT EXTRACT.

A watery solution of the extractives, etc., of lean
meat (usually beef) forms the basis of several nutrient
media. This solution is termed "meat extract," and
is prepared as follows:

1. Measure 1000 c.c. of distilled water into a large
flask (or glass beaker, or enamelled iron pot) and add
500 grammes (roughly, ij pounds) of fresh lean meat,
— e. g., beefsteak or bullock's heart, — finely minced in
a mincing machine.

2. Heat the mixture gently in a water-bath, taking
care that the temperature of the contents of the flask
does not exceed 40° C. for the first twenty minutes.
(This dissolves out the soluble proteids, extractives,
salts, etc.)

3. Now raise the temperature of the mixture to the
boiling-point, and maintain at this temperature for
ten minutes. (This precipitates some of the albumins,
the haemoglobin, etc., from the solution.)

4. Strain the mixture through sterile butter muslin
or a perforated porcelain funnel, then filter the liquid
through Swedish filter paper into a sterile " normal"
litre flask, and when cold make up to 1000 c.c. by
the addition of distilled water — to replace the loss
from evaporation.

5. If not needed at once, sterilise the meat extract in
bulk in the steam steriliser for twenty minutes on each
of three consecutive days.

Calf, sheep, or chicken flesh is occasionally substi-
tuted for the beef; or the meat extract may be pre-
pared from animal viscera, such as brain, spleen, liver,
or kidneys.

NOTE. — As an alternative method, 3 grammes of
Wyeth's beef juice, invalid bovril, or Liebig's extract
of meat may be dissolved in 1000 c.c. distilled water,
and heated and filtered as above.

128 NUTRIENT MEDIA.

Media prepared from such meat extracts are, how-
ever, eminently unsatisfactory when used for the cul-
tivation of the more highly parasitic bacteria.

Reaction of Meat Extract. — Meat extract thus pre-
pared is acid in its reaction, owing to the presence of
acid phosphates of potassium and sodium, weak acids
of the gly colic series, and organic compounds in which
the acid character predominates. Owing to the nature
of the substances from which it derives its reaction,
the total acidity of meat extract can only be estimated
accurately when the solution is at the boiling-point.
Moreover, it has been observed that prolonged boiling
(such as is involved in the preparation of nutrient
media) causes it to undergo hydrolytic changes which
increase its acidity, and the meat extract only becomes
stable in this respect after it has been maintained at
the boiling-point for forty-five minutes.

Although meat extract always reacts acid to phenol-
phthalein, it occasionally reacts neutral or even alka-
line to litmus; and again, meat extract that has been
rendered exactly neutral to litmus still reacts acid to
phenolphthalein. This peculiar behaviour depends
upon two factors:

1 . Litmus is insensitive to many weak organic acids
the presence of which is readily indicated by phenol-
phthalein.

2. Dibasic sodium phosphate which is formed during
the process of neutralisation is a salt which reacts
alkaline to litmus, but neutral to phenolphthalein.
In order, therefore, to obtain an accurate estimation
of the reaction of any given sample of meat extract,
it is essential that —

1 . The meat extract be previously exposed to a tem-
perature of 1 00° C. for forty-five minutes.

2. The estimation be performed at the boiling-point.

3. Phenolphthalein be used as the indicator.

The estimation is carried out by means of titration

METHOD OF ESTIMATING THE REACTION. 129

experiments against standard solutions of caustic soda,
in the following manner:

Method of Estimating the Reaction. —

Apparatus Required :

1. 25 c.c. burette graduated in
tenths of a centimetre.

2. I c.c. pipette graduated in
hundredths, and provided with
rubber tube, pinch-cock, and
delivery nozzle.

3. 25 c.c. measure (cylinder or
pipette, calibrated for 98° C. —
not 15° C.).

4. Several 60 c.c. conical beakers
or Erlenmeyer flasks.

5. White porcelain evaporating
basin, filled with boiling water
and arranged over a gas flame
as a water-bath.

6. Bohemian glass flask, fitted as
a wash-bottle, and filled with
distilled water, which is kept
boiling on a tripod stand.

Solutions Required :

1. loN NaOH, accurately stan-
dardised.

2. — NaOH, accurately standard-
ised.

3. — NaOH, accurately standard-
ised.

4. 0.5 per cent, solution of phenol -
phthalein in 50 per cent, alcohol.

METHOD. — Arrange the apparatus as indicated in
figure 79.

Fig- 79- — Arrangement of apparatus for titrating.

(A) i. Fill the burette with -£- NaOH.

2. Fill the pipette with -^ NaOH.

3. Measure 25 c.c. of the meat extract (previously
heated in the steamer at 100° C. for forty-five minutes)

130 NUTRIENT MEDIA.

into one of the beakers by means of the measure ; rinse
out the measure with a very small quantity of boiling
distilled water from the wash-bottle, and then add
this rinse water to the meat extract already in the
beaker.

4. Run in about 0.5 c.c. of the phenolphthalein solu-
tion and immerse the beaker in the water-bath, and
raise to the boil.

5. To the medium in the beaker run in - - NaOH
cautiously from the burette until the end-point is

a b c

Fig. 80. — a, Sample of filtered meat extract or nutrient gelatine to which
phenolphthalein has been added. The medium is acid, as evidenced by the
unaltered colour of the sample, b, The same neutralised by the addition of

-- NaOH. The production of this faint rose-pink colour indicates that the
" end-point," or neutral point to phenolphthalein, has been reached. If such
a sample is cooled down to say 30° or 20° C., the colour will be found to be-
come more distinct and decidedly deeper and brighter, resembling that shown
in c. c, Also if, after the end-point is reached, a further 0.5 c.c. or i.o c.c.
— NaOH be added to the sample, the marked alkalinity is evidenced by the
deep colour here shown.

reached, as indicated by the development of a pinkish
tinge, shown in figure 80. Note the amount of deci-
normal soda solution used in the process.

NOTE. — Just before the end-point is reached, a very
slight opalescence may be noted in the fluid, due to
the precipitation of dibasic phosphates. After the
true end-point is reached, the further addition of about
0.5 c.c. of the decinormal soda solution will produce
a deep magenta colour (Fig. 80, c), which is the so-called
"end-point" of the American Committee of Bacteri-
ologists.

METHOD OF EXPRESSING THE REACTION. 131

(B) Perform a "control" titration (occasionally two
controls may be necessary), as follows:

1. Measure 25 c.c. of the meat extract into one of the
beakers, wash out the measure with boiling water,
and add the phenolphthalein as in the first estimation.

2. Run in -y- NaOH from the pipette, just short of
the amount required to neutralise the 25 c.c. of medium.
(For example, if in the first estimation 5 c.c. of -—•
NaOH were required to render 25 c.c. of medium
neutral to phenolphthalein, only add 0.48 c.c. of -j-
NaOH.) Immerse the beaker in the water-bath.

3. Complete the titration by the aid of the •— NaOH.

4. Note the amount of -£- NaOH solution required
to complete the titration, and add it to the equivalent
of the -y- NaOH solution previously run in. Take
the total as the correct estimation.

Method of Expressing the Reaction.—

The reaction or litre of meat extract, medium,
or any solution estimated in the foregoing manner, is
most conveniently expressed by indicating the number
of cubic centimetres of normal alkali (or normal acid)
that would be required to render one litre of the solu-
tion exactly neutral to phenolphthalein.

The sign + (plus) is prefixed to this number if the
solution reacts acid, and the sign — (minus) if it reacts
alkaline.

For example, "meat extract + 10," indicates a
sample of meat extract which reacts acid to phenol-
phthalein, and would require the addition of 10 c.c.
of normal NaOH per litre, to neutralise it.

NOTE. — Such a solution would probably react alka-
line to litmus.

Conversely, if as the result of our titration experi-
ments we find that 25 c.c. of meat extract require the
addition of 5 c.c. -~ NaOH to neutralise, then 1000 c.c.

132

NUTRIENT MEDIA.

of meat extract will require the addition of 200 c.c. -^-

NaOH == 20 c.c. -f NaOH.

And this last figure, 20, preceded by the sign -f- , to

signify that it is acid,
indicates the reaction of
the meat extract.

NOTE. — The standard
soda solutions should be
prepared by accurate
measuring operations,
controlled by titrations,
from a stock solution
of loN NaOH, which
should be very care-
fully standardised. This
stock solution must be
kept in an aspirator bot-
tle to which air can only
gain access after it has
been dried and rendered
free from CO2. This may
be done by first leading
it over H2SO4 and soda
lime, or soda lime alone,
by some such arrange-
ment as is shown in
figure 8 1, which also
Fig. 81.— Soda bottle. shows a constant burette

arrangement for the de-
livery of small measured quantities of the dekanormal
soda solution.

STANDARDISATION OF MEDIA.

Differences in the reaction of the medium in which
it is grown will provoke not only differences in the rate
of growth of any given bacterium, but also well-marked
differences in its cultural and morphological characters ;
and nearly every organism will be found to affect a
definite " optimum reaction" — a point to be carefully
determined for each . Fortunately, however, the ' ' opti-

STANDARDISING NUTRIENT BOUII^ON. 133

mum" usually approximates fairly closely to -f-io;
and as experiment has shown that this reaction is the
most generally useful for laboratory work, it is the
one which may be adopted as the standard for all nu-
trient media derived from meat extract.

Briefly, the method of standardising a litre of media
to +10 consists in subtracting 10 from the initial litre
of the medium mass; the remainder indicates the
number of cubic centimetres of normal soda solution
that must be added to the medium, per litre, to render
the reaction -fio.

Standardising Nutrient Bouillon. — For example, 1000
c.c. bouillon are prepared; on titration it is found

1. 25 c.c. require the addition of 5.50 c.c. -£- NaOH
to neutralise.

2. 25 c.c. require the addition of 5.70 c.c. -~ NaOH
to neutralise.

3. 25 c.c. require the addition of 5.60 c.c. -~ NaOH
to neutralise.

2 and 3 are controls. Averaging these two controls,
25 c.c. require the addition of 5.65 c.c. -—- NaOH to
neutralise, and therefore 1000 c.c. require the addition
of 226 c.c. -£- NaOH, or 22.60 -f NaOH.

Initial titre of the bouillon = +22.6, and as such
requires the addition of (22.6 c.c. — 10 c.c.) = 12.6 c.c.
of -7- NaOH per litre to leave its finished reaction + 10.

But the three titrations, each on 25 c.c. of medium,
have reduced the original bulk of the bouillon to (1000
— 75 c.c.) = 925 c.c. The amount of -^- NaOH re-
quired to render the reaction of this quantity of medium
H-IO may be deduced thus:

1000 c.c. : 925 c.c. :: 12.6 c.c. : x.

Then x == 11.65 c.c. -f- NaOH.

Whenever possible, however, the required reaction
is produced by the addition of dekanormal soda solu-
tion, on account of the minute increase it causes in the

134

NUTRIENT MEDIA.

bulk, and the consequent insignificant disturbance of
the percentage composition of the medium. By means
of a pipette graduated to o.oi c.c. it is possible to de-
liver very small quantities; but if the calculated amount
runs into thousandth parts of a cubic centimetre, these
are replaced by corresponding quantities of normal soda.

In the above example it is necessary to add 11.65
c.c. normal NaOH or its equivalent, 1.165 c-c- deka-
normal NaOH. The first being too bulky a quantity,
and the second inconveniently small for exact measure-
ment, the total weight of soda is obtained by substi-
tuting 1. 1 6 c.c. dekanormal soda solution, and 0.05
c.c. of normal soda solution.

Standardising Nutrient Gelatine. — In the finished
medium it has been found experimentally that every
cubic centimetre of meat extract has increased in bulk
to 1.008 c.c. for every o.oi gramme of added gelatine,
and this factor must be taken into account in calcu-
lating the amount of soda solution necessary to pro-
duce the standard reaction in the finished medium.

The following expansion table shows at a glance the
increase in bulk for the varying percentages:

GELATINE EXPANSION TABLE.

AT 20° CENTIGRADE.
loo c.c. meat extract
+ 5 gms. gelatine measure
+ 6
+ 7
+ 8
+ 9
+ 10

1000 c.c. meat extract

asure iO4.oc

+ 5°gms- gelatine measure 1040 c c

104.8

+ 60

1048

105.6

+ 70

1056

106.4

+ 80

1064

107.2

+ 9Q

1072

108.0

+ 100

I080

108.8

+ 110

1088

109.6

+ I2O

1096

110.4

+ 130

1104

III. 2

+ 140

III2

II2.0

+ 150

1120

116.0

+ 2OO

1160

+ 12
+ 13

+ H

+15

+ 20

The method of standardising gelatine is very similar
to that described under bouillon.

STANDARDISING NUTRIENT AGAR. 135

For example, 1000 c.c. of meat extract are employed
in the manufacture of 1 1 per cent, gelatine.

By referring to the above table it is found that after
solution the medium mass measures 1088 c.c., and if
three titrations are performed there still remain 1013
c.c. of medium to be standardised (instead of only
925 c.c.).

If the initial reaction of the gelatine mass is + 24, it
will require the addition of 14 c.c. -7- NaOH per litre
to render the reaction of the finished medium + 10.

Therefore, 1000 c.c. : 1013 c.c. : : 14 c.c. : x.

x== 14.182 c.c. -^-NaOH.

This quantity is replaced by

1.41 c.c. dekanormal soda solution (equivalent to 14.100 — NaOH) .
0.08 " normal soda solution ( " " 0.080 y NaOH)

0.02 " decinormal soda solution ( " " 0.002 — NaOH)

14.182

giving the requisite weight of soda in a little over 1.5
c.c. of fluid.

Standardising Nutrient Agar. — Every cubic centi-
metre of meat extract increases in bulk to 1.0053 c-c-
for every o.oi gramme of added agar.

The following table gives the calculated bulk for
varying percentage of agar:

AGAR EXPANSION TABLE.

AT 20° CENTIGRADE.

100 c.c. meat extract 1000 c.c. meat extract

-(-0.5 gm. agar measures 100.265 C.c. -f- 5 agar measures 1002.65 c>c-

-f i.o " " " 100.530 " +10 " " 1005.30 "

-f-i.5gms. " " 100.795 " + 15 " " 1007.95 "

-j-2.0 " " " 101.060 " -(-20 " " 1010.60 "

The method of standardising nutrient agar is similar
to that described under nutrient gelatine, and a refer-
ence to the example there given will at once explain
the necessary steps in the calculation.

136 NUTRIENT MEDIA.

THE FILTRATION OF MEDIA.

Fluid media are usually filtered through stout Swed-
ish filter paper (occasionally through a porcelain filter
candle), and in order to accelerate the rate of filtration
the filter paper should be folded in that form which
is known as the "physiological filter," not in the ordi-
nary " quadrant" shape, as by this means a large surface
is available for filtration and a smaller area in contact
with the glass funnel supporting it.

To fold the filter proceed thus:

1. Take a circular piece of filter paper and fold it
exactly through its centre to form a semicircle.

2. Fold the semicircle exactly in half to form a quad-
rant; make the crease distinct by running the thumb-
nail along it, then open the filter out to a semicircle
again.

3. Fold each end of the semicircle in to the centre
and so form another quadrant; smooth down the two
new creases thus formed and again open out to a semi-
circle.

4. The semicircle now appears as in figure 82, a, the
dark lines indicating the creases already formed.

5. Fold the point i over to the point 3, and ia to
3a, to form the creases 4 and 4a, indicated in the dia-
gram by the light lines. Fold point i over to 3, and
i a to 3a, to form the creases 5 and 5*1.

6. Thus far the creases have all been made on the
same side of the paper. Now subdivide each of the
eight sectors by a crease through its centre on the
opposite side of the paper, indicated by the broken line
in the diagram. Fold up the filter gradually as each
crease is made, and when finished the filter has assumed
the shape of a wedge, as in figure 82, b.

When opened out the filter assumes the shape repre-
sented in figure 82, c.

The folded filter is next placed inside a glass funnel

FILTRATION OF SOLID MEDIA.

137

supported on a retort stand and moistened with hot
distilled water before the filtration of the medium is
commenced.

Solid media are filtered through a specially made
filter paper, — " papier Chardin," — which is sold in
boxes of twenty-five ready-folded filters.

Fig. 82. — Filter folding : <?, Filter folded in half, showing creases ; />,
appearance of filter on completion of folding ; c, filter opened out ready for

Gelatine, when properly made, filters through this
paper as quickly as bouillon does through the Swedish
filter paper, and does not require the use of the hot-
water funnel.

Agar, likewise, if properly made, filters readily,
although not at so rapid a rate as gelatine. If badly

138

NUTRIENT MEDIA.

"egged," and also during the winter months, it is
necessary to surround the glass funnel, in which the

filtration of the agar is
carried on, by a hot-
water jacket. This is
done by placing the glass
funnel inside a double-
walled copper funnel —
the space between the
walls being filled with
water at about 90° C. —
and supporting the lat-
ter on a ring gas burner
fixed to a retort stand
(Fig. 83). The gas is
lighted and the water
jacket maintained at a
high temperature until
filtration is completed.

Fig. 83. — Ring burner and hot-water
filter.

TUBING NUTRIENT MEDIA.

After the final filtration, the nutrient medium is
" tubed" — i. e., filled into sterile tubes in definite meas-
ured quantities, usually 10 c.c.; or "flasked" — i. e.,
filled into sterile flasks in fairly large quantities. This
process is sometimes carried out by means of a large
separator funnel fitted with a "three-way" tap which
communicates with a small graduated tube (capacity
20 c.c. and graduated in cubic centimetres) attached to
the side. The shape of this piece of apparatus, known
as Treskow's funnel, renders it particularly liable to
damage. It is better, therefore, to arrange a less ex-
pensive piece of apparatus which will serve the pur-
pose equally well (Fig. 84).

A Geissler's three-way stop-cock has the tube on
one side of the tap ground obliquely at its extremity,

TUBING NUTRIENT MEDIA.

139

the tube on the opposite side cut off within 3 c.c. of the
tap. The short tube is connected by means of a per-
forated rubber cork with a 10 c.c. length of stout glass
tubing (1.5 c.c. bore). The third channel of the three-
way tap is connected, by means of rubber tubing, with
the nozzle of an ordinary separator funnel. Finally,
the receiving cylinder above the three-way tap is gradu-
ated in cubic centimetres up to 20, by pouring into it

Fig. 84. — Three-way tap, home-made.

Fig. 85.— Gas tube
(Durham).

measured quantities of water and marking the various
levels on the outside with a writing diamond.

Fluid media containing carbohydrates are filled into
fermentation tubes (vide Fig. 14); or into ordinary
media tubes which already have smaller tubes, inverted,
inside them (Fig. 85), to collect the products of growth
of gas-forming bacteria. When first filled, the small
tubes float on the surface of the medium ; after the first
sterilisation nearly all the air is replaced by the medium,

140 NUTRIENT MEDIA.

and after the final sterilisation the gas tubes will be
submerged and completely filled with the medium.

Storing "Tubed" Media. — Media after being tubed
are best stored by packing, in the vertical position,
in oblong boxes having an internal measurement of
37 cm. long by 12 cm. wide by 10 cm. deep. Bach
box (Fig. 86) has a movable partition formed by the
vertical face of a weighted triangular block of wood,
sliding free on the bottom (Fig. 86, A); or by a flat

Fig. 86. — Medium box, showing alternative partitions A and B.

piece of wood sliding in a metal groove in the bottom
of the box, which can be fixed at any spot by tightening
the thumbscrew of a brass guide rod which transfixes
the partition (Fig. 86, B). The front of the box is pro-
vided with a handle and a celluloid label for the name
of the contained medium. These boxes are arranged
upon shelves in a dark cupboard, — or preferably an iron
safe, — which should be rendered as nearly air-tight as
possible, and should have the words "media stores"
painted on its doors.

X. CULTURE MEDIA.

Nutrient Bouillon. —

1. Measure 800 c.c. of meat extract into a litre
flask.

2. Weigh out Witte's peptone, 10 grammes (= i per
cent.), salt, 5 grammes (= 0.5 per cent.), and mix into
a smooth paste with 200 c.c. of meat extract previously
heated to 60° C. (Be careful to leave no unbroken
globular masses of peptone.)

3. Add the peptone emulsion to the meat extract in
the flask and heat the steamer for forty-five minutes
(to completely dissolve the peptone, and to render the
acidity of the meat extract stable).

4. Estimate the reaction of the medium; control the
result; render the reaction of the finished medium
+ 10 (vide page 133).

5. Heat for half an hour in the steamer at 100°
C. (to complete the precipitation of the phosphates,
etc.).

6. Filter through Swedish filter paper into a sterile
flask.

7. Fill into sterile tubes (10 c.c. in each tube).

8. Sterilise in the steamer for twenty minutes on
each of three consecutive days — i. e., by the discon-
tinuous method (vide page 41).

Inosite-free Media Bouillon (Durham).—

1. Prepare meat extract, 1000 c.c. (vide page 127), from
bullock's heart which has been "hung" for a couple of
days.

2. Prepare nutrient bouillon (-J-io), 1000 c.c. (vide supra),
from the meat extract, and store in i -litre flask.

3. Inoculate the bouillon from a pure cultivation of the
B. lactis aerogenes, and incubate at 37° C. for forty-eight
hours.

141

142 CULTURE MEDIA.

4. Heat in the steamer at 100° C. for twenty minutes to
destroy the bacilli and some of their products.

5. Estimate the reaction of the medium and if necessary
restore to -|-io.

6. Inoculate the bouillon from a pure cultivation of the
B. coli communis and incubate at 37° C. for forty-eight
hours.

7. Heat in the steamer at 100° C. for twenty minutes.
Now fill two fermentation tubes with the bouillon, tint

with litmus solution, and sterilise; inoculate with B. lactis
aerogenes. If no acid or gas is formed, the bouillon is in
a sugar-free condition; but if acid or gas is present, again
make the bouillon in the flask -f-io, reinoculate with one or
other of the above-mentioned bacteria, and incubate ; then
test again. Repeat this till neither acid nor gas appears
in the medium.

8. After the final heating, stand the flask in a cool place
and allow the growth to sediment. Filter the supernatant
broth through Swedish filter paper. If the filtrate is cloudy,
filter through a porcelain filter candle.

9. Tube, and sterilise as for bouillon.

Bouillon prepared in the above-described manner will
prove to be absolutely sugar-free; and from it may be pre-
pared nutrient sugar-free gelatine or agar, by following the
directions given on pages 145 and 149, respectively, substi-
tuting the inosite-free meat extract for the ordinary meat
extract. The most important application of inosite-free
bouillon is its use in the preparation of sugar bouillons,
whether glucose, maltose, lactose, or saccharose, of exact
percentage composition.

Glycerine Bouillon. —

1. Measure out nutrient bouillon, 1000 c.c. (vide page
141, sections i to 6).

2. Measure out glycerine, 60 c.c. (= 6 per cent.), and add
to the bouillon.

3. Tube, and sterilise as for bouillon.
Sugar Bouillon. —

1. Measure out nutrient bouillon, 1000 c.c. (vide page
141, sections i to 6).

2. Weigh out glucose (anhydrous), 20 grammes (= 2
per cent.), and dissolve in the fluid.

3. Tube, and sterilise as for bouillon.

Ordinary commercial glucose serves the purpose equally
well, but is not recommended, as during the process of
sterilisation the medium gradually deepens in colour.

NOTE. — In certain cases a corresponding percentage
of lactose, maltose, or saccharose is substituted for glu-
cose.

NUTRIENT BOUILLON. 143

Glucose Formate Bouillon (Kitasato). —

1. Measure out nutrient bouillon, 1000 c.c. (vide page
141, sections i to 6).

2. Weigh out glucose, 20 grammes (— 2 percent.), sodium
formate, 4 grammes (— 0.4 per cent.), and dissolve in the
fluid.

3. Tube, and sterilise as for bouillon.
Sulphindigotate Bouillon (Weyl).—

1. Measure out nutrient bouillon (vide page 141, sections
i to 6).

2. Weigh out glucose, 20 grammes (— 2 per cent.),
sodium sulphindigotate, i gramme (=0.1 per cent.), and
dissolve in the fluid.

3. Tube, and sterilise as for bouillon.

NOTE. — The finished medium is of a blue colour, which
during the growth of anaerobic bacteria is oxidised and
decolourised to a light yellow.

Nitrate Bouillon.—

1. Measure out nutrient bouillon, 1000 c.c. (vide page
141, sections i to 6).

2. Weigh out potassium nitrate, 5 grammes (= 0.5 per
cent.), and dissolve it in the bouillon.

3. Tube, and sterilise as for bouillon.

NOTE. — The nitrate of sodium or ammonium may be
substituted for that of potassium, or the salt may be added
in the proportion of from o. i to i per cent, to meet special
requirements.

Iron Bouillon. —

1. Measure out nutrient bouillon, 1000 c.c. (vide page
141, sections i to 6).

2. Weigh out ferric tartrate, i gramme (— o.i per cent.),
and dissolve it in the bouillon.

3. Tube, and sterilise as for bouillon.

N. B. — The lactate of iron may be substituted for the
tartrate.

Lead Bouillon. —

1. Measure out nutrient bouillon, 1000 c.c. (vide page 141,
sections i to 6).

2. Weigh out lead acetate, i gramme (— o.i percent.),
and dissolve it in the bouillon.

3i Tube, and sterilise as for bouillon.
Litmus Bouillon. —

1. Measure out nutrient bouillon, 1000 c.c. (vide page
141, sections i to 6).

2. Add sufficient sterile litmus solution to tint the medium
a dark lavender colour. (Media rendered -f-io will usually
react very faintly alkaline or occasionally neutral to litmus.)

3. Tube, and sterilise as for bouillon.

144 GUI/TURK MEDIA.

Lactose Litmus Bouillon (Lakmus Molke). —

1. Weigh out peptone, 4 grammes, and emulsify it
with 200 c.c. meat extract (vide page 127), previously
heated to 60° C.

2. Weigh out salt, 2 grammes, and lactose, 20, and
mix with the emulsion.

3. Wash the mixture into a sterile litre flask with
200 c.c. meat extract and add 600 c.c. distilled water.

4. Heat in the steamer at 100° C. for thirty minutes,
to completely dissolve the peptone, etc.

5. Neutralise carefully to litmus paper by the suc-
cessive additions of small quantities of decinormal soda
solution.

6. Replace in the steamer for twenty minutes to
precipitate phosphates, etc.

7. Filter through two thicknesses of Swedish filter
paper.

8. Add sterile litmus solution, sufficient to colour
the medium a deep purple.

9. Tube, and sterilise as* for bouillon.

Parietti's Bouillon.—

1. Measure out pure hydrochloric acid, 4 c.c., and add
to it carbolic acid solution (5 per cent.), 100 c.c. Allow
the solution to stand at least a few days before use.

2. This solution is added in quantities of o.i, 0.2, and
0.3 c.c. (delivered by means of a sterile graduated pipette)
to tubes each containing 10 c.c. of previously sterilised
nutrient bouillon (vide page 141).

3. Incubate at 37° C. for forty-eight hours to eliminate
contaminated tubes. Store the remainder for future use.

Carbolised Bouillon. —

1. Prepare nutrient bouillon (vide page 141, sections i to
6).

2. Weigh out carbolic acid, i gramme (2.5 or 5 grammes
may be needed for special purposes), and dissolve it in the
medium.

3. Tube, and sterilise as for bouillon.

NUTRIENT GELATINE. 145

Nutrient Gelatine.—

1. Measure out meat extract, 800 c.c., into a 2-litre
flask.

2. Weigh out Witte's peptone, 10 grammes (= i per
cent.), salt, 5 grammes (=0.5 per cent.), and mix
into a smooth paste with 200 c.c. meat extract pre-
viously heated to 60° C. ; add the emulsion to the meat
extract in the flask.

.3. Weigh out that quantity of best Gold Label French
gelatine necessary to secure the required percentage,—
usually 90 to 120 grammes ( = 9 per cent, to 12 per
cent.), — cut the sheets into small pieces and add to the
meat extract.

4. Place the flask in the steamer at 100° C. for one
hour.

5. Estimate the reaction of the medium mass and
control the result; then add sufficient soda solution to
render the reaction of the calculated bulk of medium
+ 10. (See Gelatine Expansion Table, page 134.)

6. Replace in the steamer for twenty minutes (to
complete the precipitation of the phosphates).

7. Allow the medium mass to cool to 60° C. Well
whip the whites of two eggs, add to the contents of the
flask, and replace in the steamer at 100° C. for about
half an hour (until the egg-albumen has coagulated
and formed large, firm masses floating on and in clear
gelatine).

8. Filter through papier Chardin into a sterile flask.

9. Tube in quantities of 10 c.c.

10. Sterilise in the steamer at 100° C. for twenty
minutes on each of three consecutive days — i. e., by
the discontinuous method.

Rapid Method of Preparing Nutrient Gelatine. —
i . Finely mince 500 grammes of lean beef and add to
800 c.c. of distilled water in a flask; place the flask
in a water-bath, and raise the temperature of its con-
tents to and keep at 45° C. for twenty minutes; then

146

GUI/TURK MEDIA.

rapidly raise the temperature to 100° C., and maintain
there for ten minutes.

2. Weigh a 2 -litre flask on a trip balance (Fig. 87)
and note the weight, or counterpoise carefully. Filter

Fig. 87. — Trip balance.

the mixture into the flask. Do not make up the fil-
trate to 1000 c.c. as in the ordinary method.

An extremely useful counterpoise is a small sheet-
brass cylinder about 38 mm. high and 38 mm. in di-
ameter, with a funnel-shaped top and provided with a
side tube by which its contents, fine "dust" shot, may
be emptied out (Fig. 88).

3. Weigh out and mix 10 grammes of peptone, 5
grammes of salt, and make into a
thick paste with 150 c.c. distilled
water; then add the emulsion to
the meat extract in the flask; also
add 100 grammes sheet gelatine
cut into small pieces, and return
the flask to the water-bath.

4. Arrange a lo-litre tin can
(with copper bottom, such as is
used in the preparation of distilled
water) by its side, fill the can with
boiling water and place a lighted
Bunsen burner under it. Fit a
long safety tube to the neck of the can and also a deliv-
ery tube, bent twice at right angles, adjusted to reach
to the bottom of the interior of the flask (Fig. 89).

Fig. 88. — Counter-
poise ; weight when emp-
,ty» 35 grammes; when
full of dust shot, 200
grammes.

NUTRIENT GELATINE.

147

5. Keep the water in the can vigourously boiling, and
so steam at 100° C., bubbling through the medium
mass, for ten minutes, by which time complete solution
of the gelatine is effected.

6. Weigh the flask and its contents; then (1115*
grammes + weight of the flask) minus (weight of the

Fig. 89. — Steam can arrangement for media.

flask and its contents) equals the weight of water re-
quired to make up the bulk to i litre. Add the requi-
site quantity of water at the boiling-point.

7. Titrate and estimate the reaction of the medium

1 This figure is obtained by adding together I litre water, 1000 grammes ;
IO per cent, gelatine, 100 grammes; I percent, peptope, IO grammes; 0.5
per cent, salt, 5 grammes ; total, 1115 grammes. Modifications of the above
process, as to quantities and percentages, will require corresponding alterations
of the figures. The average weight of I litre of IO per cent, nutrient gela-
tine when prepared in this way is 1080 grammes (compare expansion table,
page 134).

148 CULTURE MEDIA.

mass; control the result. Calculate the amount of
soda solution required to make the reaction of the
medium mass +10 (i. e., calculate for 1000 c.c., less
the quantity used for the titrations).

8. Add the necessary amount cf soda solution and
heat in the steamer at 100° C. for twenty minutes, to
precipitate the phosphates, etc.

9. Clarify with egg; filter, tube, and sterilise as for
nutrient gelatine (vide page 145).

Sugar Gelatine. —

1. Prepare nutrient gelatine (vide page 145, sections i to

7).

2. Weigh out glucose, 20 grammes (== 2 per cent.), and
dissolve in the hot gelatine.

3. Filter through papier Chardin.

4. Tube, and sterilise as for nutrient gelatine.

NOTE. — In certain cases, lactose, maltose, or saccharose,
in similar percentage, is substituted for glucose.
Glucose Formate Gelatine (Kitasato). —

1. Prepare nutrient gelatine (Me page 145, sections i to

7).

2. Weigh out glucose, 20 grammes (= 2 per cent.), and
sodium formate, 4 grammes (= 0.4 per cent.), and dissolve
in the hot gelatine.

3. Filter through papier Chardin.

4. Tube, and sterilise as for nutrient gelatine.
Sulphindigotate Gelatine (Weyl).—

1. Prepare nutrient gelatine (vide page 145, sections i

to 7).

2. Weigh out glucose, 20 grammes (= 2 per cent.), and
sodium Sulphindigotate, i gramme (=0.1 per cent.), and
dissolve in the hot gelatine.

3. Filter through papier Chardin.

4. Tube, and sterilise as for nutrient gelatine.
Litmus Gelatine. —

1. Prepare nutrient gelatine (vide page 145, sections i
to 8).

2. Add sterile litmus solution, sufficient to tint the
medium a deep lavender colour.

3. Tube, and sterilise as for nutrient gelatine.
Lactose Litmus Gelatine (Wurtz).—

1. Prepare nutrient gelatine (vide page 145, sections i to

4).

2. Render the reaction of the medium mass — 5.

NUTRIENT AGAR-AGAR. 149

3. Replace in the steamer at 100° C. for twenty minutes.

4. Clarify with egg as for gelatine.

5. Weigh out lactose, 20 grammes (= 2 per cent.), and
dissolve it in the medium.

6. Filter through papier Chardin.

7. Add sufficient sterile litmus solution to colour the
medium pale lavender.

8. Tube, and sterilise as for nutrient gelatine.
Carbolised Gelatine.—

1. Prepare nutrient gelatine (vide page 145, sections i
to 7).

2. Weigh out carbolic acid, 5 grammes (= 0.5 per cent.),
and dissolve it in the gelatine.

3. Filter through papier Chardin.

4. Tube, and sterilise as for nutrient gelatine.

One or 2.5 grammes of carbolic acid (=0.1 per cent, or
0.25 per cent.) are occasionally used in place of the 5 grammes
to meet special requirements.

Nutrient Agar=agar.—

1. Measure out meat extract, 600 c.c., into a 2-litre
flask.

2. Weigh out Witte's peptone, 10 grammes (— i
per cent.), salt, 5 grammes (= 0.5 per cent.), and mix
them into a smooth paste with 200 c.c. meat extract
previously heated to 60° C.; add the emulsion to the
meat extract in the flask.

3. Weigh out that quantity of powdered agar neces-
sary to secure the required percentage, — usually 15
to 20 grammes (— 1.5 to 2 per cent.), — and mix it into
a smooth paste with 200 c.c. of meat extract (which
must be quite cold, as the agar powder undergoes con-
siderable expansion when first mixed with hot fluids
and before solution takes place); add the emulsion to
the meat extract, etc., in the flask.

4. Place the flask in the steamer at 100° C. until the
agar is completely dissolved. This will take about
ninety minutes.

5. Estimate the reaction of the medium mass; con-
trol the result; then add sufficient soda solution to

150 CULTURE MEDIA.

render the reaction of the calculated bulk of medium
+ 10. (See Agar Expansion Table, page 135.)

6. Replace in the steamer for twenty minutes (to
complete the precipitation of the phosphates, etc.).

8. Filter through papier Chardin, by the aid of a
hot- water funnel (Fig. 83) into a sterile flask.

9. Tube, and sterilise in the steamer at 100° C. for
thirty minutes on each of three consecutive days —
i..e., by the discontinuous method.

Brain Agar; Spleen Agar. — Use meat extract pre-
pared from sheep (or ox) brain and spleen respectively,
and proceed exactly as if making nutrient agar (vide
supra).

Rapid Method of Preparing Nutrient Agar. —

1. Finely mince 500 grammes of lean beef and add
to 800 c.c. of distilled water in a flask; place the flask
in a water-bath, and raise the temperature of its con-
tents to and keep at 45° C. for twenty minutes; then
rapidly raise the temperature to 100° C., and maintain
there for ten minutes.

2. Weigh a 2 -litre flask and note the weight — or coun-
terpoise exactly. Filter the mixture into the flask
and again weigh, but do not make up the filtrate to
1000 c.c. as in the ordinary method.

3. Weigh out and mix 10 grammes of peptone, 5
grammes of salt, and 20 grammes of powdered agar,
and make into a thick paste with 150 c.c. distilled
water, and add to the meat extract in the flask; re-
turn the flask to the water-bath.

4. Arrange a lo-litre tin can (with copper bottom,
such as is used in the preparation of distilled water)

BRAIN AGAR — SPLEEN AGAR. 151

by its side, fill the can with boiling water, and place
a lighted Bunsen burner under it. Fit a long safety
tube to the neck of the can; also a delivery tube, bent
twice at right angles and adjusted to reach to the bot-
tom of the interior of the flask. (See also Rapid Method
of Preparing Gelatine, page 145.)

5. Keep the water in the can vigourously boiling, and
so steam at 100° C., bubbling through the medium
mass, for twenty-five minutes, by which time com-
plete solution of the agar is effected.

6. Now weigh the flask and its contents; then
(1035 l grammes + weight of flask) minus (weight of
flask and its contents) equals the weight of water re-
quired to make up the bulk of the medium to i litre.
Add the requisite amount.

7. Titrate, and estimate the reaction of the medium
mass; control the result. Calculate the amount of
soda solution required to make the reaction of the
medium mass +10 (i. e., calculated for 1000 c.c., less
the quantity used for the titrations).

8. Add the necessary amount of soda solution and
heat in the steamer at 100° C. for twenty minutes.

9. Clarify with egg, filter, tube, and sterilise as for
nutrient agar (vide page 150),

Glycerine Agar. —

1. Prepare nutrient agar (vide page 149, sections i to 8).
Measure out 1000 c.c.

2. Measure out pure glycerine, 60 c.c. (= 6 per cent.),
and add to the agar.

3. Tube, and sterilise as for nutrient agar.
Sugar Agar.—

1. Prepare nutrient agar (vide page 149, sections i to 8).
Measure out 1000 c.c.

2. Weigh out glucose, 20 grammes (= 2 per cent.), and
dissolve in the clear agar.

3. Tube, and sterilise as for nutrient agar.

NOTE. — In certain cases, lactose, maltose, or saccharose
(in similar percentage) is substituted for glucose.

1 Compare note on page 147. The average weight of I litre of 2 per cent,
nutrient agar when completed is 1010.5 grammes.

152 CULTURE MEDIA.

Glucose Formate Agar (Kitasato). —

1. Prepare nutrient agar (vide page 149, sections i to 8).
Measure out 1000 c.c.

2. Weigh out glucose, 20 grammes (— 2 per cent.),
sodium formate, 4 grammes (= 0.4 per cent.), and dissolve
in the agar.

3. Tube, and sterilise as for nutrient agar.
Sulphindigotate Agar.—

1. Prepare nutrient agar (vide page 149, sections i to 8).
Measure out 1000 c.c.

2. Weigh out glucose, 20 grammes (-- 2 per cent.),
sodium sulphindigotate, i gramme (— o.i percent.), and
dissolve in the hot agar.

3. Tube, and sterilise as for nutrient agar.
Lactose Litmus Agar (Wurtz). —

1. Prepare nutrient agar (vide page 149, sections i to 4).

2. Render the reaction of the medium mass — 5.

3. Replace in the steamer at 100° C. for twenty minutes.

4. Cool to 60° C. and clarify with egg as for nutrient
agar.

5. Weigh out lactose, 20 grammes (— 2 per cent.), and
dissolve it in the medium.

6. Filter through papier Chardin, using the hot- water
funnel.

7. Add sterile litmus solution, sufficient to colour the
medium a pale lavender.

8. Tube, and sterilise as for nutrient agar.
Carbolised Agar. —

i; Prepare nutrient agar (vide page 149, sections i to 8).
Measure out 900 c.c.

2. Weigh out i gramme pure phenol and dissolve in the
medium.

3. Tube, and sterilise as for nutrient agar.

Gelatine Agar. — This medium is prepared by adding to
nutrient gelatine sufficient agar to ensure the solidity of
the medium when incubated at temperatures above 22° C.
If it is intended to employ an incubating temperature of
30° C., 10 per cent, gelatine and 0.5 per cent, agar must be
dissolved in the meat extract before the addition of the pep-
tone and salt; while for incubating at 37° C., 12 per cent,
gelatine and 0.75 per cent, agar must be used. Avoid the
addition of more agar than is absolutely necessary, other-
wise the action upon the medium of such organisms as elabo-
rate a liquefying ferment may be retarded or completely
inhibited.

i. Measure out 600 c.c. meat extract into a 2-litre flask,
and add to it gelatine, 100 grammes (or 120, as may be
necessary) .

BLOOD-SERUM. 153

2. Weigh out powdered agar, 5 grammes (or 7.5 grammes,
as required), emulsify with 200 c.c. meat extract, and add
to the contents of the flask.

3. Heat in the steamer at 100° C. for ninety minutes to
completely dissolve the agar and gelatine.

4. Weigh out peptone, 10 grammes, salt, 5 grammes ; emul-
sify with 200 c.c. meat extract previously heated to 60° C.,
and add to the contents of the flask.

5. Replace in the steamer for fifteen minutes.

6. Estimate the reaction; control the result. Then add
sufficient caustic soda solution to render the reaction -j-io.

7. Replace in the steamer at 100° C. for twenty minutes.

8. Cool to 60° C. Clarify with egg as for nutrient gela-
tine.

9. Filter through papier Chardin, using the hot- water
funnel.

10. Tube, and sterilise in the steamer at 100° C. for
thirty minutes on each of three consecutive days.

Agar Gelatine (Guarniari).—

1. Measure out meat extract, 750 c.c., into a 2-litre flask,
and add to it gelatine, 50 grammes.

2. Weigh out powdered agar, 3 grammes; emulsify with
cold distilled water, 50 c.c., and add to the contents of the
flask.

3. Heat in the steamer at 100° C. for sixty to ninety
minutes to completely dissolve the agar and gelatine.

4. Weigh out Witte's peptone, 25 grammes, salt, 5
grammes, and emulsify with 200 c.c. meat extract pre-
viously heated to 60° C., and add to the contents of the
flask.

5. Replace in the steamer for fifteen minutes.

6. Neutralise carefully to litmus paper by the successive
additions of small quantities of normal soda solution.

7. Replace in the steamer at 100° C. for twenty minutes.

8. Cool to 60° C. Clarify with egg as for nutrient gela-
tine.

9. Filter through papier Chardin, using the hot- water
funnel.

10. Tube, and sterilise in the steamer at 100° C. for thirty
minutes on each of three consecutive days.

Blood=serum. —

i. vSterilise cylindrical glass jar (Fig. 90) and its
cover by dry heat, or by washing first with ether and
then with alcohol.

154

CULTURE MEDIA.

2. Collect blood from sheep or ox in the sterile cylin-
der.

3. Allow the vessel to stand for fifteen minutes for
the blood to coagulate. (This must be done before
leaving the slaughter-house, otherwise the serum will
be stained with haemoglobin.)

4. Separate the clot from the sides of the vessel by
means of a sterile glass rod (the yield of serum is much
smaller when this is not done), and place the cylinder
in the ice-chest for twenty-four hours.

Fig. 90. — Blood- serum jar with wicker basket for transport.

5. Remove the serum with sterile pipettes, or syphon
it off, and fill into sterile tubes (5 c.c. in each) or flasks.

6. Sterilise the serum by the fractional method —
that is, by exposure in a water-bath to a temperature
of 56° C. for half an hour on each of six consecutive
days; store in the fluid condition.

6a. Or heat tubes containing serum to 56° C. in a
water-bath for half an hour on each of two successive
days.

GLYCERINE BLOOD-SERUM.

7. On the third day, heat the tubes, in a sloping
position, in a serum inspissator to about 72° C. (A
coagulum is formed at this temperature which is fairly
transparent; above 72° C., a thick- turbid coagulum is
formed.)

The serum inspissator (Fig. 9 1 ) in its simplest form
is a double-walled rectangular copper box, closed
in by a loose glass lid, and cased in felt or asbestos—
the space between the walls is filled with water. The
inspissator is supported on adjustable legs so that the

Fig. 91. — Serum inspissator.

serum may be solidified at any desired " slant," and
is heated from below by a Bunsen burner. The more
elaborate forms resemble the hot-air oven (Fig. 18)
in shape and are provided with thermo-regulators.

8. Place the tubes in the incubator at 37° C. for
forty-eight hours in order to eliminate those that have
been contaminated. Store the remainder in a cool
place for future use.

Glycerine Blood-serum.—

i. Prepare blood-serum as described above, sections i
to 4.

156 CULTURE MEDIA.

2. Add 6 per cent, pure glycerine.

3. Complete as described above for ordinary blood-serum,
sections 5 to 7.

NOTE. — Different percentages of glycerine — from 4 per
cent, to 8 per cent.-1— are used for special purposes. Five
per cent, is that usually employed.

Blood-serum (Lbffler).—

1. Prepare nutrient bouillon (vide page 141), using meat
extract made from veal instead of beef.

2. Add i per cent, glucose to the bouillon, and allow it
to dissolve completely.

3. Now add 300 c.c. clear blood-serum (vide page 153,
sections i to 4) to every 100 c.c. of this bouillon.

4. Fill into sterile tubes and complete as for ordinary
blood-serum.

Blood-serum (Lorrain Smith). —

1. Collect blood-serum (vide page 153, sections i to 4),
as free from haemoglobin as possible.

2. Weigh out 0.15 per cent, sodium hydrate and dissolve
it in the fluid (or add 0.375 c.c. of dekanormal soda solution
for every 100 c.c. of serum).

3. Tube, and stiffen at 100° C. in the serum inspissator.

4. Incubate at 37° C. for forty-eight hours to eliminate
any contaminated tubes. Store the remainder for future
use.

Ascitic Bouillon (Serum Bouillon).—

1. Collect ascitic fluid (pleuritic fluid, hydrocele fluid,
etc., by aspiration directly into sterile flasks, under strict
aseptic precautions.

2. Mix the serum with twice its bulk of sterile nutrient
bouillon (vide page 141).

3. If considered necessary (on account of the presence of
blood, crystals, etc.), filter the serum bouillon through porce-
lain filter candle.

4. Tube, and sterilise by the fractional method for half
an hour on each of five consecutive days.

5. Incubate at 37° C. for forty-eight hours and eliminate
contaminated tubes. Store for future use.

Serum Agar (Wertheimer). —

1. Prepare nutrient agar (vide page 149), to the following
formula: agar, 2 per cent.; peptone, 2 per cent.; salt, 0.5
per cent. ; meat extract, quantum sufficit.

2. Make reaction of medium -f- 10.

3. Filter; tube in quantities of 5 c.c.

4. Sterilise by the discontinuous method.

5. After the last sterilisation cool to 42° C., then add
5 c.c. sterile blood-serum from human placenta (sterilised,

SERUM AGAR. 157

if necessary, by the fractional method) to each tube; slope
the tubes.

6. When set, incubate at 37° C. for forty-eight hours, to
eliminate any contaminated tubes. Store the remainder for
future use.

Serum Agar (Heiman).—

1. Prepare nutrient agar (vide page 149), to follow-
ing formula: agar, 2 per cent.; peptone, 1.5 per cent.;
salt, 0.5 per cent.; glucose, 2 per cent; meat extract,
quantum sufficit.

2. Make reaction of medium +10.

3. Filter; tube in quantities of 6 c.c.

4. Sterilise by the discontinuous method.

5. After the third sterilisation cool the tubes to
42° C., and add to each 3 c.c. of sterile hydrocele fluid,
ascitic fluid, or pleuritic effusion (previously sterilised,
if necessary, by the fractional method); allow the
tubes to solidify in a sloping position.

6. When solid, incubate at 37° C. for forty-eight
hours, and eliminate any contaminated tubes. Store
the remainder for future use.

Serum Agar (Kanthack and Stevens). —

1. Collect ascitic, pleuritic, or hydrocele fluid in sterile
flasks and allow it to stand in the ice-chest for twelve hours
to sediment.

2. Decant the clear fluid into a measuring cylinder.

3. Add 0.5 c.c. dekanormal NaOH solution for every 100
c.c. serum, and mix thoroughly.

4. Heat in the steamer for sixty minutes.

5. Weigh out 15 grammes agar, emulsify with about 200
c.c. of the alkaline fluid, and add to the remainder of the
fluid.

6. Heat in the steamer ninety minutes to dissolve the
agar.

7. Filter through papier Chardin, using a hot- water funnel.

8. Weigh out glucose, i per cent., and dissolve it in the
clear agar.

8a. If desired, add glycerine, 5 per cent., to the clear
agar.

9. Tube, and sterilise in the steamer at 100° C. for thirty
minutes on each of three consecutive days.

158 CULTURE MEDIA.

Blood Agar (Washbourn).—

1. Melt up several tubes of nutrient agar (vide page
149) and allow them to solidify in the oblique position.

2. Place the tubes, in the horizontal position, in
the " hot" incubator for forty-eight hours, to evaporate
off some of the condensation water.

3. Kill a small rabbit with chloroform and nail it out
on a board (as for a necropsy). Moisten the hair thor-
oughly with 2 per cent, solution of lysol.

4. Sterilise several pairs of forceps, scissors, etc., by
boiling.

5. Reflect the skin over the thorax with sterile in-
struments.

6. Open the thoracic cavity by the aid of a fresh set
of sterile instruments.

7. Open the pericardium with another set of sterile
instruments.

8. Sear the surface of the left ventricle with a red-
hot iron and remove fluid blood from the heart by
means of sterile pipettes (e. g., those shown in Fig.
10, c).

9. Deliver a small quantity of the blood on the
slanted surface of the agar in each of the tubes, and
allow it to run over the entire surface of the medium.

10. Place the tubes in the horizontal position and
allow the blood to coagulate.

11. Return the " blood agar" to the hot incubator
for forty-eight hours and eliminate any contaminated
tubes. Store the remainder for future use.

Urine Media.—

1. Collect freshly passed urine in sterile flask or flasks.

2. Place the flask in the steamer at 100° C. for thirty
minutes.

3. Filter through two thicknesses of Swedish filter paper.

4. Tube, and sterilise by the discontinuous method.
(Leave the reaction unaltered.)

Urine Gelatine. —

i. Collect freshly passed urine in sterile flask.

URINE AGAR. 159

2. Take the specific gravity, and, if above 1010, dilute
with sterile water until that point is reached.

3. Estimate (with control) at the boiling-point, and note
the reaction of the urine.

4. Weigh out gelatine, 10 per cent., and add to the urine
in the flask.

5. Heat in the steamer at 100° C. for one hour to dissolve
the gelatine.

6. Estimate the reaction and add sufficient caustic soda
solution to restore the reaction of the medium mass to the
equivalent of the original urine.

7. Cool to 60° C. and clarify with egg as for nutrient
gelatine (vide page 145).

8. Filter through papier Chardin.

9. Tube, and sterilise as for gelatine.
Urine Gelatine (Heller).—

1. Collect freshly passed urine in sterile flask.

2. Filter through animal charcoal to remove part of the
colouring matter.

3. Take the specific gravity, and if above 1010, dilute
with sterile water till this gravity is reached.

4. Add Witte's peptone, i per cent.; salt, 0.5 per cent.;
gelatine, 10 per cent.

5. Heat in the steamer at 100° C. for one hour, to dissolve
the gelatine, etc.

6. Add normal caustic soda solution in successive small
quantities, and test the reaction from time to time with
litmus paper, until the fluid reacts faintly alkaline.

7 Cool to 60° C. and clarify with egg as for nutrient
gelatine (vide page 145).

8. Filter through papier Chardin.

9. Tube, and sterilise as for nutrient gelatine.

Urine Agar. —

1. Collect freshly passed urine in sterile flask.

2. Weigh out 1.5 per cent, or 2 per cent, powdered
agar, and add it to the urine.

3. Heat in the steamer ninety minutes to dissolve
the agar.

4. Cool to 60° C. and clarify with egg as for nutrient
agar (vide page 150).

5. Filter through papier Chardin, using the hot-
water funnel.

6. Tube, and sterilise as for nutrient agar.
(Leave the reaction unaltered.)

160 CULTURE MEDIA.

Egg=albumen. —

1. Break several fresh eggs (hens', ducks', or turkeys'
eggs), and collect the " whites" in a graduated cylinder,
taking care to avoid admixture with the yolks.

2. Add 40 per cent, distilled water, and incorporate
the mixture thoroughly by the aid of an egg-whisk.

3. Weigh out 0.15 per cent, sodium hydrate and
dissolve it in the fluid (or add the amount of dekanormal
caustic soda solution calculated to yield the required
percentage of soda in the total bulk of the fluid— i. e.,
0.375 c-c- °f dekanormal NaOH solution per 100 c.c.
of the mixture).

3<z. Glucose to the extent of i to 2 per cent, may now
be added, if desired.

4. Strain the mixture through butter muslin and
filter through a porcelain filter candle into a sterile
filter flask.

5. Tube, and stiffen at 100° C. in the serum inspis-
sator.

6. Incubate at 37° C. for forty-eight hours and
eliminate any contaminated tubes ; store the remainder
for future use.

Egg-albumen (Tarchanoff and Kolesnikoff). —

1. Place unbroken hens' eggs in dekanormal caustic soda
solution for ten days. (After this time the white becomes
firm like gelatine.)

2. Carefully remove the shell and cut the egg into fine
slices.

3. Wash for two hours in running water.

4. Place the egg slices in a large beaker and sterilise in
the steamer at 100° C. for one hour.

5. Transfer each slice of egg by means of a pair of steril-
ised forceps to a Petri dish or large capsule.

6. Sterilise in the steamer at 100° C. for twenty minutes
on each of three consecutive days.

Milk.-

1. Pour i litre of fresh milk into a large separator
funnel, and heat in the steamer at 100° C. for one hour.

2. Remove from the steamer and estimate the re-

WHEY GELATINE. l6l

action of the milk (normal cows' milk averages +17).
If of higher acidity than +20, or lower than +10, re-
ject this sample of milk and proceed with another litre
of milk from a different source.

Carefully eliminate milk to which antiseptics have
been added as preservatives.

3. Allow the milk to cool, when the fat or cream
will rise to the surface and form a thick layer.

4. Draw off the subnatant fat-free milk into sterile
tubes (10 c.c. in each).

5. Sterilise in the steamer at 100° C. for twenty
minutes on each of five successive days.

6. Incubate at 37° C. for forty-eight hours and
eliminate any contaminated tubes. Store the re-
mainder for future use.

Litmus Milk.—

1. Prepare milk as described above, sections i to 3.

2. Draw off the fat-free milk into a flask.

3. Add sterile litmus solution, sufficient to colour
the milk a deep lavender.

4. Tube, sterilise, etc., as for milk.

Litmus Whey.—

1. Curdle fresh milk by adding rennet (or by acidifying
with hydrochloric acid).

2. Filter off the whey into a sterile flask.

3. Heat in the steamer at 100° C. for one hour.

4. Filter into a sterile flask.

5. Tint the whey with litmus solution to a deep purple
red.

6. Tube, and sterilise as for milk.
Whey Gelatine.—

1. Curdle fresh milk by adding rennet; filter off the:
whey into a sterile flask.

2. Estimate and note the reaction of the whey.

3. Weigh out gelatine, 10 per cent., and add it to the
whey in the flask.

4. Heat in the steamer at 100° C. for one hour to dissolve
the gelatine.

5. Estimate the reaction of the medium mass; then add
sufficient caustic soda solution to restore the reaction of the
medium mass to the equivalent of the original whey.

1 62 CULTURE MEDIA.

6. Cool to 60° C. and clarify with egg as for nutrient
gelatine (vide page 145).

7. Filter through papier Chardin.

8. Tube, and sterilise as for nutrient gelatine.
Whey Agar.—

1 . Curdle fresh milk by adding rennet ; filter off the whey
into a sterile flask.

2. Weigh out agar, 1.5 or 2 per cent., and add it to the
whey in the flask.

3. Heat in the steamer at 100° C. for one hour, to dis-
solve the agar.

4. Cool to 60° C. ; clarify with egg as for nutrient agar
(vide page 150).

5. Filter through papier Chardin, using the hot-water
funnel.

6. Tube, and sterilise as for nutrient agar.
Fish Bouillon.—

1 . Weigh out herring, mackerel, or cod, 500 grammes, and
place in a large porcelain beaker (or enamelled iron pot).

2. Weigh out sodium chloride, 26.5 grammes; potassium
chloride, 0.75 gramme; magnesium chloride, 3.25 grammes;
and dissolve in 1000 c.c. distilled water. Add the solution
to the fish in the beaker.

3. Place the beaker in a water-bath and proceed as if
preparing meat extract — i. e., heat gently at 40° C. for
twenty minutes, then rapidly raise the temperature to, and
maintain at, the boiling-point for ten minutes.

4. Strain the mixture through butter muslin into a clean
flask.

5. Weigh out peptone, 5 grammes, and emulsify with
about 200 c.c. of the hot fish water; incorporate thoroughly
with the remainder of the fish water in the flask.

6. Heat in the steamer at 100° C. for twenty minutes
to complete the solution of the peptone.

7. Filter through Swedish filter paper.

8. When the fish bouillon is cold, make up to 1000 c.c.
by the addition of distilled water (to replace the loss from
evaporation).

As an alternative method " Marvis " fish food (16 grammes) may be sub-
stituted for the 5°o grammes of fresh fish.

Fish Gelatine.—

1. Measure out fish bouillon, 1000 c.c., into a 2 -litre flask.

2. Add sheet gelatine, 100 grammes, cut into small pieces.

3. Heat in the steamer at 100° C. for one hour.

4. Cool to below 60° C., and clarify with egg.

5. Filter through papier Chardin.

POTATO.

I63

6. Tube, and sterilise as for nutrient gelatine.
Shake well after the final sterilisation, to aerate the
medium.

Fish Gelatine— Agar.—

1. Weigh out powdered agar, 5 grammes, and emulsify it
with 200 c.c. fish bouillon.

2. Wash the emulsion into a 2 -litre flask with 800 c.c.
fish bouillon.

3. Weigh out sheet gelatine, 70 grammes, cut it into small
pieces and add it to the contents of the flask.

4. Heat in the steamer at 100° C. for one hour.

5. Cool to below 60° C. and clarify with egg.

6. Filter through papier Chardin.

7. Tube, and sterilise as for fish gelatine.

Shake well after the final sterilisation, to aerate the
medium.

Potato.-

1. Choose fairly large potatoes, wash them well, and
scrub the peel with a stiff nail-brush.

2. Peel and take out the eyes.

3. Remove cylinders from the longest
diameter of each potato by means of an
apple-corer or a large cork-borer (i. e., one
of about 1.4 cm. diameter).

3a. The reaction of the fresh potato is
strongly acid to phenolphthalein. If, there-
fore, the potatoes are required to approxi-
mate + 10, as for the cultivation of some
of the vibrios, the cylinders should be
soaked in a i per cent, solution of sodium
carbonate for thirty minutes.

4. Cut each cylinder obliquely from end
to end, forming two wedge-shaped por-
tions.

5. Place a small piece of sterilised cot-
ton-wool, moistened with sterile water, at
the bottom of a sterile test-tube; insert
the potato wedge into the tube so that its

base rests upon the cotton- wool. Now plug the tube
with cotton- wool (Fig. 92).

Fig. 92.— Po-
tato tube.

164 CULTURE MEDIA.

6. Sterilise in the steamer at 100° C. for twenty
minutes on each of five consecutive days.

Beet-root, carrot, turnip, and parsnip are prepared,
tubed, and sterilised as potato.
Glycerinated Potato. —

1. Prepare ordinary potato wedges (vide page 163, sec-
tions i to 4).

2. Soak the wedges in 25 per cent, solution of glycerine
for fifteen minutes.

3. Moisten the cotton- wool pads at the bottom of the
potato tubes with a 25 per cent, solution of glycerine instead
of plain water.

4. Insert a wedge of potato in each tube and replug the
tubes.

5. Sterilise in the steamer at 100° C. for twenty minutes
on each of five consecutive days.

Glycerine Potato Broth.—

1. Take i kilo of potatoes, wash thoroughly in water,
peel, and grate finely on a bread-grater.

2. Weigh the potato gratings, place them in a 2 -litre
flask, and add distilled water in the proportion of i c.c.
for every gramme weight of potato. Allow the flask to
stand in the ice-chest for twelve hours.

3. Strain the mixture through butter muslin and filter
through Swedish filter paper into a graduated cylinder.
Note the amount of the filtrate.

4. Place the filtrate in a flask, add an equal quantity
of distilled water, and heat in the steam steriliser for sixty
minutes.

5. Add glycerine, 4 per cent., mix thoroughly, and again
filter.

6. Tube and sterilise in the steamer at 100° C. for twenty
minutes on each of three consecutive days.

Potato Gelatine (Eisner).—

1. Take i kilo of potatoes, wash thoroughly in water,
peel, and finally grate finely on a bread-grater.

2. Weigh the potato gratings, place them in a 2 -litre
flask, and add distilled water in the proportion of i c.c. for
every gramme weight of potato. Allow the flask to stand
in the ice-chest for twelve hours.

3. Strain the mixture through butter muslin, and filter
through Swedish filter paper into a graduated cylinder.

4. Add 15 per cent, gelatine to the potato decoction and
heat in the steamer for sixty minutes.

5. Estimate the reaction and render the reaction of the
medium mass +25.

BEER WORT. 165

6. Cool the medium to below 60° C. ; clarify with egg as
for nutrient gelatine (vide page 145).

7. Add i per cent, potassium iodide (powdered) to the
medium.

8. Filter through papier Chardin.

9. Tube in quantities of 10 c.c.

10. Sterilise in the steamer at 100° C. for twenty minutes
on each of three consecutive days.

Potato Gelatine (Goadby). —

1. Prepare glycerine potato broth as above, sections i
to 5.

2. Add 10 per cent, gelatine to the potato decoction and
heat in the steamer at 100° C. for sixty minutes.

3. Estimate the reaction and render -j~5-

4. Cool the medium to below 60° C., clarify with egg as
for nutrient gelatine.

5. Filter through papier Chardin.

6. Tube, and sterilise as for nutrient gelatine.
Hay Infusion. —

1. Weigh out dried hay, 10 grammes, chop it up into
fine particles and place in a flask.

2. Add 1000 c.c. distilled water, heated to 70° C. ; close
the flask with a solid rubber stopper.

3. Macerate in a water-bath at 60° C. for three hours.

4. Replace the stopper by a cotton-wool plug, and heat
in the steamer at 100° C. for one hour.

5. Filter through Swedish filter paper.

6. Tube, and sterilise in the steamer at 100° C. for one
hour on each of three consecutive days.

Beer Wort. — Wort is chiefly used as a medium for
the cultivation of yeasts, etc., both in its fluid form
and also when made solid by the addition of gelatine
or agar. The wort is prepared as follows:

1. Weigh out 250 grammes crushed malt and place
in a 2 -litre flask.

2. Add 1000 c.c. distilled water, heated to 70° C.,
and close the flask with a rubber stopper.

3. Place the flask in a water-bath regulated to 60°
C. and allow the maceration to continue for one hour.

4. Strain through butter muslin into a clean flask
and heat in the steamer for thirty minutes.

5. Filter through Swedish filter paper.

6. Tube in quantities of 10 c.c. or store in flasks.

1 66 CULTURE MEDIA.

7. Sterilise in the steamer at 100° C. for twenty
minutes on each of three consecutive days.

The natural reaction of the wort should not be inter-
fered with.

NOTE. — It is sometimes more convenient to obtain
" unhopped " l beer wort direct from the brewery. In
this case it is diluted with an equal quantity of dis-
tilled water, steamed for an hour, filtered, filled into
sterile flasks or tubes, and sterilised by the discon-
tinuous method.

Wort Gelatine.—

1. Measure out wort (prepared as above), 1000 c.c.,
into a sterile flask.

2. Weigh out gelatine, 100 grammes (= 10 percent.),
and add it to the wort in the flask.

3. Heat in the steamer at 100° C. for one hour, to
dissolve the gelatine.

4. Cool to 60° C.; clarify with egg as for nutrient
gelatine (vide page 145).

5. Filter through papier Char din.

6. Tube, and sterilise as for nutrient gelatine.
Wort Agar. —

1. Measure out wort (as above), 800 c.c., into a
sterile flask.

2. Weigh out powdered agar, 20 grammes; mix into
a smooth paste with 200 c.c. of cold wort and add to
the wort in the flask.

3. Heat in the steamer at 100° C. for ninety minutes,
to dissolve the agar.

4. Cool to 60° C.; clarify with egg as for nutrient
agar (vide page 150).

5. Filter through papier Char din, using the hot-
water funnel.

6. Tube, and sterilise as for nutrient agar.

1 " Hopped" wort exerts a toxic effect upon many bacteria, including the
lactic acid bacteria.

MILK RICE. 167

Wine Must. — (Wine must is obtained from Sicily, in
hermetically sealed tins, in a highly concentrated form, —
as a thick syrup, — but not sterilised.)

1. Weigh out "wine must," 200 grammes, place in a
2 -litre flask and add distilled water, 800 c.c.

2. Weigh out ammonium tartrate, 5 grammes, and add
to the dilute must.

3. Place the flask in a water-bath regulated to 60° C.
for one hour and incorporate the mixture thoroughly by
frequent shaking.

4. Filter through papier Chardin.

5. Tube, and sterilise by the discontinuous method (three
days).

Wheat Broth (Gasperini).—

1 . Weigh out and mix wheat flour, 1 50 grammes ; magne-
sium sulphate, 0.5 gramme; potassium nitrate, i gramme;
glucose, 15 grammes.

2. Dissolve the mixture in 1000 c.c. of water heated to
100° C.

3. Filter through papier Chardin.

4. Tube, and sterilise by the discontinuous method.
Bread Paste.—

1. Grate stale bread finely on a bread-grater.

2. Distribute the crumbs in sterile Erlenmeyer flasks,
sufficient to form a layer about half an inch thick over the
bottom of each.

3. Add as much distilled water as the crumbs will soak
up, but not enough to cover the bread.

4. Plug the flasks and sterilise in the steamer at 100° C.
for thirty minutes on each of jour consecutive days.

Milk Rice (Eisenberg). —

1. Measure out nutrient bouillon, 70 c.c., and milk, 2 10 c.c.,
and mix thoroughly.

2. Weigh out rice powder, 100 grammes, and rub it up
in a mortar with the milk and broth mixture.

3. Fill the paste into sterile capsules, spreading it out
so as to form a layer over the bottom of each.

4. Heat over a water-bath at 100° C. until the mixture
solidifies.

5. Replace the lids of the capsules. Sterilise in the
steamer at 100° C. for twenty minutes on each of three con-
secutive days.

(A solid medium of the colour of cafe au lait is thus pro-
duced.)

Milk Rice (Soyka).—

i. Measure out nutrient bouillon, 50 c.c., and milk, 150
c.c., and mix thoroughly.

1 68 CUI/TURE MEDIA.

2. Weigh out rice powder, 100 grammes, and rub it up in
a mortar with the milk and broth mixture.

3. Fill the paste into sterile capsules, to form a layer over
the bottom of each.

4. Replace the lids of the capsules.

5. Sterilise in the steamer at 100° C. for twenty minutes
on each of three consecutive days.

(A pure white, opaque medium is thus formed.)

Peptone Water (Dunham).—

1. Weigh out Witte's peptone, 10 grammes, and
salt, 5 grammes, and emulsify with about 250 c.c. of
distilled water previously heated to 60° C.

2. Pour the emulsion into a litre flask and make up
to 1000 c.c. by the- addition of distilled water.

3. Heat in the steamer at 100° C. for thirty minutes.

4. Filter through Swedish filter paper.

5. Tube in quantities of 10 c.c. each.

6. Sterilise in the steamer at 100° C. for twenty
minutes on each of three consecutive days.

Peptone Rosolic Acid Water. —

1. Weigh out rosolic acid (coralline), 0.5 gramme, and
dissolve it in 80 per cent, alcohol, 100 c.c. Keep this as a
stock solution.

2. Measure out peptone water (Dunham), 100 c.c., and
rosolic acid solution, 2 c.c., and mix.

3. Heat in the steamer at 100° C. for thirty minutes.

4. Filter through Swedish filter paper.

5. Tube, and sterilise as for peptone water.
Iron Peptone Solution (Pakes). —

1. Weigh out peptone, 30 grammes, and emulsify it with
200 c.c. tap water, previously heated to about 60° C.

2. Wash the emulsion into a litre flask with 80 c.c. tap
water.

3. Weigh out salt, 5 grammes, and sodium phosphate, 3
grammes, and dissolve in the mixture in the flask.

4. Heat the mixture in the steamer at 100° C. for thirty
minutes, to complete the solution of the peptone, and filter
into a clean flask.

5. Fill into tubes in quantities of 10 c.c. each.

6. Add to each tube o.i c.c. of a 2 per cent, neutral solu-

SAI/T AGAR. 169

tion of ferric tartrate. (A yellowish- white precipitate
forms.)

7. Sterilise as for peptone solution.

NOTE. — A similar quantity of a i per cent, neutral solution
of lead acetate may be substituted for the iron salt.

Nitrate Water (Pakes). —

1. Weigh out Witte's peptone, 10 grammes, and
emulsify it with 200 c.c. ammonia-free distilled water
previously heated to 60° C.

2. Wash the emulsion into a flask and make up to
looo c.c., with similar water.

3. Heat in the steamer at 100° C. for twenty minutes.

4. Weigh out sodium nitrate, i gramme, and dis-
solve in the contents of the flask.

5. Filter through Swedish filter paper.

6. Tube, and sterilise as for peptone water.

Bile Salt Broth (MacConkey). —

1. Weigh out Witte's peptone, 20 grammes (= 2 per cent.),
and emulsify with 200 c.c. distilled water previously
warmed to 60° C.

2. Weigh out sodium taurocholate (commercial), 5
grammes (=0.5 per cent.), and glucose, 5 grammes (=0.5
percent.), and dissolve in the peptone emulsion.

3. Wash the peptone emulsion into a flask with 800 c.c.
distilled water, and heat in the steamer at 100° C. for twenty
minutes.

4. Filter through Swedish filter paper into a sterile flask.

5. Add sterile litmus solution sufficient to colour the
medium to a deep purple.

6. Fill, in quantities of 10 c.c., into tubes containing
small gas tubes (vide Fig. 85, page 139), and sterilise in the
steamer at 100° C. for twenty minutes on each of three con-
secutive days.

Bile Salt Agar (MacConkey).—

1. Weigh out powdered agar, 15 grammes (= 1.5 per
cent.), and emulsify with 200 c.c. cold distilled water.

2. Weigh out peptone, 20 grammes (== 2 per cent.), and
emulsify with 200 c.c. distilled water previously warmed to
60° C.

3. Mix the peptone and agar emulsions thoroughly.

4. Weigh out sodium taurocholate, 5 grammes (— 0.5

170 CULTURE MEDIA.

per cent.), dissolve it in 600 c.c. distilled water, and use
the solution to wash the agar- pep tone emulsion into a 2 -litre
flask.

5. Heat in the steamer at 100° C. for sixty minutes.

6. Cool to 60° C. and clarify with egg as for nutrient
agar (vide page 150).

7. Filter through papier Chardin, using the hot-water
funnel.

8. Weigh out lactose, 10 grammes (— i per cent.), and
dissolve it in the agar.

9. If desired, add saturated aqueous solution of neutral
red, 2 c.c.

10. Tube, and sterilise in the steamer at 100° C. for
twenty minutes on each of three consecutive days.

French Proof Agar (Sabouraud). —

1. Weigh out peptone (Chassaing), 7 grammes, and
emulsify it with 200 c.c. distilled water previously heated
to 60° C.

2. Weigh out powdered agar, 13 grammes, and emulsify
with 200 c.c. cold distilled water.

3. Mix the two emulsions and wash into a 2 -litre flask
with 600 c.c. distilled water.

4. Heat in the steamer for ninety minutes, to dissolve
the agar.

5. Cool to 60° C. and clarify with egg as for nutrient
agar (vide page 150).

6. Filter through papier Chardin, using the hot-water
funnel.

7. Weigh out maltose, 38 grammes, and dissolve in the
agar.

8. Tube, and sterilise as for nutrient agar.

English Proof Agar (Blaxall).— Substitute Witte's pep-
tone for that of Chassaing, and proceed as for French proof
agar.

Pasteur's Solution. —

1. Weigh out and mix the ash from 10 grammes of
yeast; ammonium tartrate, 10 grammes; cane sugar, 100
grammes.

2. Dissolve the mixture in distilled water, 1000 c.c.

3. Tube or flask, and sterilise by the discontinuous method
(three days).

Yeast Water (Pasteur).—

1. Weigh out pressed yeast, 75 grammes; place in a 2-
litre flask and add 1000 c.c. distilled water.

2. Heat in the steamer at 100° C. for thirty minutes.

3. Filter through papier Chardin.

4. Tube or flask, and sterilise as for Pasteur's solution.

ASPARAGIN MEDIUM. iyi

Cohn's Solution. —

1. Weigh out and mix

Acid potassium phosphate 5.00 grammes

Tribasic calcium phosphate 0.50 gramme

Magnesium sulphate 5.00 grammes

Ammonium tartrate 10.00 "

and dissolve in

Distilled water . 1000 c.c.

2. Tube, or flask and sterilise as for Pasteur's solution.
Naegelfs Solution. —

1. Weigh out and mix

Dibasic potassium phosphate I.o gramme

Magnesium sulphate 0.2 "

Calcium chloride o. i "

Ammonium tartrate 10.0 grammes

and dissolve in

Distilled water looo c.c.

2. Tube or flask; sterilise as for Pasteur's solution.
Asparagin Medium (Uschinsky).—

1. Weigh out and mix

Asparagin 3.4 grammes

Ammonium lactate 10.0 "

Sodium chloride 5.0 "

Magnesium sulphate 0.2 gramme

Calcium chloride o. I "

Acid potassium phosphate I.o "

2. Dissolve the mixture in distilled water, 1000 c.c.

3. Add glycerine, 40 c.c.

4. Tube, and sterilise by the discontinuous method
(three days).

Asparagin Medium (Frankel and Voges). —

1. Weigh out and mix

Asparagin 4 grammes

Sodium phosphate (neutral) 2 "

Ammonium lactate 6 "

Sodium chloride 5 "

and dissolve in

Distilled water 1000 c.c.

2. Tube, and sterilise by the discontinuous method (three
days).

NOTE. — Either of the above asparagin media, after the
addition of 10 per cent, gelatine or 1.5 per cent, agar, may
be advantageously employed in the solid condition.

172 CULTURE MEDIA.

Winogradsky's Solution (for Nitric Organisms). —

1. Weigh out and mix

Potassium phosphate l.o gramme

Magnesium sulphate 0.5 "

Calcium chloride o.oi "

Sodium chloride 2.00 grammes.

and dissolve in

Distilled water . . . , looo c.c.

2. Fill into flasks, in quantities of 20 c.c., and add to
each a small quantity of freshly washed magnesium car-
bonate.

3. Sterilise in the steamer at 100° C. for twenty minutes
on each of three consecutive days.

4. Add to each flask containing 20 c.c. solution, 2 c.c.
of a sterile 2 per cent, solution of ammonium sulphate.

5. Incubate at 37° C. for forty-eight hours and eliminate
any contaminated culture flasks.

Winogradsky's Solution (for Nitrous Organisms) —

1. Weigh out and mix

Ammonium sulphate I gramme

Potassium sulphate I "

and dissolve in

Distilled water looo c.c.

2. Add magnesium carbonate, previously sterilised by
boiling.

3. Fill into flasks and sterilise as for previous solution.
Silicate Jelly (Winogradsky). —

1. Weigh out and mix

Ammonium sulphate 0,40 gramme

Magnesium sulphate 0.05 "

Calcium chloride o.oi "

and dissolve in

Distilled water 50 c.c.

Label — Solution A.

2. Weigh out and mix

Potassium phosphate o. 10 gramme

Sodium carbonate 0.60 "

and dissolve in

Distilled water 50 c.c.

Label — Solution B.

PLASTER-OF-PARIS DISCS. 173

3. Weigh out

Silicic acid 3.4 grammes

and dissolve in

Distilled water loo c.c.

4. Pour the silicic acid solution into a large porcelain
basin.

5. Mix equal quantities of the solutions A and B; then
add successive small quantities of the mixed salts to the
silicic acid solution, stirring continuously with a glass rod,
until a jelly of sufficiently firm consistence has been formed.

6. Spread a layer of this jelly over the bottom of each
of several large capsules or "plates."

7. Sterilise in the steamer for twenty minutes on each
of three consecutive days.

Plaster-of-Paris Discs.—

1. Take large corks, 2.5 cm. diameter, and roll a piece
of stiff note-paper round each, so that about a centimeter
projects as a ridge above the upper sur-
face of the cork, and secure in position

with a pin (Fig. 93).

2. Mix plaster-of-Paris into a stiff
paste with distilled water, and fill each
of the cork moulds with the paste.

3. When the plaster has set, remove
the paper from the corks, and lift up the
plaster discs.

4. Place the plaster discs on a piece of Fig. 93. — Cork and
asbestos board and sterilise by exposing paper mould for plas-
in the hot-air oven to 150° C. for half ter-of- Paris disc.

an hour.

5. Remove the sterile discs from the oven by means
of sterile forceps, place each inside a sterile capsule, and
moisten with a little sterile water.

6. Sterilise in the steamer at 100° C. for twenty minutes
on each of three consecutive days.

XI. INCUBATORS.

AN incubator consists essentially of a chamber for
the reception of cultivations, etc., surrounded by a
water jacket, the walls of which are of metal, usually
copper, and outside all an asbestos or felt jacket, or

Fig. 94. — Incubator.

wooden casing. The water in the jacket is heated
by gas and maintained at some constant temperature
by a thermo-regulator.

Two incubators at least are required for the culti-
vation of bacteria in the laboratory, the one regulated

174

THERMOREGULATORS. 1 75

to maintain a temperature of 37° C., and known as
the "hot" incubator; the other, 22° C., and known as
the "cool" or "cold" incubator.

Thermo=regulators. — The thermo-regulator is the
most essential portion of the incubator, as upon its
efficient working depends the maintenance of a con-
stant temperature in the cultivation chamber. It is
also used in the fitting up of water and paraffin baths,
and for many other purposes.

Of the many forms and varieties of thermo-regulator
(other than electrical), two only are of sufficiently
general use to need mention. In one of these the flow
of gas to the gas-jet is controlled by
the expansion or contraction of mer-
cury within a glass bulb ; in the other,
by alterations in the position of the
walls of a hollow metallic capsule.
They are:

(a) Reichert's (Fig. 95), which con-
sists of a bulb containing mercury.
Gas enters at A, and passes out to
the jet by B. As the temperature
rises the mercury expands and cuts

rr ^1 • 1 j_i 1 Fig. 95. — Reichert's

off the mam gas supply, thereby fhermo-reguiator.
reducing the temperature. As the
temperature falls the mercury contracts and reopens
the narrow tube C. By means of a thumbscrew D
(which mechanically raises or lowers the column of
mercury irrespective of the temperature) and the aid
of a thermometer the apparatus can be set to keep the
incubator at any desired temperature. With this form
a special gas burner is required, with separate supply
of gas to a pilot jet at the side.

(b) Capsule regulator consists of a metal capsule filled
with a liquid which boils at the required temperature,
and hermetically sealed; this is adjusted in the interior
of the incubator. Soldered to the upper side of the

1 76 INCUBATORS.

capsule is a thick piece of metal having a central de-
pression which receives the lower end of a rigid rod,
by means of which the movements of the walls of the
capsule are transmitted to the gas valve fixed outside
the incubator.

Figure 96 represents a well-known form of capsule
regulator. A is the inlet for gas, C the outlet to
burner heating the water jacket, B D a lever pivoted
to standards at G, and acted upon by the capsule,
through the needle which enters the socket below
the screw P.

The construction of the valve is such that, when-
ever the end B of the lever B D presses on the disc

below the end B, the
main supply of gas
is entirely cut off.
At such times, how-
ever, a very small
portion of gas
passes from A to C,
through an aperture

Fig. 96.— Capsule thermo- regulator. inside the Valve, the

size of which aper-
ture can be adjusted by the screw needle S, hence the
gas flame below the incubator is never extinguished.

The expansion of the metal walls of the capsule,
owing to the boiling of its contents, provides the
motive force for acting upon the lever B D, and as this
expansion only takes place at a predetermined tempera-
ture, the lever will only be acted upon when the
critical temperature is reached, no sensible effect being
produced at even i° C. below that at which the capsule
is desired to act.

W is a weight sliding on the lever rod D, by means
of which the boiling-point of the liquid in the capsule
can be slightly retarded, and a range of about two de-
grees obtained with any particular capsule.

XIL METHODS OF CULTIVATION.

CULTIVATIONS of micro-organisms are usually pre-
pared in the laboratory in one of three ways :
Tube cultures.
Plate cultures.
Hanging=drop cultures.

These may be incubated either aerobically (i. e.,
in the presence of oxygen) or anaerobically (i. e., in
the absence of oxygen, or in the presence of an in-
different gas, such as hydrogen, nitrogen, or carbon
dioxide).

With regard to the temperature at which the culti-
vations are grown, it may be stated as a general rule
that all media rendered solid by the addition of gela-
tine are incubated at 20° C., or at any rate at a tem-
perature not exceeding 22° C. (that is, in the "cold"
incubator) ; whilst fluid media and all other solid media
are incubated at 37° C. (that is, in the "hot" incu-
bator). Exceptions to this rule are numerous. For
instance, in studying the growth of the psychrophylic
bacteria, the yeasts and the moulds, the cold incubator
is employed for all media.

Tube cultivations are usually packed in the incubator
in small tin cylinders, such as those in which American
cigarettes are sold. Beakers or tumblers may be
used for the same purpose, but are not so convenient.

AEROBIC

The Preparation of Tube Cultivations.

The preparation of a tube cultivation consists in:
(a) Inoculating a tube of sterile nutrient medium
with a portion of the material to be examined.
12 177

178 METHODS OF CULTIVATION.

(6) Incubating the inoculated tube at a suitable
temperature.

The details of the first of these processes must be
varied somewhat according to whether the tubes of
nutrient media are inoculated or ''planted" from—

1. Pre-existing cultivations.

2. Previously collected morbid material.

3. The animal body direct.

The method of preparing tube cultivations from
pre-existing cultivations is as follows:

1. Fluid Media (e. g., Nutrient Bouillon). —

i. Flame the cotton- wool plug of the tube contain-

Fig. 97. — Inoculating tubes, seen from the front.

ing the cultivation and also that of the tube of sterile
bouillon.

2. Hold the two tubes, side by side, between the
left thumb and the first and third fingers, allowing
the sealed ends to rest on the dorsum of the hand,
and separating the mouths of the tubes (which are
pointed to the right) by the tip of the second finger.
Keep the tubes as nearly horizontal as is possible
without allowing the fluid in the bouillon tube to reach
the cotton- wool plug.

3. Sterilise the platinum loop and allow it to cool.1

4. Grasp the plug of the tube containing the culti-
vation between the little finger and palm of the hand
and remove it from the tube.

1 See also method of opening and closing culture tubes, page 69.

SOLID MEDIA.

179

5. Grasp the plug of the bouillon tube between the
fourth finger and the ball of the thumb and remove
it from the tube.

6. Pass the platinum loop into the tube containing
the culture, — do not allow the loop to

touch the sides of the tube, or the
handle to touch the medium, — and re-
move a small portion of the growth;
withdraw the loop from the tube, keep-
ing the infected side of the loop down-
wards.

7. Pass the loop into the bouillon
tube almost down to the level of the
fluid, reverse the loop so that the in-
fected side faces upwards, emulsify the
portion of the growth in the moisture
adhering to the side of the tube which
is uppermost. Withdraw the loop.

8. Replug both tubes.

9. Sterilise the platinum loop.

10. Label the bouillon tube with (a)
the name of the organism and (b) the
date of inoculation.

11. Incubate.

2. Solid Media. — Solid media are
stored in tubes in one of two ways :

i. Oblique tube or slanted tube (Fig.
98), in which the medium has been
allowed to solidify whilst the tube was
retained in an inclined position, so forming an exten-
sive surface of medium extending from the bottom of
the tube almost to its mouth.

This is employed for "streak" or "smear" cultiva-
tions (Strichcultur) .

NOTE. — Gelatine and agar oblique tubes should be
freshly "slanted" before use.

Fig. 98. — Streak
tube.

i8o

METHODS OF CULTIVATION.

2. Straight tube (Fig. 99), in which the medium
forms a cylindrical mass in the lower portion of the
tube and presents an upper surface which is at right
angles to the long axis of the tube.

This is employed for "stab" or ''stick" cultivations
(Stichcultur\ or by inoculating the medium whilst
fluid, and allowing to solidify in this
position, for "shake" cultivations.
Streak Culture. —

1. Flame the plugs, sterilise the plat-
inum loop (or spatula). Open the tubes
and charge the loop as in previous in-
oculation.

2. Pass the infected loop to the bot-
tom of the tube to be inoculated and
draw it, as lightly as possible, along the
centre of the surface of the medium,
terminating the "streak" over the thin
layer of medium near the mouth of the
tube.

3. Replug the tubes, sterilise the
platinum loop.

4. Label the newly inoculated tube
and incubate.

Smear Culture. — Proceed generally as
in streak culture, but rub the infected
loop all over the surface of the medium,
instead of restricting the inoculation to
a narrow line.
Stab Culture. —
i. Flame the plugs, open the tubes,
sterilise the platinum needle and charge it with the
inoculum as in the previous cultivations.

2. Pass the platinum needle into the tube to be
inoculated until it touches the centre of the surface of
the medium. Now thrust it deeply into the sub-
stance of the medium, keeping the needle as nearly as

Fig. 99- — Straight
tube.

THE PREPARATION OF PLATE CULTURES. l8l

possible in the axis of the cylinder of medium. Then
withdraw the needle.

3. Replug the tubes. Sterilise the platinum needle.

4. Label the newly planted tube and incubate.
Shake Culture. —

1. Liquefy a tube of nutrient gelatine (or agar, or
other similar medium), by heating in a water-bath
(Fig. i oo).

2. Inoculate the liquefied
medium and label it, etc.,
precisely as if dealing with a
tube of bouillon.

3. Place the newly planted
tube in the upright position
(e. g., in a test-tube rack) and
allow it to solidify.

4. Label the tube; when
solid, incubate.

The Preparation of Plate Cul=
tures.

If a small number of bac-
teria are suspended in lique-
fied gelatine, agar, or other
similar medium, and the in-
fected medium spread out in
an even layer over a flat sur-
face and allowed to solidify,
each individual micro-organ-
ism becomes fixed to a cer-
tain spot and its further development is restricted to
tho vicinity of this spot. After a variable interval the
growth of this organism becomes visible to the naked

Fig. 100. — Handy form of
water-bath for melting tubes of
agar and gelatine previous to
slanting them ; or to making
shake cultures.

eye as a "colony." This is the principle upon which
tho method of plate cultivation is based. The method
itself is as follows:

182

METHODS OF CULTIVATION.

Apparatus Required. —

1. Tripod levelling stand.

2. Large shallow glass dish, with a square sheet of plate

glass to cover it.

3. Spirit level.

4. Case of sterile Petri dishes.

5. Tubes of sterile nutrient media, gelatine (or agar) pre-

viously liquefied by heating in the water-bath and
cooled to 42° C., otherwise the heat of the medium
would destroy many, if not all, of the bacteria intro-
duced.

6. Tube of cultivation to be planted from.

7. Platinum loop.

8. Bunsen burner.

9. Grease pencil.

Fig. 101. — Plate-levelling stand.

Method of "Pouring" Plates.—

1. Place the glass dish on the levelling tripod (Figs.
10 1, 102), fill it with ice water if gelatine plates are to
be poured, or with water at 50° C. if agar is to be
used; cover it with the square sheet of glass.

2. Place the spirit level on the sheet of glass and by
means of the levelling screws adjust the surface of
the glass to the horizontal.

3. Place three sterile Petri dishes in a row on the
surface of the glass plate and number them 1,2, and
3, from left to right.

4. Number, the previously liquefied tubes of nutrient
media i, 2, and 3. Flame the plugs and see that
each plug can be readily removed from the mouth
of its tube.

METHOD OF " POURING" PLATES.

5. Add one loopful of the inoculum to tube No. i,
treating the liquefied medium as bouillon. After re-
plugging, grasp the tube near its mouth by the thumb
and first finger of the right hand, and with an even
circular movement of the whole arm, diffuse the inocu-

Fig. 1 02. — Plate-levelling stand, side view.

lum throughout the medium; avoid jerky movements,
as these cause bubbles of air to form in the medium.

6. Sterilise the platinum loop, and add two loopfuls
of diluted inoculum to tube No. 2, and mix as before.

7. In a similar manner transfer three loopfuls of

Fig. 103. — Pouring plates.

and

liquefied medium from tube No. 2 to tube No. 3,
mix thoroughly.

8. Flame the plug of tube No. i, remove it, then
flame the lips of the tube; slightly raise the cover of
Petri dish No. i, introduce the mouth of the tube;
then, elevating the bottom of the tube, pour the lique-
fied medium into the Petri dish, to form a thin layer.

184 METHODS OF CULTIVATION.

Remove the mouth of the tube and close the "plate."
If the medium has failed to flow evenly over the bottom
of the plate, raise the plate from the levelling platform
and by tilting in different directions rectify the fault.

9. Pour plates No. 2 and No. 3, in a similar manner,
from tubes Nos. 2 and 3.

10. Label the plates with the distinctive name or
number of the inoculum, the number of the dilution,
also the date.

11. Place in the cool incubator for three or more
days, as may be necessary.

In this way colonies may be obtained quite pure and
separate from each other.

In plate No. i, probably, the colonies will be so
numerous and crowded, and therefore so small, as to
render it useless. In plate No. 2 they will be more
widely separated, but usually No. 3 is the plate reserved
for careful examination, as in this the colonies are usu-
ally widely separated, few in number, and large in size.

A gar plates are poured in a similar manner, but the
agar must be melted in boiling water and then allowed
to cool to 45° C. or 42° C. in a carefully regulated
water-bath before being inoculated, and the entire pro-
cess must be carried out very rapidly, otherwise the agar
will have solidified before the operation is completed.

NOTE. — In pouring plates, tube No. i (for the first
dilution) very rarely gives a plate that is of any prac-
tical value; consequently it is frequently replaced by
a tube of bouillon or sterile salt solution, and plate
No. i is not poured.

Hanging=drop Cultivation.

(a) Fluid Media.—

i . Prepare first and second dilutions of the inoculum
as directed for plate cultivations (vide page 182,
sections 4 to 6), substituting tubes of nutrient broth
for the liquefied gelatine.

HANGING-DROP CULTIVATION. 185

2. Sterilise a hanging-drop slide by washing thor-
oughly in water and drying, then plunging it into a
beaker of absolute alcohol, draining off the greater
part of the spirit, grasping the slide in a pair of forceps,
and burning off the remainder of the alcohol in the
flame.

3. Place the hanging-drop slide on a piece of blotting
paper moistened with 2 per cent, lysol solution and
cover it with a small bell glass that has been rinsed
out with the same solution and not dried.

4. Raise the bell glass slightly and smear sterile
vaseline around the rim of the metal cell by means
of a sterile spatula of stout platinum wire.

5. Remove a clean cover- slip from the alcohol pot
with sterile forceps and burn off the alcohol; again
raise the bell glass and place the sterile cover-slip on
the blotting paper by the side of the hanging-drop slide.

6. Remove a drop of the broth from the second
dilution tube with a large platinum loop; raise the
bell glass and deposit the drop on the centre of the
cover-slip. Sterilise the loop.

7. Raise the bell glass sufficiently to allow of the
cover-slip being grasped with forceps, inverted, and
adjusted over the cell of the hanging-drop slide. Re-
move the bell glass altogether and press the cover-slip
firmly on to the cell.

8. Either incubate and examine at definite intervals,
or observe continuously with the microscope, using
a warm stage if necessary (Fig. 40) .

(b) Solid Media. — Observing precisely similar tech-
nique, a few drops of liquefied gelatine or agar from
the second dilution tube may be run over the surface
of the sterile cover-slip and a hanging-drop plate cul-
tivation thereby prepared.

This method is extremely useful in connection with
the study of yeasts, and in this connection the circular
cell on the hanging-drop slide is replaced by a rectangu-

1 86 METHODS OF CULTIVATION.

lar cell some 38 by 19 mm., and the gelatine spread
over a cover-slip of similar size. After sealing down
the preparation, the upper surface of the cover-slip
may be ruled into squares by the aid of the grease
pencil or a writing diamond.

ANAEROBIC CULTIVATIONS.

Numerous methods have been devised for the culti-
vation of anaerobic bacteria, the majority requiring
the employment of special apparatus. The principle
upon which any method is based and upon which it
depends for its success falls under one or another of
the following headings:

(a) Exclusion of air from the cultivation.

(b) Exhaustion of air from the vessel containing the
cultivation by means of an air pump — i. e., cultiva-
tion in vacua.

(c) Absorption of oxygen from the air in contact
with the cultivation by means of pyrogallic acid
rendered alkaline with caustic soda — i. e., cultivation
in an atmosphere of nitrogen.

(d) Displacement of air by an indifferent gas, such
as hydrogen or coal gas — i. e., cultivation in an atmos-
phere of hydrogen.

(e) A combination of two or more of the above
methods.

A selection of the simplest and most generally useful
methods is given here.

Whenever possible, the nutrient media that are em-
ployed in any of the processes should contain some
easily oxidisable substance, such as sodium formate
(0.4 per cent.) or sodium sulphindigotate (o.i per
cent.), which will absorb all the available oxygen held
in solution by the medium. The further addition of
glucose, 2 per cent., favors the growth of anaerobic
bacteria (vide glucose formate bouillon, page 142;
sulphindigotate bouillon, page 143, etc.).

ANAEROBIC CULTIVATIONS. 187

Further, it is advisable to seal all joints between
india-rubber stoppers and tubulures or the mouths
of the tube with melted paraffin.

(A) Method I (Hesse's Method).—

1. Make a stab culture in gelatine or agar, choosing
for the purpose a straight tube containing a deep
column of medium, and thrusting the inoculating
needle to the bottom of the tube.

2. Pour a layer of sterilised oil (olive oil, vaseline,
or petroleum), i or 2 cm. deep, upon the surface of
the medium.

3. Incubate.
Method II.-

1. Make an Esmarch's roll cultivation in the usual
way.

2. Fill the lumen of the tube with sterile gelatine
that has been liquefied by heat and cooled in the
water-bath to 15° C. (At this temperature the gela-
tine will remain fluid for only a few minutes.)

3. Incubate.

NOTE. — This method is but rarely employed.

Method III. — This method is only available when
dealing with pure cultivations.

1. Liquefy a tube of gelatine (or agar) by heat,
pour it into a Petri dish, and allow it to solidify.

2. Inoculate the surface of the medium in one spot
only.

3. Remove a cover-slip from the pot of absolute
alcohol with sterile forceps; burn off the alcohol in the
gas flame.

4. Lower the now sterile cover-slip carefully on to
the inoculated surface of the medium, carefully ex-
cluding air bubbles, and press it down firmly with the
points of the forceps. (A sterile disc of mica may
be substituted for the cover-slip.)

5. Incubate.

i88

METHODS OF CULTIVATION.

Method IV (Roux's Physical Method).—

1. Prepare tube cultures of fluid media (or solid
media rendered fluid by heat) in the usual way.

2. Aspirate some of the inoculated media into capil-
lary pipettes.

3. Seal both ends of each pipette in the blowpipe
flame.

4. Incubate.

Method V (Roux's Biological Method).—

1. Plant a deep stab, as in method I.

2. Pour a layer, i or 2 cm. deep, of broth cultiva-
tion of an aerobe — e. g., B. aquatilis sulcatus or B.

prodigiosus — upon the surface of the
medium; or an equal depth of lique-
fied gelatine, which is then inoculated
with the aerobic organism.

3. Incubate.

The growth of the aerobe will use
up all the oxygen that reaches it and
will not allow any to pass through to
the medium below, which will con-
sequently remain in an anaerobic
condition.

(B) Method VI.-

1. Prepare tube or flask cultiva-
tions in the usual way.

2. Replace the cotton- wool plug
by an india-rubber stopper perfor-
ated with one hole and fitted with a
length of glass tubing which has a

constriction about 3 cm. above the stopper and is then
bent at right angles (Fig. 104). The stopper and glass
tubing are sterilised by being boiled in a beaker of
water for five minutes.

3. Connect the tube leading from the culture vessel
with a water or air pump, interposing a Wulff's bottle
fitted as a wash-bottle and containing sulphuric acid.

Fig. 104. — Vacuum
culture.

ANAEROBIC CULTIVATIONS.

189

4. Exhaust the air from the culture vessel.

5. Before disconnecting the apparatus, seal the glass
tube from the culture vessel at the constriction, using
the blowpipe flame.

6. Incubate.

Apparatus and Solutions Required. —

Buchner's tube (a stout glass test-tube 23 cm. long and
4 cm. in diameter, fitted with india-rubber stopper,
Fig. 105).

Ten per cent, aqueous solution of pyrogallic acid. l

Dekanormal solution of caustic soda.

METHOD.—

1. Prepare the tube cultivation in the usual way.

2. Moisten the india-rubber stopper of the Buchner's
tube with water and see that it fits the

mouth of the tube accurately.

3. Remove the stoppers from the py-
rogallic acid and caustic soda bottles.

4. Run about 10 c.c. of the pyro-
gallic solution into the Buchner's tube
(roughly, use 5 c.c. pyrogallic solution
for every 100 c.c. air capacity of the
receiving vessel).

5. Add about i c.c. of the soda solution.

6. Place the inoculated tube inside
the Buchner's tube.

7. Fit the india-rubber stopper tightly
into the mouth of the Buchner's tube.

Fig. 105. — Buch-
ner's tube.

NOTE. — Sections 4 to 7 must be per-
formed as quickly as possible.

8. Restopper the pyrogallic acid and
caustic soda bottles.

9. Place Buchner's tube in a wire support, and incu-
bate.

1 One and a half cubic centimetres of hydrochloric acid should be added to
every looo c.c. of the stock pyrogallic solution to prevent oxidation.

METHODS OF CULTIVATION.

Method VIII (Wright's Method).—

1. Prepare tube cultivation in the usual way.

2. Cut off that portion of the cotton- wool plug
projecting above the mouth of the tube with scissors,
then push the plug into the tube for a distance of 2 or
3 cm.

3. By means of a pipette drop about i c.c. of the
pyrogallic acid solution on to the plug. It will imme-
diately be absorbed by the cotton- wool.

4. With another pipette run in an equal quantity
of the caustic soda solution.

5. Quickly close the mouth of the tube with a tightly
fitting india-rubber stopper.

6. Incubate.

(D) Method IX.-

Apparatus Required. —

Small Ruffer's or Woodhead's flask (Fig. 23).
Sterile india-rubber stopper.
India-rubber tubing.
Glass tubing.
Metal screw clips.

Cylinder of compressed hydrogen or Kipp's hydrogen
apparatus.

METHOD.—

1. Sterilise a glass vessel, shaped as in a Ruffer's or
Woodhead's flask, in the hot-air oven. (The tubulure
and the side tubes are plugged with cotton- wool.)
After sterilisation, fix a short piece of rubber tubing
occluded by a metal clip to each side tube.

2. Inoculate a large quantity (e. g., 200 c.c.) of the
medium. Where solid media are employed they must
first be liquefied by heat.

3. Remove the cotton- wool plug from the tubulure
and pour the inoculated medium into the glass vessel.

4. Close the tubulure by means of an india-rubber
stopper previously sterilised by boiling in a beaker
of water.

ANAEROBIC CULTIVATIONS.

191

5. Connect up the india-rubber tubing on one of
the- side tubes with a cylinder of compressed hydrogen
(or the delivery tube of a Kipp's hydrogen apparatus,
Fig. 1 06), interposing a short piece of glass tubing;
and in like manner connect a long piece of rubber
tubing which should be led into a basin of water, to
the opposite side tube.

6. Open both metal clips and pass hydrogen through
the vessel until the atmospheric air is replaced by

Fig. 106. — Kipp's hydrogen apparatus with two washing bottles containing
lead nitrate and silver nitrate solutions respectively to remove impurities.

hydrogen. This is determined by collecting some of
the gas which bubbles through the water in the
basin in a test-tube and testing it by means of a
lighted taper.

7. Close the metal clip on the tube through which
the gas is entering; close the clip on the exit tube.

8. Disconnect the gas apparatus.

9. Incubate.

192

METHODS OF CULTIVATION.

Method X (Botkin's Method).—

Apparatus Required. —

Large glass dish 20 cm. diameter and 8 cm. deep. Flat
leaden cross slightly shorter than the internal diameter
of the glass dish.

Bell glass about 15 cm. diameter and 20 to 25 cm. high.

Metal frame for plate cultivations.

Or, glass battery jar for tube cultivations.

Cylinder of compressed hydrogen.

Rubber tubing.

Two pieces of U-shaped glass tubing (each arm 8 cm. in
length).

Half a litre of glycerine.

METHOD.—

1. Place the leaden cross inside the glass dish, rest-
ing on the bottom.

2. Prepare the cultivations in the usual way.

3. Place the tube cultivations in a glass battery jar

(or the plate culti-
vations on a metal
frame), resting on
the centre of the
leaden cross.

4. Cover the cul-
tivations with the
bell jar.

5. Adjust the (J-
shaped pieces of

•*- glass tubing in a
vertical position on
opposite side of the
bell jar, one arm of
each inside the jar,
the other outside.

Fig. 107.— Botkin's apparatus. Fix a short length

of rubber tubing

clamped with a metal clip to each of the outside arms.
6. Fill the glass dish with glycerine to a depth of
about 5 cm. (Fig. 107).

ANAEROBIC CULTIVATIONS.

193

7. Connect up one U-shaped tube with the hydrogen
cylinder by means of rubber tubing. Replace the
atmospheric air by hydrogen, as in method IX.

8. Clamp the tubes and disconnect the gas apparatus.

9. Incubate.

Method XI (Novy's Method). -

Apparatus Required. —

Jar for plate cultivations (Fig. 108).

Or, jar for tube cultivations (Fig. 109).

Lubricant for stopper of jar (beeswax i part, olive oil 4

parts) .

Rubber tubing.
Cylinder of compressed hydrogen.

Fig. 108. — Novy jar for
plate cultivations.

Fig. 109. — Novy's jar for
tube cultivations.

METHOD.—

1. Prepare cultivations in the usual way.

2. Place these inside the jar.

3. Lubricate the stopper and insert it in the mouth
of the jar, with the handle in a line with the two side
tubes.

4. Connect up the delivery tube a with the hydrogen
gas supply by means of rubber tubing.

5. Attach a piece of rubber tubing to the exit tube
b and collect samples of the issuing gas (over water)
and test from time to time.

194 METHODS OF CULTIVATION.

6. When the air is completely displaced by hydrogen,
turn the handle of the stopper at right angles to the
line of the entry and exit tubes; this seals the orifice
of both tubes.

7. Disconnect the gas apparatus and incubate.
(E) Method XII (Bullock's Method).—

Apparatus Required. —

Bullock's jar.

Pot of resin ointment.

Small glass dish 14 cm. diameter by 5 cm. deep.

Vessel for tube cultures or metal rack for plate cultures.

Pyrogallic acid powder.

Cylinder of compressed hydrogen.

Geryk or other air pump.

Rubber tubing.

Glass tubing.

Small beaker of dekanormal caustic soda.

Small beaker of water.

METHOD.—

1. Prepare the cultivations in the usual way.

2. Place the glass dish in the centre of the glass
slab, and stand the cultivations inside this.

3. Place a quantity of dry pyrogallic acid in a heap
at one side of the glass dish.

4. Smear the flange of the bell jar with resin ointment
and apply the jar firmly to the glass slab, covering
the cultivations, — so arranged that the long tube passes
with its lower end into the glass dish at a point directly
opposite to the pyrogallic acid powder. (This is to
prevent the tube getting blocked with pyrogallic acid
during the next step.) Lubricate the two stop-cocks
with resin ointment.

5. Connect up the short tube a with the gas-supply
by means of rubber tubing and open both stop-cocks.

6. When the air is displaced, shut off the stop-cock
of the entry tube, then that of the exit tube b.

7. Connect a long, straight piece of glass tubing to
the long tube b by means of a piece of rubber tubing;

ANAEROBIC CULTIVATIONS.

195

and connect up the short tube a to the air pump by
means of pressure tubing.

8. Open the stop-cock of tube a and aspirate a small
quantity, say 100 c.c., of gas by means of the air pump,
so creating a slight vacuum. Then shut off the stop-
cock and disconnect the air pump.

9. Dip the long glass tube (connected with 6) into
the beaker of soda solution ; open the stop-cock and
the alkali will run

down the long tube
and come into con-
tact with the dry
pyrogallic acid.

10. When 2 .or 3
c.c. of soda solution
have been run in,
shut off the stop-
cock, remove the
glass tube from the
soda solution, and
place it in the
beaker of water.

11. In a similar
manner run in a few
cubic centimetres of
water and again

shut the stop-cock. (This serves to wash out the tubes
and prevents the alkali collecting at the stop-cock and
exerting a corrosive action on the glass.)

12. Incubate.

This last method is the most satisfactory for anae-
robic cultivations, as by its means complete anaerobi-
osis can be obtained with the least expenditure of time
and trouble.

Fig. no. — Bullock's jar.

XHI. METHODS OF ISOLATION.

THE work in the preceding sections, arranged to
demonstrate the chief biological characters of bacteria
in general, is intended to be carried out by means of
pure cultivations of various organisms. But before
undertaking a systematic study of selected bacteria,
it is necessary to indicate the chief methods by which
one or more organisms may be isolated in a state of
purity from a mixture; whether that mixture exists
as an impure cultivation, in pus and other morbid
exudations, infected tissues, or water or food-stuffs.

Fig. III. — Haematocytometer cell, showing, a, section through the centre of
the cell, and b, a magnified image of the cell rulings.

Before the introduction of solid media the only
method of obtaining pure cultivations was by "dilu-
tion"— by no means a reliable method. " Dilution"
consisted in estimating approximately the number of
bacteria present in a given volume of fluid (by means
of a graduated-celled slide resembling a haemato-
cytometer, Fig. in.), diluting the fluid by the addi-
tion of sterile water or bouillon until a given volume
(usually i c.c.) of the dilution should contain but one
organism. By planting this volume of the fluid into
several tubes or flasks of nutrient media, it occasion-

196

PLATE CULTIVATIONS. 197

ally happened that the resulting growth was the pro-
duct of one individual microbe. A method so uncer-
tain is now fortunately replaced by many others,
both reliable and convenient, and in those methods
selected for description here, the isolation of the re-
quired bacteria is effected—

1. By plate cultivation:

(a) Gelatine.

(b) Agar.

(d) Blood agar.

2. By Bsmarch's roll cultivation:

(a) Gelatine.

(b) Agar.

3. By serial cultivation.

4. By differential media.

(a) Selective.

(b) Deterrent.

5. By differential incubation.

6. By differential sterilisation.

7. By differential atmosphere cultivation.

8. By animal inoculation.

The selection of the method to be employed in any
specific instance will depend upon a variety of cir-
cumstances, and often a combination of two or more
will ensure a quicker and more reliable result than
a rigid adherence to any one method. Experience is
the only reliable guide, but as a general rule the use
of either the first or the third method will be found
most convenient, affording as they do an opportunity
for the simultaneous isolation of more than one of the
bacteria present in a mixture.

1. Plate Cultivations.—

(a) Gelatine (vide page 145).

(b) Agar (vide page 149).

These plates are poured in a manner precisely

198 METHODS OF ISOLATION.

similar to that adopted for nutrient gelatine and nu-
trient agar plates (vide page 182).
(c') Serum Agar.—

1. Melt three tubes of nutrient agar, label them 1,2,
and 3, and place them, with three tubes of sterile
fluid serum, also labelled 1,2, and 3, in a water-bath
regulated at 45° C. ; allow sufficient time to elapse for
the temperature of the contents of each tube to reach
that of the water-bath.

2. Make three dilutions of the inoculum in the three
liquid serum tubes, treating them exactly as if they
were tubes of liquefied gelatine (vide page 183) ; replace
them in the water-bath.

3. Take serum tube No. i and agar tube No. i.
Flame the plugs and remove them from the tubes
(retaining the plug of the agar tube in the hand);
flame the mouths of the tubes, pour the serum into
the tube of liquefied agar and replace the plug of the
agar tube.

4. Mix thoroughly and pour plate No. i secundum
artem.

5. Treat the remaining dilutions in a similar fashion,
and pour plates Nos. 2 and 3 in the usual way.

6. Label and incubate.
(d) Human Blood Agar. —

1. Melt a tube of sterile agar and pour it into a
sterile plate; let it set.

2. Collect a few drops of human blood, under all
aseptic conditions, in a sterile capillary pipette.

3. Raise the cover of the Petri dish very slightly,
insert the extremity of the capillary pipette, and de-
posit the blood on the centre of the agar surface.
Close the dish.

4. Charge a platinum loop (or a sterilised camel's
hair brush) with a small quantity of the inoculum.
Raise the cover of the plate, introduce the loop, mix

ESMARCH'S ROLL CULTIVATION. 199

its contents with the drop of blood, and finally smear
the mixture over the surface of the agar.

5. Withdraw the loop and close the plate.

6. Label and incubate.

(If considered necessary, two, three, or more similar
plates may be inoculated in series.)
2. Esmarch's Roll Cultivation.-
(a) Gelatine.—

1. Liquefy three tubes of gelatine by heat.

2. Prepare three dilutions of the inoculum (as de-
scribed for plate cultivations).

3. Roll the tubes, held almost horizontally, in a
groove made in a block of ice, until the gelatine has

Fig. 112. — Esmarch's roll culture on block of ice.

set in a thin film on the inner surface of tube (Fig. 112);
or under the cold-water tap.

(6) Agar roll cultures are made in precisely the same
way as gelatine roll cultures, but in order that the
medium may adhere firmly to the glass, the agar used
for the purpose should have i per cent, gelatine or i
per cent, gum arabic added to it before sterilisation.

Roll cultivations, which served a most important
purpose in the days before the introduction of Petri
dishes for plate cultivations, are now seldom prepared,
and are in point of fact practically obsolete.

3. Serial Cultivations. — These are usually made

200 METHODS OF ISOLATION.

upon agar or blood-serum, although gelatine may also
be used. The method is as follows:

1. Take at least six "slanted" tubes of media and
number them consecutively.

2. Flame all the plugs and see that each can be
readily removed.

3. Charge the platinum loop with a small quantity
of the inoculum, observing the usual routine, and plant
tube No. i, smearing thoroughly all over the surface.
If any water of condensation has collected at the
bottom of the tube, use this as a diluent before smear-
ing the contents of the loop over the surface of the
medium.

4. Without sterilising or recharging the loop, inocu-
late tube No. 2.

5. In like manner plant the remainder of the tubes
in the series.

6. Label with distinctive name or number, and
date; incubate.

The growth that ensues in the first two or three
tubes of the series will probably be so crowded as to be
useless. Towards the end of the series, however, dis-
crete colonies will be found, each of which can be
transferred to a fresh tube of nutrient medium without
risk of contamination from the neighboring colonies.

4. Differential Media.—

(a) Selective. — Some varieties of media are specially
suitable for certain species of bacteria and enable them
to overgrow and finally choke out other varieties; e.g.,
wort is the most suitable medium-base for the growth
of torulae and yeasts and must always be employed
when pouring plates for the isolation of these organisms.
To obtain a pure cultivation of yeast from a mixture
containing bacteria as well, it is sufficient to inoculate
wort from the mixture and incubate at 37° C. for
twenty-four hours. Plant a fresh tube of wort from
the resulting growth and incubate. Repeat the pro-

DIFFERENTIAL STERILISATION. 2OI

cess once more, and from the growth in this third
tube plant a streak on wort gelatine, and incubate
at 20° C. The resulting growth will almost certainly
be a pure culture of the yeast.

(b) Deterrent. — The converse of the above also
obtains. Certain media possess the power of inhibiting
the growth of a greater or less number of species.
For instance, media containing carbolic acid to the
amount of i per cent, will inhibit the growth of prac-
tically everything but the Bacillus coli communis.

5. Differential Incubation. — In isolating certain bac-
teria, advantage is taken of the fact that different
species vary in their optimum temperature. A mix-
ture containing the Bacillus typhosus and the Bacillus
aquatilis sulcatus, for example, may be planted on
two slanted agar tubes, the one incubated at 40° C.,
and the other at 12° C. After twenty-four hours'
incubation the first will show a pure cultivation of the
Bacillus typhosus, whilst the second will be an almost
pure culture of the Bacillus aquatilis.

6. Differential Sterilisation.—

(a) Non-sporing Bacteria. — Similarly, advantage may
be taken of the varying thermal death-points of bac-
teria. From a mixture of two organisms whose ther-
mal death-points differ by, say, 4° C. — e. g., Bacillus
pyocyaneus, thermal death-point 55° C., and Bacillus
mesentericus vulgatus, thermal death-point 60° C. — a
pure cultivation of the latter may be obtained by heat-
ing the mixture in a water-bath to 58° C. and keeping
it at that point for ten minutes. The mixture is then
planted on to fresh media and incubated, when the re-
sulting growth will be found to consist entirely of the
B. mesentericus.

(b) Sparing Bacteria. — This method is found to be
of even greater practical value when applied to the
differentiation of a spore-bearing organism from one
which does not form spores. In this case the mixture

202

METHODS OF ISOLATION.

is heated in a water-bath at 80° C. for fifteen to twenty
minutes. At the end of this time the non-sporing
bacteria are dead, and cultivations made from the
mixture will only yield a growth resulting from the
germination of the spores only.

Differential sterilisation at 80° C. is most conveni-
ently carried out in a water-bath of special construc-
tion, designed by Balfour Stewart (Fig. 113). It con-
sists of a double- walled copper vessel
mounted on legs, and provided with
a tubulure communicating with the
space between the walls. This space
is nearly filled with benzole (boiling-
point 80° C.), and to the tubulure is
fitted a long glass tube, some 2 metres
long and about 0.75 cm. diameter,
serving as a condensing tube. The
interior of the vessel is partly filled
with water and covered with a lid
which is perforated for a thermom-
eter. This latter dips into the water
and records its temperature. A very
small Bunsen flame under the appa-
ratus suffices to keep the benzole
boiling and the water within at a
\ constant temperature of 80° C. The
bath is thus always ready for use.

Fig. 113. — Benzole ,, /^ ...

bath. METHOD. — To use the apparatus,

i. Place some of the mixture it-
self, if fluid, containing the spores, or an emulsion of
the same if derived from solid material, in a test-tube.

2. Immerse the test-tube in the water contained
in the benzole bath, taking care that the upper level
of the liquid in the tube is at least 2 cm. beneath
the surface of the water in the copper vessel.

3. The temperature of the water, of course, falls a
few degrees after opening the bath and introducing

DIFFERENTIAL ATMOSPHERE CULTIVATION. 203

a tube of colder liquid, but after a few minutes the
temperature will have again reached 80° C.

4. When the thermometer again records 80° C.,
note the time, and fifteen minutes later remove the
tube containing the mixture from the bath.

5. Make cultures upon suitable media; incubate.

7. Differential Atmosphere Cultivation.—

(a) By adapting the atmospheric conditions to the
particular organism it is desired to isolate, it is com-
paratively easy to separate a strict aerobe from a
strict anaerobe, and vice -versa. In the first case,
however, it is important that the cultivations should
be made upon solid media, for if carried out in fluid
media the aerobes multiplying in the upper layers of
fluid render the depths completely anaerobic, and
under these conditions the growth of the anaerobes will
continue unchecked.

(b) When it is desired to separate a facultative
anaerobe from a strict anaerobe, it is generally suffi-
cient to plant the mixture upon the sloped surface
agar, incubate aerobically at 37° C., and examine
carefully at frequent intervals. At the first sign of
growth, subcultivations must be prepared and treated
in a similar manner. As a result of these rapid sub-
cultures, the facultative anaerobe will be secured in
pure culture at about the third or fourth generation.

(c) If, on the other hand, the strict anaerobe is the
organism required from a mixture of facultative and
strict anaerobes, pour plates of glucose formate agar
(or gelatine) in the usual manner, place them in a
Bullock's or Novy's jar, and incubate at a suitable
temperature. Pick off the colonies of the required
organism when the growth appears, and transfer to
tubes of the various media.

Incubate under suitable conditions as to tempera-
ture.

8. Animal Inoculation. — Finally, when dealing with

204 METHODS OF ISOLATION.

pathogenic organisms, it is often advisable to inoculate
some of the impure culture (or even some of the original
materies morbi) into an animal specially chosen on ac-
count of its susceptibility to the particular pathogenic
organism it is desired to inoculate. Indeed, with some
of the more sensitive and strictly parasitic bacteria this
method of animal inoculation is practically the only
method that will yield a satisfactory result.

XIV. METHODS OF IDENTIFICATION.

IN order to identify an organism after nutrient
media have been inoculated, and tube, plate, and
other cultivations prepared, these are incubated under
suitable conditions as to temperature and environ-
ment, are examined from time to time (a) macroscopi=
cally, (b) by microscopical methods, (c) by chemical
methods, (d) by physical methods, (e) by inoculation
methods, and the results of these examinations duly
recorded.

It must be stated definitely that no micro-organism
can be identified by any one character or property,
whether microscopical, biological, or chemical, but
that on the contrary its entire life history must be
carefully studied and then its identity established
from a consideration of the sum total of these observa-
tions.

In order to give to the recorded results their maxi-
mum value it is essential that they should be exact
and systematical, therefore some such scheme as the
following should be adhered to ; and especially is this
necessary in describing an organism not previously
isolated and studied.

SCHEME OF STUDY.

Designation :

Originally isolated by in 1 8 . . . , from

. Cultural Characters. — (Vide Macroscopical Exam-
ination of Cultivation, page 207.)
Gelatine plates,

Gelatine streak,

r\ 1 .• . •• at 20 C.

Gelatine stab,

Gelatine shake,

205

206 METHODS OF IDENTIFICATION.

Agar plates,

Agar streak or smear,

Agar stab,

Inspissated blood-serum,

at 20° C. and 37° C.

Bouillon,
Litmus milk,
Potato,

Special media for the purpose of demonstrating
characteristic reactions.

2. Morphology. — (Vide Microscopical Examination of

Cultivations, page 218.)
Vegetative forms:

Shape.

Size.

Motility.

Flagella (if present).

Capsule (if present).

Involution forms.

Pleomorphism (if observed).
Sporing forms (if observed). Of which class?
Staining reactions.

3. Biology. — (Vide Physical Examination of Cultures,

page 238.)
Vitality.

Resistance to lethal agents:
Physical :
Light.
Colours.

Chemical germicides.
Atmosphere.
Temperature.

Reaction of nutrient media.
Agglutination reaction.

4. Chemical Products of Growth. — (Vide Chemical Ex-

amination of Cultivations, page 221.)
Chromogenesis.
Photogenesis.

MACROSCOPICAI, EXAMINATION. 207

Enzyme formation.

Fermentation of carbohydrates:

In glucose gelatine shake cultivation.
In saccharose gelatine or bouillon.
In lactose gelatine or bouillon.
In maltose gelatine or bouillon.
In glycerine bouillon or bouillon.
Acid formation.
Alkali formation (if present).
Indol formation.
Phenol formation.
Reducing and oxidising agents.
Gas formation.
5. Pathogenicity:

Susceptible animals.

Immune animals.

Experimental inoculation, symptoms of disease.

Post-mortem appearances.

Virulence :

Length of time maintained.
Upon what medium?
Minimal lethal dose.

Is virulence readily exalted and attenuated ?
Toxin formation.

MACROSCOPICAL EXAMINATION OF CULTIVATIONS.

In describing the naked-eye and low-power appear-
ances of the bacterial growth the descriptive terms
introduced by Chester (and included in the following
scheme) should be employed.

Solid Media.

Plate Cultures. —

Gelatine. — Note the presence or absence of lique-
faction of the surrounding medium. If liquefaction
is present, note shape and character (vide page 215,
''stab" cultures).

208 METHODS OF IDENTIFICATION.

A gar. — No liquefaction takes place in this medium.
The liquid found on the surface of the agar (or at the
bottom of the tube in agar tube cultures) is merely
water which has been expressed during solidification
and has subsequently condensed.

Gelatine and Agar. — Examine the colonies at various
intervals —

(a) With the naked eye.

(b) Under a low power (i inch) of the microscope,
or by means of a small dissecting microscope.

Distinguish superficial from deep colonies and note
the characters of the individual colonies.

a b c

Fig. 114. — Types of colonies: a, Cochleate ; 3, amoeboid; c> mycelioid.

(A) Size. — The diameter in millimetres, at the various
ages.

(B) Shape.-

Punctiform : Dimensions too slight for defining form
by naked eye; minute, raised, hemispherical.

Round: Of a more or less circular outline.

Elliptical: Of a more or less oval outline.

Irregular.

Fusiform: Spindle-shaped, tapering at each end.

Cochleate : Spiral or twisted like a snail shell (Fig.
1 14, a).

MACROSCOPICAL EXAMINATION. 2Og

Amoeboid: Very irregular, streaming (Fig. 114, b).

Mycelioid: A filamentous colony, with the radiate
character of a mould (Fig. 114, c).

Filamentous: An irregular mass of loosely woven
filaments (Fig. 115, a).

Floccose: Of a dense woolly structure.

Rhizoid: Of an irregular, branched, root-like char-
acter (Fig. 115, 6).

Conglomerate: An aggregate of colonies of similar
size and form (Fig. 115, c).

a b c d

Fig. 115. — Types of colonies: a, Filamentous; b, rhizoid ; <:, conglomerate;

d, toruloid.

Toruloid: An aggregate of colonies, like the budding
of the yeast plant (Fig. 115, d).

Rosulate: Shaped like a rosette.

i. General Character of Surface as a Whole:

Flat: Thin, leafy, spreading over the surface (Fig.
116, a).

Effused: Spread over the surface as a thin, veily
layer, more delicate than the preceding.

Raised: Growth thick, with abrupt terraced edges
(Fig. 1 1 6, b).

Convex: Surface the segment of a circle, but very
flatly convex (Fig. 116, c).
14

210

METHODS OF IDENTIFICATION.

Pulvinate: Surface the segment of a circle, but de-
cidedly convex (Fig. 116, d).

Capitate : Surface hemispherical
(Fig. 1 1 6, e).

Umbilicate: Having a central pit
or depression (Fig. 116, /).

Umbonate: Having a central
convex nipple-like elevation (Fig.
116, g).

2. Detailed Characters of Surface:
Smooth: Surface even, without
any of the following distinctive char-
acters.

Alveolate: Marked by depressions
separated by thin walls so as to re-
semble a honeycomb (Fig. 117).

Punctate: Dotted with punctures
like pin-pricks.

Bullate: Like a blistered surface,
rising in convex prominences, rather
coarse.

Vesicular: More or less covered
with minute vesicles due to gas
formation; more minute than bul-
late.

Verrucose : Wart-like, bearing wart-like prominences.
Squamose: Scaly, covered
with scales.

Echinate : Beset with
pointed prominences.

Papillate: Beset with nip-
ple or mamma-like pro-
cesses.

Rugose : Short irregular
folds, due to shrinkage of
surface growth.

Corrugated: In. long folds, due to shrinkage.

Fig. 1 1 6. — Surface
elevation of colonies :
a, Flat ; b, raised ;
r, convex ; d, pulvin-
ate ; ^, capitate ; f,
umbilicate ; g, um-
bonate.

Fig. 117. — Types of colonies —
alveolate.

MACROSCOPICAL EXAMINATION.

211

Contoured : An irregular but smoothly undulating
surface, like the surface of a relief map.

Rimose: Abounding in chinks, clefts, or cracks.

(D) Internal Structure of Colony (Microscopical). —

Refraction Weak: Outline and surface of relief not
strongly denned.

Refraction Strong: Outline and surface of relief
strongly defined; dense, not filamentous colonies.

i. General:

Amorphous: Without definite structure, as below
specified.

a b c

Fig. 1 1 8. — Types of colonies: a, Grumose ; £, moruloid ; cy clouded.

Hyaline: Clear and colourless.

Homogeneous: Structure uniform throughout all
parts of the colony.

Homochromous : Colour uniform throughout.

2. Granulations or Blotchings:

Finely granular.

Coarsely granular.

Grumose: Coarser than the preceding, with a
clotted appearance, and particles in clustered grains
(Fig. 1 1 8, a).

Moruloid : Having the character of a mulberry, seg-

212

METHODS OF IDENTIFICATION.

mented, by which the colony is divided in more or
less regular segments (Fig. 118, b).

Clouded: Having a pale ground, with ill-defined
patches of a deeper tint (Fig. 118, c).

3. Colony Marking or Striping:

Reticulate: In the form of a network, like the veins
of a leaf (Fig. 119, a).

a . o c

Fig. 119. — Types of colonies : «, Reticulate; bt gyrose ; c, marmorated.

Areolate: Divided into rather irregular, or angular,
spaces by more or less definite boundaries.

Gyrose: Marked by wavy lines, indefinitely placed
(Fig. 119, b).

Marmorated : Showing faint, irregular stripes, or tra-
versed by vein-like markings, as in
marble (Fig. 119, c).

Rivulose : Marked by lines like the
rivers of a map.

Rimose: Showing chinks, cracks,
or clefts.

4. Filamentous Colonies:
Filamentous: As already defined.
Floccose: Composed of filaments,
densely placed.

Curled: Filaments in parallel strands, like locks or
ringlets, as in agar colonies of B. anthracis.

Fig. 1 20. — Types of
colonies — curled.

MACROSCOPICAL EXAMINATION. 213

(E) Edges of Colonies. —

Entire : Without toothing or division (Fig. 121, a) .

Undulate: Wavy (Fig. 121, b).

Repand : Like the border of an open umbrella (Fig.

121, C).

Erose: As if gnawed, irregularly toothed (Fig.
121, d).

Fig. 121. — Edges of colonies: a, Entire; b, undulate; c, repand ; d, erose.

Lobate.

Lobulate: Minutely lobate (Fig. 122, e).
Auriculate: With ear-like lobes (Fig. 122, /).
Lacerate: Irregularly cleft, as if torn (Fig. 122, g).
Fimbriate: Fringed (Fig. 122, h}.
Ciliate: Hair-like extensions, radiately placed (Fig.
122,7).
Tufted.

Filamentous: As already defined.
Curled: As already defined.

Fig. 122. — Edges of colonies : <?, Lobar-lobulate ; /, auriculate ; g, lacerate ;
^, fimbriate ; /, ciliate.

(F) Optical Characters (after Shuttleworth).—
i. General Characters:
Transparent: Transmitting light.
Vitreous: Transparent and colourless.

214 METHODS OF IDENTIFICATION.

Oleaginous: Transparent and yellow; olive to lin-
seed-oil coloured.

Resinous: Transparent and brown, varnish or resin-
coloured.

Translucent: Faintly transparent.

Porcelaneous : Translucent and white.

Opalescent: Translucent; greyish- white by reflected
light.

• Nacreous: Translucent, greyish-white, with pearly
lustre.

Sebaceous: Translucent, yellowish or greyish- white.

Butyrous: Translucent and yellow.

Ceraceous: Translucent and wax-coloured.

Opaque.

Cretaceous: Opaque and white, chalky.

Dull: Without lustre.

Glistening: Shining.

Fluorescent.

Iridescent.

2. Chromogenicity :

Colour of pigment.

Pigment restricted to colonies.

Pigment restricted to medium surrounding colonies.

Pigment present in colonies and in medium.

Streak or Smear Cultures. —

Gelatine and A gar. — Note general points as indi-
cated under plate cultivations.

Inspissated Blood-serum. — Note the presence or
absence of liquefaction of the medium. (The presence
of condensation water at the bottom of the tube must
not be confounded with liquefaction of the medium.)

All Oblique Tube Cultures.—

1. Colonies Discrete: Size, shape, etc., as for plate
cultivations (vide page 208).

2. Colonies Confluent: Surface elevation and char-
acter of edge, as for plate cultivations (vide page 209).

Chromogenicity: As for plate cultures (supra).

GELATINE STAB CULTURES. 215

Gelatine Stab Cultures.—

(A) Surface Growth. — As for individual colonies in
plate cultures (vide page 209).

(B) Line of Puncture. —

Filiform: Uniform growth, without special char-
acters (Fig. 123, a).

Fig. 123. — Stab cultivations — types of growth : a, Filiform; &, beaded; c,
echinate ; d, villous ; <?, arborescent.

Nodose: Consisting of closely aggregated colonies.
Beaded: Consisting of loosely placed or disjointed
colonies (Fig. 123, b).

Papillate: Beset with papillate extensions.
Echinate : Beset with acicular extensions (Fig. 1 23, c) .

21 6 METHODS OF IDENTIFICATION.

Villous: Beset with short, undivided, hair-like
extensions (Fig. 123, d). .

Plumose: A delicate feathery growth.

Arborescent: Branched or tree-like, beset with
branched hair-like extensions (Fig. 123, e).

Fig. 124. — Stab cultivations — types of growth: /, Crateriform ; g, saccate;
k, infundibuliform ; j, napiform ; k, fusiform ; /, stratiform.

Crateriform: A saucer-shaped liquefaction of the
gelatine (Fig. 124, /).

Saccate: Shape of an elongated sack, tubular,
cylindrical (Fig. 124, g).

Infundibuliform: Shape of a funnel, conical (Fig.
124, h).

UTMUS MILK CULTIVATIONS. 217

Napiform: Shape of a turnip (Fig. 124, ;).

Fusiform: Outline of a parsnip, narrow at either
end, broadest below the surface (Fig. 124, k).

Stratiform: Liquefaction extending to the walls of
the tube and downwards horizontally (Fig. 1 24, /) .

(D) Character of the Liquefied Gelatine. —

1. Pellicle on surface.

2. Uniformly turbid.

3. Granular.

4. Mainly clear, but containing flocculi.

5. Deposit at apex of liquefied portion.

(E) Production of Gas Bubbles.
Shake Cultures.—

1. Presence or absence of liquefaction.

2. Production of gas bubbles.

3. Bulk of growth at the surface — aerobic.

4. Bulk of growth in depths — anaerobic.

Fluid Media.

1. Surface of the Liquid. —

Presence or absence of froth due to gas bubbles.
Presence or absence of pellicle formation.
Character of pellicle.

2. Body of the Liquid.—
Uniformly turbid.
Flocculi in suspension.
Granules in suspension.

Clear, with precipitate at bottom of tube.
Colouration of fluid, presence or absence of.

3. Precipitate. —
Character.
Amount.
Colour.

Litmus Milk Cultivations. —

{Unaltered.
Acid.
Alkaline.

2l8 METHODS OF IDENTIFICATION.

2. Odour.

3. Formation of gas.

c Unaltered.

4. Consistency: < Digested (? character of solution)

I Coagulated.

rA1 ^, ( hard: solid.

5. Clot: Character^ ...

\ soft: flocculent.

(a) Coagulum undissolved.
(6) Coagulum finally digested, completely: in-
completely.

Resulting solution, clear: turbid.
f Abundant.

nru Scanty.

6. Whey: 1 J

Clear.

I Turbid.

BY MICROSCOPICAL METHODS.

Preparations must be made from the cultivations
at intervals of, say, twenty-four hours, during the
period they are under observation, and examined —

(A) Living. — 1. In hanging drop, to determine mo-
tility or non-motility.

In this connection it must be remembered that
under certain conditions as to environment (e. g., cold,
heat, light, unsuitable medium, etc.) motile bacilli
may fail to exhibit activity. No organism, therefore,
should be recorded as non-motile from one observation
only; a series of observations at different ages and
under varying conditions should form the basis of an
opinion as to the absence of true locomotion.

Size. — In the case of non-motile or sluggishly motile
organisms, endeavour to measure several individuals in
each hanging drop by means of the eyepiece microm-
eter, and average the results.

If the organism is one which forms spores, ob-
serve—

BY MICROSCOPICAL METHODS. 2 19

(a) Spore Formation. — Prepare hanging-drop culti-
vations (vide page 69) from vegetative forms of the
organism, adding a trace of magenta solution (0.5 per
cent.) to the drop, on the point of the platinum needle,
to facilitate the observation of the phenomenon by
rendering the bacilli more distinct.

Place the preparation on the stage of the micro-
scope; if necessary, using a warm stage.

Arrange illumination, etc., and select a solitary
bacillus for observation, by the help of the ^-inch
lens.

Substitute the ^-inch oil-immersion lens for the
sixth, and observe the formation of the spore; if
possible, measure any alteration in size which may
occur by means of the Ramsden micrometer.

(b) Spore Germination. — In a similar manner prepare
hanging-drop cultivations from old cultivations in
which no living vegetative forms are present, and
observe the process of germination.

The comfort of the microscopist is largely enhanced
in those cases where the period of observation is at all
lengthy, by the use of some form of eye screen before
the unemployed eye, such as is figured on page 59
(Fig. 41).

If it is impossible to carry out the method suggested
above, proceed as follows:

(a) Spore Formation. — Plant the organism in broth
and incubate under optimum conditions.

At regular intervals, say every thirty minutes, re-
move a loopful of the cultivation and prepare a cover-
slip film preparation.

Fix, while still wet, in the corrosive sublimate fixing
solution.

Stain with aniline gentian violet, and partially de-
colourise with 2 per cent, acetic acid.

Mount and number consecutively.

(b) Spore Germination. — Expose a thick emulsion of

220 METHODS OF IDENTIFICATION.

the spores to a temperature of 80° C. for ten minutes
in the differential steriliser (vide page 202).

Transfer the emulsion to a tube of sterile nutrient
broth and incubate.

Remove specimens from the tube culture at intervals
of, say, five minutes.

Fix, stain, etc., as under (a), and examine.

(B) Fixed. — 2. In stained preparations.

(a) To determine points in morphology:

Shape (vide classification, page in).

Size:

(a) Prepare cover-slip film preparations at the
various ages, and fix by exposure to a temperature
of 115° C. for twenty minutes (vide page 75).

(b) Stain the preparations by Gram's method (if
applicable) or with dilute carbol-fuchsin, and mount
in the usual way.

(c) Measure (vide page 63) some twenty-five indi-
viduals in each film by means of the Ramsden's or
the stage micrometer and average the result.

Pleomorphism : If noted, record —
The predominant character of the variant forms.
On what medium or media they are observed.
(b) To demonstrate details of structure:
Flagella: If noted, record-
Method of staining (vide page 87).
Position and arrangement (vide page 115).
Number.

Spores: If noted, record —
Method of staining.
Shape.
Size.

Position within the parent cell.
Condition, as to shape, of the parent cell (vide
page 74).

On what medium they are best observed.
Age of medium.

BY CHEMICAL METHODS. 221

Conditions of environment as to temperature, atmos-
phere.

Method of germination (vide page 119).

Involution Forms: If noted, record —

Method of staining.

Character (e. g., if living or dead).

Shape.

On what medium they are observed.

Age of medium.

Environment.

Metachromatic Granules: If noted, record —

Method of staining.

Character of granules.

Number of granules.

Colour of granules.

3. Staining Reactions. —

1. Gram's Method. — Decolourised or retain the stain.

2. Neisser's Method. — If granules are noted, record —

1. Position.

2. Number.

3. Ziehl-Neelsen's Method. — Decolourised or acid-
fast.

4. Simple Aniline Dyes. — (Noting those giving the
best results, with times of staining.)

Methylene-blue

Fuchsm aiid the.r modifications>

Gentian violet

Thionine blue

BY CHEMICAL METHODS.

Test cultivations of the organism for the presence of —

Soluble enzymes — proteolytic, diastatic, invertin.

Organic acids — (a) quantitatively — i. e., estimate
the total acid production; (6) qualitatively for
formic, acetic, propionic, butyric, lactic.

Ammonia.

222 METHODS OF IDENTIFICATION.

Alcohol — ethyl alcohol, aldehyde, acetone.

Aromatic products — indol, phenol.

Soluble pigments.

Test the power of reducing (a) colouring matters,
(6) nitrates to nitrites.

Investigate the gas production — H2S, CO2, H. Esti-
mate the ratio between the last two gases.

Prepare all cultivations for these methods of ex-
amination under optimum conditions, previously deter-
mined for each of the organisms it is intended to
investigate, as to

(a) Reaction of medium;
(6) Incubation temperature;
(c) Atmospheric environment.

and keep careful records of these points, and also of
the age of the cultivation used in the final examina-
tion.

Examine the cultivations for the various products
of bacterial metabolism after forty-eight hours' growth,
and never omit to examine " control " (uninoculated)
tube or flask of medium kept for a similar period
under identical conditions. If the results are nega-
tive, test further cultivations at three days, five days,
and ten days.

1. Enzyme Production. —

(A) Proteolytic Enzymes. — (Convert proteids into
peptones and propeptones; e. g., B. pyocyaneus.)

Media Required:

Blood-serum and milk-serum which have been carefully

filtered through a porcelain candle.
Reagents Required:

Ammonium sulphate.

^ caustic soda solution.

Copper sulphate, i per cent, aqueous solution.

METHOD. —

i. Prepare cultivations in bulk (50 c.c.) in a flask
and incubate.

BY CHEMICAL METHODS. 223

2. Add 60 grammes of ammonium sulphate to 40
c.c. of the cultivation, and warm to 50° C. for half
an hour. (This precipitates the proteid bodies.)

3. Filter.

Test the filtrate for propeptones and peptones.
Make the filtrate strongly alkaline with caustic soda.
Add a few drops of copper sulphate solution.
Violet colour = peptones.

(B) Diastatic Enzymes. — (Convert starch into sugar;
e. g., B. subtilis.)

Medium Required:

Inosite-free bouillon.
Reagents Required:

Starch.

Thymol.

Fehling's solution.

METHOD.—

1. Prepare tube cultivation and incubate.

2. Prepare a thin starch paste and add 2 per cent,
thymol to it.

3. Mix equal parts of the cultivation to be tested
and the starch paste, and place in the incubator at
37° C. for six to eight hours.

4. Filter.

Test the filtrate for sugar.

Boil some of the Fehling's solution in a test-tube.

Add the filtrate drop by drop until, if necessary, a
quantity has been added equal in amount to the
Fehling's solution employed, keeping the mixture at
the boiling-point during the process.

Yellow or orange precipitate = sugar.

Medium Required:

Inosite-free bouillon.
Reagents Required:

Cane sugar, 2 per cent, aqueous solution.

Carbolic acid.

224 METHODS OF IDENTIFICATION.

METHOD. —

1. Prepare tube cultivations and incubate.

2. Add 2 per cent, of carbolic acid to the sugar
solution.

3. Mix equal quantities of the carbolised sugar solu-
tion and the cultivation in a test-tube; allow the
mixture to stand for several hours.

4. Filter.

Test the nitrate for glucose as in the preceding
section.

(D) Rennet and "Lab " Enzymes. — (Coagulate milk
independently of the action of acids; e. g., B. pro-
digiosus.)

Media Required:

Inosite-free bouillon.
Litmus milk.

METHOD. —

1. Prepare tube cultivations and incubate.

2. After incubation heat the cultivation to 55° C.
for half an hour, to sterilise.

3. By means of a sterile pipette run 5 c.c. of the
cultivation into each of three tubes of litmus milk.

4. Place in the cold incubator at 22° C. and examine
each day for ten days.

Absence of coagulation at the end of that period
will indicate absence of rennet ferment formation.
2. Acid Production. —
(a) Quantitative. —

Medium Required:

Sugar (glucose) bouillon (vide page 142) of known " opti-
mum " reaction.
Apparatus and Reagents Required:

As for estimating reaction of media (vide page 129).

METHOD.—

1. Prepare cultivation in bulk (100 c.c.) in a flask.

2. After suitable incubation, heat in the steamer at
1 00° C. for thirty minutes to sterilise.

BY CHEMICAL METHODS. 225

3. Determine the litre of the medium as described
in the preparation of nutrient media (vide page 130).

4. The difference between the original titre of the
medium and that now estimated gives the total acid
production in terms of normal NaOH.

NOTE. — If the growth is very heavy it may be a
difficult matter to determine the end-point. The
cultivation should then be filtered through a Berkfeld
filter candle previous to step 2, and the filtrate em-
ployed in the titration.

(b) Qualitative (of all the organic acids present).—

Medium Required:

Sugar (glucose or lactose) bouillon as in quantitative ex-
amination.
Reagents Required:

Hydrochloric acid, concentrated.

Sulphuric acid, concentrated (pure).

Sulphuric acid, 25 per cent, solution.

Ammonia.

Ammonium sulphide.

Baryta water.

Sodium carbonate, saturated aqueous solution.

Absolute alcohol.

Ether.

Calcium chloride.

Zinc oxide.

Permanganate of potash, 4 per cent, aqueous solution.

Schiff's reagent.

Arsenious oxide.

Ferric chloride, 4 per cent, aqueous solution.

Cobalt nitrate, 2 per cent, aqueous solution.

Silver nitrate, i per cent, aqueous solution.

Lugol's iodine (vide page 94).

Cane sugar, 10 per cent, aqueous solution.

Hard paraffin wax (melting-point about 52° C.).

METHOD. —

1. Prepare cultivation in bulk (500 c.c.) in a litre
flask and add sterilised precipitated chalk, 10 grammes.
Incubate at the optimum temperature.

2. After incubation throw a piece of paraffin wax
(about a centimetre cube) into the cultivation and
connect up the flask with a condenser.

226

METHODS OF IDENTIFICATION.

The paraffin, which liquefies and forms a thin
layer on the surface of the fluid, is necessary to pre-
vent the cultivation frothing up and running un-
altered through the condenser during the subsequent
process of distillation.

3. Distil over 200 to 300 c.c.

Use a rose-top burner to minimise the danger of

Fig. 125. — Arrangement of distillation apparatus for acids, etc.

cracking the flask; and to the same end, well agitate
the contents of the flask to prevent the chalk settling.

The distillate "A" will contain alcohol, etc. (vide
page 229); the residue "a" will contain the volatile
and fixed acids.

4. Disconnect the flask and filter the chalk from the

contained residue; add 10 c.c.
acid to the filtrate; mix well.

cone, hydrochloric

BY CHEMICAL METHODS.

227

5. Precipitate the calcium by adding sodium car-
bonate solution, until alkaline.

6. Boil thoroughly (to ensure complete precipitation
of lime) and filter.

7. Add 20 c.c. sulphuric acid (25 per cent.) to the
filtrate (this liberates the volatile acids), and distil
as completely as possible.

DISTILLATE "B."

(Volatile Acids.)

1. Saturate with baryta water to alkalinity,

and evaporate to dryness.

2. Add 20 c.c. absolute alcohol and allow

to stand, with frequent stirring, for
two to three hours.

3. Filter and wash with alcohol.

FILTRATE

may contain barium propionate,
barium butyrate.

1. Evaporate to dryness.

2. Dissolve residue in 150 c.c. water.

3. Saturate with calcium chloride.

4. Distil.

5. Test distillate for butyric acid :

Add 3 c. c. alcohol and 4 drops con-
centrated sulphuric acid.
Smell of pineapple = butyric

acid.

Propionic acid in small quantities can-
not be distinguished from butyric
acid by tests within the scope of
the bacteriological laboratory.

RESIDUE

may contain barium acetate,
barium formate.

1. Evaporate off a cohol and dissolve

up the residue on the filter in hot
water.

2. Divide the solution into four portions :

(a) Add ferric chloride solution.

Brown colour = acetic or for-
mic acids.

(b) Add silver nitrate solution.

White flocculent precipitate
(soluble in hot water and sep-
arating in spangles when
cool) = acetic acid.
Add silver nitrate solu^n ; then
add one drop ammonia water,
and boil.

Black precipitate of metallic
silver = formic acid.

equal quantity of arsenious
oxide and heat on platinum
foil.

Unpleasant smell of cacodyl
= acetic acid.

(d) Add a few drops of mercuric

chloride solution in test-tube,
and heat to 70° C.
Precipitate of mercurous chlo-
ride and the formation, after
a long time, of a metallic
mirror = formic acid.

The distillate "B" may contain acetic, propionic,
formic, or butyric acid.

228

METHODS OF IDENTIFICATION.

The residue "b" may contain lactic, oxalic, succinic,
glycocholic, taurocholic, cholic, benzoic, hippuric,
tannic, or gallic acid.

RESIDUE «b.'
(Fixed Acids.)

1. Evaporate the remainder of the residue

to a syrup.

2. Extract with ether by agitation in a

separatory funnel. (This dissolves
out the fixed organic acids.)

3. 'Evaporate ethereal extract to a syrup.

(No residue = absence of lactic,
oxalic, succinic acids.)

4. Add 100 c.c. water and mix thoroughly.

5. Add excess of zinc oxide and heat nearly

to boiling. Filter.

FILTRATE

(a) Test for cholic acid series :

1. To 6 c.c. filtrate add 4 c.c. cone.

sulphuric acid ; then add i
drop cane sugar solution
and warm to 75° C.
Crimson colour = glyco-
cholic, taurocholic, or
cholic acids.

2. To a portion of the filtrate add

Lugol's iodine.
Blue colour (resembling
that of the starch -iodo
compound) = cholic acid.

(b) Test for lactic acid :

1. Acidify with hydrochloric acid.

2. Add ammonia water in slight ex-

cess and boil off the excess.

3. Add cobalt nitrate solution.

Violet colour (or if in suffi-
cient quantity crystalline
precipitate) = lactic acid.
Or—

1. Evaporate bulk of filtrate to dry-

ness.

2. Dissolve in 10 c.c. hot water.

3. Allow to crystallise — with con-

centration if necessary.
Crystals of zinc lactate =
lactic acid.

( Do not confuse with zinc
sulphate, which will
probably be present
also.)

RESIDUE.

Dissolve in hydrochloric acid from off
the filter.

(a) Test for oxalic acid :

1. Neutralise with ammonia till

faintly alkaline.

2. Add calcium chloride solution.

White precipitate = cal-
cium oxalate. No precipi-
tate = absence of oxalic
acid.

(b) Test for succinic, benzoic, or hip-

puric, salicylic, tannic, or gallic
acid :

1. Neutralise with ammonia and

boil off the excess.

2. Add ferric chloride solution on a

glass rod.

Red»brown colouration or
precipitate = succinic acid.

Buff colouration or precipi-
tate = benzoic or hippuric
acid.

Violet colouration or pre-
cipitate = salicylic acid.

Inky-black colouration or
precipitate = tannic or
gallic acid.

8. Use 50 c.c. of the distillate "B" for titrations.
This will give the amount of volatile acid formation.

BY CHEMICAI, METHODS. 229

3. Ammonia Production. —

Medium Required:

Nutrient bouillon.
Reagent Required:

Nessler reagent.

METHOD.—

1. Prepare cultivation in bulk (100 c.c.) in a 250
c.c. flask and incubate together with a control flask.

Test the cultivation and the control for ammonia
in the following manner:

2. To each flask add 2 grammes of calcined mag-
nesia, then connect up with condensers and distil.

3. Collect 50 c.c. distillate, from each, in a Nessler
glass.

4. Add to each i c.c. Nessler reagent by means of a
clean pipette.

A yellow colour = ammonia.

The depth of colour is proportionate to the amount
present.

4. Alcohol, etc., Production. — Divide the distillate
"A" obtained in the course of a previous experiment
(vide page 226, step 3) into four portions and test for
the production of alcohol, acetaldehyde, acetone.

1. Add Lugol's iodine, then a little NaOH solution,
and stir with a glass rod.

Pale yellow crystalline. precipitate of iodoform, with
its characteristic smell, indicates alcohol, aldehyde,
or acetone.

The precipitate may be absent even when the odour
is pronounced.

2. Add Schiff's reagent.

Violet or red colour = aldehyde.
.3. To 10 c.c. of solution add 5 c.c. cone, sulphuric
acid, and i c.c. of potassium permanganate solution.
After an interval of five minutes add Schiff's reagent.
Red colour (due to oxidation of magenta in reagent)
= aldehyde from alcohol.

230 METHODS OP IDENTIFICATION.

4. Make the solution strongly alkaline with ammonia.
Add gradually a solution of iodine in ammonium
iodide. A black precipitate of nitrogen iodide forms,
which quickly disappears on shaking. As soon as the
precipitate tends to become permanent, it will change
to iodoform if acetone is present.

5. Indol Production. —

Media Required:

Inosite-free bouillon (vide page 141).

Or peptone water (vide page 168).
Reagents Required:

Sulphuric acid, concentrated pure.

Sodium nitrite, o.oi per cent, aqueous solution.

METHOD. —

1. Prepare several test-tube cultivations of the
organism to be tested, and incubate.

Test for indol by means of the nitroso-indol reaction,
in the following manner. (If the culture has been
incubated at 37° C., it must be allowed to cool to the
room temperature before applying the test.)

2. Remove the cotton- wool plug from the tube, and
run in i c.c. sulphuric acid by means of a sterile pipette.
Place the tube upright in a rack, and allow it to stand,
if necessary, for ten minutes.

A rose-pink or red colour = indol (plus a nitrite).

3. If the colour of the medium remains unaltered,
add 2 c.c. sodium nitrite solution, and again allow
the culture to stand for ten minutes.

Red colouration — indol.

NOTE. — In place of performing the test in two
stages as given above, 2 c.c. concentrated commercial
sulphuric, hydrochloric, or nitric acid, all of which
hold a trace of nitrite in solution, may be run into
the cultivation. The development of a red colour
within twenty minutes will indicate the presence of
indol.

BY CHEMICAL METHODS. 231

5a. Phenol Production. —

Medium Required:

Nutrient bouillon.
Reagents Required:

Hydrochloric acid, concentrated.

Millon's reagent.

Ferric chloride, i per cent, aqueous solution.

METHOD. —

1. Prepare cultivation in a Bohemian flask contain-
ing at least 50 c.c. of medium, and incubate.

Test for phenol in the following manner :

2. Add 5 c.c. hydrochloric acid to the cultivation
and connect up the flask with a condenser.

3. Distil over 15 to 20 c.c. Divide the distillate
into two portions.

4. Test one portion by adding 0.5 c.c. Millon's re-
agent and boiling.

A red colour — phenol.

5. Test the other portion by adding about 0.5 c.c.
ferric chloride solution.

A violet colour = phenol.

NOTE. — If both indol and phenol appear to be present
in cultivations of the same organism, it is well to
separate them before testing. This may be done in
the following manner:

1. Prepare inosite-free bouillon cultivation, say 200
or 300 c.c., in a flask as before.

2. Add 50 to 60 c.c. hydrochloric acid and connect
up the flask with a condenser.

3. Distil over 50 to 70 c.c.

Distillate will contain both indol and phenol.

4. Render the distillate strongly alkaline with caustic
potash and redistil.

Distillate will contain indol ; residue will contain
phenol.

5. Test the distillate for indol (vide ante).

232 METHODS OF IDENTIFICATION.

6. Saturate the residue, when cold, with carbon
dioxide and redistil.

7. Test this distillate for phenol (vide ante).

6. Pigment Production. —

1. Prepare tube cultivations upon the various media
and incubate under varying conditions as to tem-
perature (at 37° C. and at 20° C.), atmosphere (aerobic
and anaerobic), and light (exposure to and protection
from).

Note the conditions most favorable to pigment
formation.

2. Note the solubility of the pigment in various
solvents, such as water, hot and cold, alcohol, ether,
chloroform, benzol, carbon bisulphide.

3. Note the effect of acids and alkalies respectively
upon the pigmented cultivation, or upon solutions of
the pigment.

4. Note spectroscopic reactions.

7. Reducing Agent Formation. —

(a) Colour Destruction. —

1. Prepare tube cultivations in nutrient bouillon
tinted with litmus, rosolic acid, neutral red, and
incubate.

2. Examine the cultures each day and note whether
any colour change occurs.

(b) Nitrates to Nitrites.—

Medium Required:

Nitrate bouillon (vide page 143).

Or nitrate water (vide page 169).
Reagents Required:

Sulphuric acid (25 per cent.).

Metaphenylene diamine, 5 per cent, aqueous solution.

METHOD.—

i. Prepare tube cultivations and incubate together
with control tubes (i. e.t uninoculated tubes of the
same medium, placed under identical conditions as to
environment).

BY CHEMICAL METHODS. 233

This precaution is necessary as the medium is liable
to take up nitrites from the atmosphere, and an
opinion as to the absence, of nitrites in the cultiva-
tion is often based upon an equal colouration of the
medium in the control tube.

Test both the culture tube and the control tube
for the presence of nitrites.

2. Add a few drops of sulphuric acid to the medium
in each of the tubes.

3. Then run in 2 or 3 c.c. metaphenylene diamine
into each tube.

A brownish-red colour = nitrites.
The depth of colour is proportionate to the amount
present.

8. Gas Production. —

(A) Carbon Dioxide and ( Hydrogen. —

Apparatus Required:

Fermentation tubes (vide page 24) containing sugar
bouillon (glucose, lactose, etc.)- The medium should be
prepared from inosite-free bouillon (vide page 141).
Reagent Required:
^ caustic soda.

METHOD.—

1. Inoculate the surface of the medium in the bulb
of a fermentation tube and incubate.

2. Mark the level of the fluid in the closed branch
of the fermentation tube, at intervals of twenty-four
hours, and when the evolution of gas has ceased,
measure the length of the column of gas with the
millimetre scale.

Express this column of gas as a percentage of the
entire length of the closed branch.

3. To analyse the gas and to determine roughly
the relative proportions of CO2 and H, proceed as
follows :

Fill the bulb of the fermentation tube with caustic
soda solution.

234 METHODS OF IDENTIFICATION.

Close the mouth of the bulb with a rubber stopper.

Alternately invert and revert the tube six or eight
times, to bring the soda solution into intimate contact
with the gas.

Return the residual gas to the end of the closed
branch, and measure.

The loss in volume of gas = carbon dioxide.

The residual gas = hydrogen.

Transfer gas to the bulb of the tube, and explode it
by applying a lighted taper.

(B) Sulphuretted Hydrogen.—

Media Required:

Peptone iron solution (vide page 168).
Peptone lead solution.

1. Inoculate tubes of media, and incubate together
with control tubes.

2. Examine from day to day, at intervals of twenty-
four hours.

The liberation of the H2S will cause the yellowish-
white precipitate to darken to a brownish-black, or
jet black, the depth of the colour being proportionate to
the amount of sulphuretted hydrogen present.

Quantitative : For exact quantitative analyses of the
gases produced by bacteria from certain media of
definite composition, the methods devised by Pakes
must be employed, as follows:

Apparatus Required:

Bohemian flask (300 to 1500 c.c. capacity) containing
from 100 to 400 c.c. of the medium. The mouth of
the flask is fitted with a perforated rubber stopper, car-
rying an L -shaped piece of glass tubing (the short arm
passing just through the stopper). To the long arm of
the tube is attached a piece of pressure tubing some
8 cm. in length, plugged at its free end with a piece of
cotton- wool. Measure accurately the total capacity of
the flask and exit tube, also the amount of medium con-
tained. Note the difference.

Gas receiver. This is a bell jar of stout glass, 14 cm. high
and 9 cm. in diameter. At its apex a glass tube is fused

BY CHEMICAL METHODS.

235

in. This rises vertically 5 cm., and is then bent at right
angles, the horizontal arm being 10 cm. in length. A
three-way tap is let horizontally into the vertical tube
just above its junction with the bell jar.

An iron cylinder just large enough to contain the bell jar.

About 15 kilos of mercury.

Melted paraffin.

An Orsat-Lunge working with mercury instead of water,
provided with two gas tubes of extra length (capacity
1 20 and 60 c.c. respectively and graduated throughout,
both being water-jacketed) or other gas analysis appa-
ratus, capable of deal-
ing with CO, CO,, O,
H, and N.

METHOD.—

1. Inoculate the me-
dium in the flask in the
usual manner, by means
of a platinum needle,
taking care that the neck
of the flask and the rub-
ber stopper are thor-
oughly heated in the
flame before and after
the operation.

2. Fill the iron cylinder with mercury.

3. Place the bell jar mouth downwards in the
mercury — first seeing that there is free communication
between the interior of the jar and the external air—
and suck up the mercury into the tap; then shut off
the tap.

4. Plug the open end of the three-way tap with
melted wax.

5. Connect up the horizontal arm of the culture
flask with that of the gas receiver by means of the
pressure tubing (after removing the cotton-wool plug
from the rubber tube), as shown in Fig, 127.

6. Give the three-way tap a half turn to open com-
munication between flask and receiver, and seal all

Fig. 126. — Orsat-Lunge gas analysis
apparatus.

236

METHODS OF IDENTIFICATION.

joints by coating with a film of melted wax. When
the tap is turned, the mercury in the receiver will
naturally fall.

7. Place the entire apparatus in the incubator.
(Two hours later, by which time the temperature of
the apparatus is that of the incubator, mark the
height of the mercury on the receiver.)

8. Examine the apparatus from day to day and mark
the level of the mercury in the receiver at intervals
of twenty-four hours.

9. When the evolution of gas has ceased, remove

Fig. 127. — Gas-collecting apparatus.

the apparatus from the incubator; clear out the wax
from the nozzle of the three-way tap (first adjusting
the tap so that no escape of gas shall take place) and
connect it with the Orsat.

10. Remove, say, 100 c.c. of gas from the receiver,
reverse the tap and force it into the culture flask.
Remove 100 c.c. of mixed gases from the culture
flask and replace in the receiver.

Repeat these processes three or four times to ensure
thorough admixture of the contents of flask and re-
ceiver.

BY CHEMICAI, METHODS. 237

1 1 . Now withdraw a sample of the mixed gases into
the Orsat and analyse.

In calculating the results be careful to allow for the
volume of air contained in the flask at the commence-
ment of the experiment.

For the collection of gases formed under anaerobic
conditions a slightly different procedure is adopted:

1. Fix a culture flask (500 c.c. capacity) with a per-
forated rubber stopper carrying an L-shaped piece of
manometer tubing, each arm 5 cm. in length.

2. Prepare a second L-shaped piece of tubing, the
short arm 5 cm. and the long arm 20 cm., and connect
its short arm to the horizontal arm of the tube in the
culture flask by means of a length of pressure tubing,
provided with a screw clamp.

3. Fill the culture flask completely with boiling
medium and pass the long piece of tubing through the
plug of an Erlenmeyer flask (150 c.c. capacity) which
contains 100 c.c. of the same medium.

4. Sterilise these coupled flasks by the discontinuous
method, in the usual manner.

Immediately the last sterilisation is completed,
screw up the clamp on the pressure tubing which con-
nects them, and allow them to cool.

As the fluid cools and contracts it leaves a vacuum
in the neck of the flask below the rubber stopper.

5. To inoculate the culture flask, withdraw the long
arm of the bent tube from the Erlenmeyer flask and
pass it to the bottom of a test-tube containing a young
cultivation (in a fluid medium similar to that con-
tained in the culture flask) of the organism it is desired
to investigate.

6. Slightly release the clamp on the pressure tubing
to allow 4 or 5 c.c. of the culture to enter the flask.

7. Clamp the rubber tube tightly; remove the bent
glass tube from the culture tube and plunge it into a

238 METHODS OF IDENTIFICATION.

flask containing recently boiled and quickly cooled dis-
tilled water.

8. Release the clamp again and wash in the remains
of the cultivation until the culture flask and tubing
are completely filled with water.

9. Clamp the rubber tubing tightly and take away
the long-armed glass tubing.

10. Prepare the gas receiver as in the previous
method (in this case, however, the mercury should be
warmed slightly) and fill the horizontal arm of the
receiver with hot water.

1 1 . Connect up the culture flask with the horizontal
arm of the gas receiver.

12. Remove the screw clamp from the rubber tubing,
adjust the three-way tap, seal all joints with melted
wax, and incubate.

13. Complete the investigation as described for the
previous method.

BY PHYSICAL METHODS,

Examine cultivations of the organism with reference
to the following points:
Atmosphere :

(a) In the presence of oxygen.

(b) In the absence of oxygen.

(a) Range.

(b) Optimum.

Moist: Vegetative forms.

Spores.
Dry: Vegetative forms.

Spores.

Reaction of medium.
Resistance to lethal agents:
(a) Desiccation.

BY PHYSICAL METHODS. 239

(b) Light: Diffuse.

Direct.
Primary colours.

(d) Chemical antiseptics and disinfectants.
Vitality in artificial cultures.
Agglutination reaction.

I. Atmosphere. — The question as to whether the
organism under observation is (a) an obligate aerobe,
(b) a facultative anaerobe, or (c) an obligate anaerobe
is roughly decided by the appearance of cultivations
in the fermentation tubes. Obvious growth in the
closed branch as well as in the bulb will indicate that
it is a facultative anaerobe; whilst growth only occur-
ring in the bulb or in the closed branch shows that it
is an obligate aerobe or anaerobe respectively. This
method, however, is not sufficiently accurate for the
present purpose, and the examination of an organism
with respect to its behaviour in the absence of oxygen
is carried out as follows:

Apparatus Required:

Buchner's tubes.

Bulloch's apparatus.

Exhaust pump.

Pyrogallic acid.

Dekanormal caustic soda.
Media Required:

Glucose formate agar.

Glucose formate gelatine.

Glucose formate bouillon.

METHOD. —

i. Prepare four sets of cultivations:

(A) Oblique glucose formate agar and incubate
aerobically at 37° C.

Oblique glucose formate gelatine and incubate
aerobically at 20° C.

(B) Oblique glucose agar and incubate anaerobically
at 37° C.

240 METHODS OF IDENTIFICATION.

Oblique glucose formate gelatine and incubate
anaerobically at 20° C.

Glucose formate bouillon and incubate anaerobi-
cally at 37° C.

(D) Oblique glucose formate gelatine and incubate
anaerobically at 20° C.

Glucose formate bouillon and incubate anaerobi-
cally at 20° C.

2. Seal the cultures forming set B in Buchner's
tubes (vide page 189).

3. Seal the cultures forming sets C and D in Bulloch's
apparatus; exhaust the air by means of a vacuum
pump, and provide for the absorption of any residual
oxygen by the introduction of pyrogallic acid and
caustic soda solution (vide page 194).

4. Observe the cultivations macroscopically and
microscopically at intervals of twenty-four hours until
the completion, if necessary, of seven days' incubation.

5. Control these results.
Gases Other than Oxygen.—

Apparatus Required:
Bulloch's apparatus.
Sterile gas filter (vide page 43).
Gasometer containing the gas it is desired to test or gas

generator for the production of SO2, N2O, NO, CO2,

coal gas, etc.

METHOD. —

1. Prepare tube cultivations upon the surface of
solid media and deposit them in Bulloch's apparatus.

2. Connect up the inlet tube of the Bulloch's jar
with the sterile gas filter, and this again with the
delivery tube of the gasometer or gas generator.

3. Open both stop-cocks of the Bulloch's apparatus
and pass the gas through until it has completely re-
placed the air in the bell jar.

BY PHYSICAL METHODS. 24!

4. Incubate under optimum conditions as to tem-
perature.

5. Examine the cultivations at intervals of twenty-
four hours, until the completion of seven days.

6. Remove one tube from the interior of the appa-
ratus each day. If no growth is visible, incubate the
tube under optimum conditions as to temperature
and atmosphere, and in this way determine the length
of exposure to the action of the gas necessary to kill
the organisms under observation.

7. Control these results.
II. Temperature. —

(A) Range.—

1. Prepare a series of ten tube cultivations, in fluid
media, of optimum reaction.

2. Arrange a series of incubators at fixed tempera-
ture, varying 5° C. and including temperatures between
5° C. and 50° C.

(Or utilise the water-bath employed in testing the
thermal death-point of vegetative forms.)

3. Incubate one tube cultivation of the organism
aerobically or anaerobically, as may be necessary, in
each incubator, and examine at half-hour intervals
for from five to eighteen hours.

4. Note that temperature at which growth is first
observed macroscopically.

5. Continue the incubation until the completion of
seven days. Note the extremes of temperature at
which growth takes place (Range of temperature).

6. Control these results.

(B) Optimum. —

1. Prepare a second series of ten tube cultivations
under similar conditions as to atmosphere and reaction
of medium.

2. Incubate in a series of incubators in which the
temperature is regulated at intervals of i° C. for five

242

METHODS OF IDENTIFICATION.

degrees on either side of optimum temperature ob-
served in the previous experiment, step 4.

3. Observe again at half -hour intervals and note
that temperature at which growth is first visible to
the naked eye = Optimum temperature.

Moist — Vegetative Forms:

Apparatus Required :

Water-bath. For the purpose of observing the thermal
death-point a special water-bath is necessary. The
temperature of this piece of apparatus is controlled by

-^-To water
blower.

Fig. 128. — Hearson's water-bath.

means of a capsule regulator and can be regulated for
intervals of half a degree centigrade through a range of
30°, from 50° C. to 80° C. by means of a spring, actuated
by the handle a, which increases the pressure in the
interior of the capsule. A hole is provided for the
reception of the nozzle of a blast pump, so that a cur-
rent of air may be blown through the water whilst the
bath is in use, and thus ensure a uniform temperature
of its contents.

BY PHYSICAL METHODS. 243

Sterile capsules.

Flask containing 250 c.c. sterile normal saline solution.
Case of sterile pipettes, 10 c.c. (in tenths of a cubic centi-
metre).
Special loop.
Test-tubes, 18 by 1.5 cm., of thin German glass.

METHOD. —

1. Prepare several tube cultivations on solid media
of optimum reaction and incubate for forty-eight hours
under optimum conditions as to temperature and
atmosphere.

2. Examine preparations from the cultivation micro-
scopically to determine the absence of spores.

3. Pipette 5 c.c. salt solution into each of twelve
capsules.

4. Suspend three loopfuls of the growth from the
surface of the medium (using a carefully made platinum
loop especially reserved for this purpose) in the normal
saline solution in each capsule.

5. Transfer each suspension to a sterile test-tube
and number consecutively from i to 12.

6. Adjust the first tube in the water-bath, regulated
at 40° C., by means of two rubber rings around the
tube, one above and the other below the perforated
top of the bath, so that the upper level of the fluid
in the tube is about 4 cm. below the surface of the
water in the bath, and the bottom of the tube is a
similar distance above the bottom of the bath.

7. Arrange a control test-tube containing 5 c.c.
water under similar conditions. Plug the tube with
cotton- wool and pass a thermometer through the plug
so that its bulb is immersed in the water.

8. Close the unoccupied perforations in the lid of
the water-bath by means of glass balls.

9. Watch the thermometer in the test-tube until it
records a temperature of 40° C. Note the time.
Ten minutes later remove the tube containing the sus-

244 METHODS OF IDENTIFICATION.

pension, and cool rapidly by immersing its lower
end in a stream of running water.

10. Pour three gelatine (or agar) plates containing
respectively 0.2, 0.3, and 0.5 c.c. of the suspension,
and incubate.

11. Pipette the remaining 4 c.c. of the suspension
into a culture flask containing 250 c.c. of nutrient
bouillon, and incubate.

12. Observe these cultivations from day to day.
"No growth" must not be recorded as final until after
the completion of seven days' incubation.

13. Extend these observations to the remaining
tubes of the series, but varying the conditions so that
each tube is exposed to a temperature 2° C. higher
than the immediately preceding one — i. e., 42° C., 44°
C., 46° C., and so on.

14. Note that temperature, after exposure to which
no growth takes place up to the end of seven days'
incubation, = the thermal death-point.

15. If greater accuracy is desired, a second series
of tubes may be prepared and exposed for ten minutes
to fixed temperatures varying only 0.5° C., through
a range of 5° C. on either side of the previously observed
death-point.

Moist — Spores : The thermal death-point in the case
of spores is that time exposure to a temperature of
1 00° C. necessary to effect the death of all the spores
present in a suspension.

It is determined in the following manner

Apparatus Required:

Steam can be fitted with a delivery tube and a large bore

safety-valve tube.
Water-bath at 100° C.

Brlenmeyer flask, 500 c.c. capacity, containing 140 c.c.
sterile normal saline solution and fitted with rubber
stopper perforated with four holes.
The rubber stopper is fitted as follows :

(a) Thermometer to 120° C., its bulb immersed in the
normal saline.

BY PHYSICAL METHODS.

245

(b) Straight entry tube, reaching to the bottom of

the flask, the upper end plugged with cotton-
wool.

by means of rubber tubing and fitted with
pinch-cock.

The nozzle is protected from accidental contami-
nation by passing it through the cotton-wool
plug of a small test-tube.

Fig. 129. — Apparatus arranged for the determination of the death-point of

spores.

(d) A sickle-shaped piece of glass tubing passing
just through the stopper, plugged with cotton-
wool, to act as a vent for the steam.
Sterile plates.
Sterile pipettes.

Sterile test-tubes graduated to 5 c.c.
Media Required:
Gelatine or agar.
Culture flasks containing 200 c.c. nutrient bouillon.

246 METHODS OF IDENTIFICATION.

METHOD. —

1. Prepare twelve tube cultivations upon the sur-
face (or two cultures in large flat culture bottles—
-vide page 19) of nutrient agar and incubate under
the optimum conditions (previously determined), for
the formation of spores.

Examine preparations from the cultures micro-
scopically to determine the presence of spores.

2. Pipette 5 c.c. sterile normal saline into each cul-
ture tube or 30 c.c. into each bottle and by means of a
sterile platinum spatula emulsify the entire surface
growth with the solution.

3. Add the 60 c.c. emulsion to 140 c.c. normal saline
contained in the fitted Erlenmeyer flask.

4. Place the flask in the water-bath of boiling water.

5. Connect up the straight tube, after removing the
cotton- wool plug, with the delivery tube of the steam
can; remove the plug from the vent tube.

6. When the thermometer reaches 100° C., syphon
off 5 c.c. of the suspension into the sterile gradu-
ated test-tube and pour plates and prepare flask cul-
tures as in the previous experiments.

7. Repeat this process at intervals of twenty-five
minutes' steaming.

8. Control these experiments, but in this instance
syphon off portions of the suspension at intervals of
one-half or one minute during the five or ten minutes
preceding the previously determined death-point.

Thermal Death-point. —
Dry — Vegetative Forms:

Apparatus Required:

Hot-air oven, provided with thermo-regulator.
Sterile cover-slips.

Flask containing 250 c.c. sterile normal saline solution.
Case of sterile pipettes, 10 c.c. (in tenths of a cubic centi-
metre).

Case of sterile capsules.
Crucible tongs.

BY PHYSICAL METHODS. 247

METHOD.—

1. Prepare an emulsion with three loopfuls from an
optimum cultivation in 5 c.c. normal saline in a ster-
ile capsule and examine microscopically to determine
the absence of spore forms.

2. Make twelve cover-slip films on sterile cover-slips;
place each in a sterile capsule to dry.

3. Expose each capsule in turn in the hot-air oven
for ten minutes to a different fixed temperature, vary-
ing 5° C. between 60° C. and 120° C.

4. Remove each capsule from the oven with crucible
tongs immediately the ten minutes are completed;
remove the cover-glass from its interior with a sterile
pair of forceps.

5. Deposit the film in a flask containing 200 c.c.
nutrient bouillon.

6. Prepare subcultivations from such flasks as show
evidence of growth, to determine that no contamination
has taken place.

7. Control the result of these experiments.
Dry — Spores :

Apparatus Required:
As for vegetative forms.

METHOD.—

1. Prepare an oblique agar tube cultivation and in-
cubate under optimum conditions as to spore forma-
tions.

2. Pipette 5 c.c. sterile normal saline into the culture
tube and emulsify the entire surface growth in it. Ex-
amine microscopically to determine the presence of
spores in large numbers.

3. Spread thin even films on twelve sterile cover-
slips and place each cover-slip in a separate sterile
capsule.

4. Expose each capsule in turn for ten minutes to a
different fixed temperature, varying 5° C., between
100° C. and 160° C.

248 METHODS OF IDENTIFICATION.

5. Complete the examination as for vegetative forms.
III. Reaction of Medium.—

(A) Range. —

1 . Prepare a bouillon culture of the organism and in-
cubate, under optimum conditions as to temperature
and atmosphere, for twenty-four hours.

2. Pipette o.i c.c. of the cultivation into a sterile
capsule; add 9.9 c.c. sterile bouillon and mix thor-
oughly.

3. Prepare a series of tubes of nutrient bouillon of
varying reactions, from +25 to — 30 (vide page 133),
viz.: +25, +20, +15, +10, +5, neutral, — 5, — 10,
—15, —20, —25, —30.

4. Inoculate each of the bouillon tubes with o.i
c.c. of the diluted cultivation and incubate under opti-
mum conditions.

5. Observe the cultures at half -hourly intervals
from the third to the twelfth hours. Note the reaction
of the tube or tubes in which growth is first visible
macroscopically (probably optimum reaction).

6. Continue the incubation until the completion of
forty-eight hours. Note the extremes of acidity and
alkalinity in which macroscopical growth has developed
(Range of reaction) .

7. Control the result of these observations.

(B) Optimum Reaction. — The optimum reaction has
already been roughly determined whilst observing the
range. It can be fixed within narrower limits by
inoculating in a similar manner a series of tubes of
bouillon which have a smaller variation in reaction than
those previously employed (say, i instead of 5) for five
points on either side of the previously observed opti-
mum. For example, optimum reaction in the set of
experiments to determine the range was + 10. Now
plant tubes having reactions of +15, +14, +13, +12,
+ 11, +10, +9, +8, + 7, +6, +5, and observe as
before.

BY PHYSICAL METHODS. 249

IV. Resistance to Lethal Agents. —

(A) Desiccation. —

Apparatus Required:

Miiller's desiccator. This consists of a bell glass fitted
with an exhaust tube and stop-cock (d), which can be
secured to a plate-glass base (c) by means of wax or
grease. It contains a cylindrical vessel of porous clay
(a) into which pure sulphuric acid is poured whilst the
material to be dried is placed within its walls on a
glass shelf (b). The air is exhausted from the interior
and the acid rapidly converts the clay vessel into a
large absorbing surface.

^ <*

Fig. 130. — Muller's desiccator.

Exhaust pump.

Pure concentrated sulphuric acid.

Sterile cover-slips.

Sterile forceps.

Culture flask containing 200 c.c. nutrient bouillon.

Sterile ventilated Petri dish. This is prepared by bending
three short pieces of aluminium wire into V shape and
hanging these on the edge of the lower dish and resting
the lid upon them (Fig. 131).

250 METHODS OF IDENTIFICATION.

METHOD. —

1. Prepare a surface cultivation on nutrient agar in
a culture bottle and incubate under optimum condi-
tions for forty-eight hours.

2. Examine preparations from the cultivation,
microscopically, to determine the absence of spores.

3. Pipette 5 c.c. sterile nor-
mal saline solution into the
flask and suspend the entire
growth in it.

4- Spread the suspension in
thin, even films on sterile
cover-slips and deposit inside sterile " plates" to dry.

5. As soon as dry, transfer the cover-slip films to the
ventilated Petri dish by means of sterile forceps.

6. Place the Petri dish inside the M tiller's desiccator;
fill the upper chamber with pure sulphuric acid, cover
with the bell jar, and exhaust the air from its interior.

7. At intervals of five hours admit air to the appara-
tus, remove one of the cover-slip films from the Petri
dish, and transfer it to the interior of a culture flask,
with every precaution against contamination. Re-
seal the desiccator and exhaust.

8. Incubate the culture flask under optimum condi-
tions until the completion of seven days, if necessary.

9. Pour plates from those culture flasks which grow,
to determine the absence of contamination.

10. Repeat these observations at hourly intervals for
the five hours preceding and succeeding the death
time, as determined in the first set of experiments.

(B) Light.—

(a) Diffuse Daylight:

1. Prepare a tube cultivation in nutrient bouillon,
and incubate under optimum conditions, for forty-eight
hours.

2. Pour twenty plate cultivations, ten of nutrient

BY PHYSICAL METHODS. 251

gelatine and ten of nutrient agar, each containing o.i
c.c. of the bouillon culture.

3. Place one agar plate and one gelatine plate into
the hot and cold incubators, respectively, as controls.

4. Fasten a piece of black paper, cut the shape of a
cross or star, on the centre of the cover of each of the
remaining plates (Fig. 132).

5. Expose these plates to the action of diffuse day-
light (not direct sunlight) in the laboratory for one,
two, three, four, five,

six, eight, ten, twelve
hours.

6. After exposure
to light, incubate un-
der optimum condi-
tions.

7. Examine the
plate cultivations af-

. 1 Fig. 132. — Plate with star for testing effect

ter twenty-four and Of iight.

forty-eight hours' in-
cubation, and compare with the two controls. Record
results. If growth is absent from that portion of the
plate unprotected by the black paper, continue the incu-
bation and daily observation until the end of seven days.

8. Control the results.
(b) Direct Sunlight:

1. Prepare plate cultivations precisely as in the
former experiments and place the two controls in the
incubators.

2. Arrange the remaining plates upon a platform in
the direct rays of the sun.

3. On the top of each plate stand a small glass dish
14 cm. in diameter and 5 cm. deep.

4. Fill a solution of potash alum (2 per cent, in dis-
tilled water) into each dish to the depth of 2 cm. to
absorb the heat of the sun's rays and so eliminate possi-
ble effects of temperature on the cultivations.

252 METHODS OF IDENTIFICATION.

5. After exposures for periods similar to those em-
ployed in the preceding experiment, incubate and com-
plete the observation as above.

1. Prepare plate cultivations, as in the previous
"light" experiments, and incubate controls.

2. Fasten a strip of black paper, 3 cm. wide, across
one diameter of the cover of each plate.

3. Coat the remainder of the surface of the cover
with a film of pure photographic collodion which con-
tains 2 per cent, of either of the following aniline dyes,
as may be necessary:

Chrysoidin (for red) .
Aurantia (for orange).
Naples yellow (for yellow).
Malachite green (for green).
Eosin, bluish (for blue).
Methyl violet (for violet).

4. Expose the plates, thus prepared, to bright day-
light (but not direct sunlight) for varying periods, and
complete the observations as in the preceding experi-
ments.

5. Control the results.

(D) Antiseptics and Disinfectants. — (Vide Testing
Germicides, page 359.) Testing the organism under
observation against, for example,

Bichloride of mercury;

Formaldehyde ;

Carbolic acid;

noting (a) strength of solution; (b) duration of ex-
posure necessary to produce death.

The Agglutination Reaction. — This test, which is
variously known as the agglutination reaction, clump-
ing reaction, or Gruber's reaction, depends upon the fact
that the blood-serum of an animal immunised against

BY PHYSICAL METHODS. 253

a certain micro-organism possesses the power of agglu-
tinating (or collecting together in clumps and masses)
watery suspensions of that particular microbe. It was
first applied by Durham and Gruber in the identifica-
tion of races of cholera vibrios, and was afterwards
extended by various workers to other species of bac-
teria, such as B. typhosus, B. pestis, M. melitensis,
etc.

The converse of the test — viz., the diagnosis of dis-
ease by the determination of the particular pathogenic
organism that is agglutinated by the blood-serum of
the patient — is often termed Widal's reaction, and is
extensively used in the diagnosis of enteric fever, when
the serum from a suspected case of typhoid is tested
against a bouillon cultivation of an authentic Bacillus
typhosus.

It is now generally agreed that the reaction is unre-
liable unless performed under certain conditions.

1. As to the period of time the suspension of the
organism is in contact with specific serum. This must
not exceed thirty minutes.

2. As to the strength of the solution of specific serum
employed in the test. This must not exceed 5 per cent.

The method of employing the test and the prepara-
tion of the serum solution are best considered sepa-
rately, and the test itself illustrated by an example such
as the confirmation of the identity of a bacillus pro-
visionally regarded as the B. typhosus.

(A) Preparation.—

Collection of the Specific Serum:

Apparatus Required:
Razor.
Liquid soap.
Cotton-wool.

Two per cent, lysol solution in drop bottle.
Absolute alcohol in drop bottle.
Hare-lip pin or pointed scalpel.
Blood pipette (vide page 22).

254 METHODS OF IDENTIFICATION.

METHOD. —

1 . Select a rabbit which has been immunised to the B.
typhi abdominalis, and have it firmly held by an assist-
ant.

2. Shave the dorsal surface of the ear, over the poste-
rior auricular vein.

NOTE. — The serum may also be obtained from the
lobe of the ear of a patient suffering or convalescent
from enteric fever, by carrying out the succeeding
steps (see Fig. 133).

3. Sterilise the skin by washing with lysol.

The lysol should be applied with sterile cotton-wool

Fig. 133. — Collecting blood.

and the ear vigourously rubbed, not only to remove
superficial scales of epithelium, but also to render the
ear hyperaemic and the vein prominent.

4. Remove the lysol with absolute alcohol.

5. Dry the sterilised area of skin with sterile cotton-
wool.

6. Puncture the vein with the sterile hare-lip pin
and collect the issuing blood in the blood pipette, thus :

Hold one of the narrow .tube-ends in contact with
the blood and depress the other end. The blood will
run into the pipette by gravity. When the tube is full
to the shoulder, remove the pipette, place the clean end

BY PHYSICAL, METHODS.

255

to the lips, and aspirate gently, so that the blood flows
into the barrel of the pipette.

Hold the pipette horizontally, and seal the ends in a
Bunsen flame.

Rest the pipette in the horizontal position, by its
ends on the rim of a tumbler or beaker, so that its bar-
rel is suspended, and allow the blood to coagulate
(Fig. 134, a). This will probably take about twenty
to thirty minutes.

Place the pipette in the vertical position, clean end
downwards, in a beaker or wire stand, and set it in

Fig. 134. — Collecting serum : a, formation of clot ; l>, separation of serum.

the ice-chest for an hour or so, for the clear serum to
separate and collect in the clean end of the pipette
(Fig. 134, b).

Dilution of the Specific Serum:

Apparatus Required:

Sterile graduated capillary pipettes (to contain 10 c.mm.).

Sterile graduated capillary pipettes (to contain 90 c.mm.).

Small sterile test-tubes (5 cm. by 0.5 cm.).

Tube of nutrient bouillon.

Pipette of specific serum.

Three-square file.

Glass capsule, nearly full of dry silver sand.

Grease pencil.

256 METHODS OF IDENTIFICATION.

METHOD. —

1. Take three sterile test-tubes and number them i,
2, and 3.

2. Pipette 90 c.mm. sterile bouillon into each tube,
and stand it upright in the sand in the capsule.

3. Make a file scratch on the blood pipette above the
upper level of .the clear serum, and snap off the narrow
tube containing the serum.

4. Remove 10 c.mm. of the serum from the blood
pipette tube, and mix it thoroughly with the bouillon
in tube No. i ; = specific serum solution, 10 per cent.

5. Remove 10 c.mm. of the solution from tube No. i
by means of a fresh pipette, and mix it with the con-
tents of tube No. 2; = specific serum solution, i per
cent.

6. Remove 10 c.mm. of the solution from tube No. 2
by means of a fresh pipette, and mix it with the con-
tents of tube No. 3 ; — specific serum solution, o. i per
cent.

(B) Application. —

The Microscopical Reaction:

Apparatus Required:

Five hanging-drop slides (or preferably two slides, with

two cells mounted side by side on each (Fig. 47, a),

and one slide with one cell only).
Vaseline.
Cover-slips.
Platinum loop.
Grease pencil.
Eighteen- to twenty-four-hour-old bouillon cultivation of

the organism to be tested (Bacillus typhi abdominalis?).
Pipette end with the remainder of the undiluted serum.
Tubes containing the three solutions of the specific serum,

10, i, and o.i per cent, respectively.

METHOD. —

i. Make five hanging-drop preparations, thus:
(a) One loopful of bouillon cultivation -f one loopful
sterile bouillon; label "Control."

BY PHYSICAL METHODS. 257

(b) One loopful culture + one loopful undiluted
serum; label 50 per cent.

Mount these two cover-slips on a double-celled slide.

Mount this on single-cell slide.

(d) One loopful bouillon culture + one loopful i
per cent, serum; label 0.5 per cent.

(e) One loopful bouillon culture + one loopful o.i
per cent, serum; label 0.05 per cent.

Mount these two cover-slips on a double-celled
slide.

2. Note the time: Examine the control to deter-
mine that the bacilli are motile and uniformly scattered
over the field — not collected into masses.

3. Next examine the 50 per cent, preparation.

If the test is giving a positive reaction, the bacilli
•will be collected in large clumps.

If the test is giving a negative reaction, the bacilli
'may be collected in large clumps.

4. Observe the 5 per cent, preparation microscopi-
cally.

If the bacilli are aggregated into clumps, positive re-
action.

If the bacilli are not aggregated into clumps, observe
until thirty minutes from the time of preparation be-
fore recording a negative reaction.

5. Examine the 0.5 and 0.05 per cent, preparations^
These may or may not show agglutination when the-
result of the examination of the 5 per cent, prepara-
tion is positive, according to the potency of the specific:
serum; and by dilutions such as these a quantitative:
comparison of the valency of specific sera may be ob-
tained.

NOTE. — The graduated pipettes supplied with
Thoma's haematocytometer (intended for the collection
of the specimen of blood required for the enumeration
17

258 METHODS OF IDENTIFICATION.

of leucocytes), giving a dilution of i in 10, — i. e., 10 per
cent., — may be substituted for the graduated capillary
pipettes referred to above, if the vessel in which the
serum has been separated is of sufficiently large diam-
eter to permit of their use.

A handy, though somewhat crude, method of apply-
ing this microscopical test is carried out as follows:

Apparatus Required:

Pipette containing immune serum.

Eighteen- to twenty-four-hour-old broth cultivation of the

organism to be tested.
Tube of sterile broth (or sterile water).
Cover-slips.
Platinum loop.
Hanging-drop slides.
Vaseline.
Grease pencil.

METHOD. —

1. Flame a clean cover-slip and rest it on a piece of
blotting paper.

2. By means of a sterile platinum loop place nine
as nearly as possible equal loopfuls of sterile broth
(or sterile water) on the surface of the cover-slip.

3. Sterilise the loop, fill it once with the serum to
be tested, and mix thoroughly with the nine drops
of diluent. This gives approximately a 10 per cent,
solution of serum.

4. Flame a second clean cover-slip and place it by
the side of the first.

5. Sterilise the loop, charge it with the serum solu-
tion, and deposit a drop on the surface of the second
cover-slip.

6. Sterilise the loop, and add a loopful of the broth
cultivation to the drop of diluted serum, and mix
thoroughly.

7. Mount the cover-slip as a hanging drop, label
5 per cent., and examine microscopically.

Higher dilutions can be prepared in a similar man-
ner.

PATHOGENESIS. 259

The Macroscopical Reaction :

Apparatus Required:

Sterile graduated capillary pipettes to contain 90 c.mm.

Eighteen- to twenty-four-hour-old bouillon cultivation of
the organism to be tested.

Three test-tubes containing the 10, i, and o.i per cent, so-
lutions of specific serum (about 90 c.mm. remaining in
each).

Sedimentation tubes ('vufepage 23).

METHOD.—

1. Pipette 90 c.mm. of the bouillon culture into each
of the tubes containing the diluted serum.

2. Fill a sedimentation tube (by aspirating) from
the contents of each tube. Seal off the lower ends of
the sedimentation tubes in the Bunsen flame.

3. Label each tube with the dilution of serum that it
contains — viz., 5, 0.5, and 0.05 per cent.

4. Place the pipettes in a vertical position, in a
beaker, in the incubator at 37° C., for one or two
hours.

5. Observe the granular precipitate which is thrown
down when the reaction is positive, and the uniform
turbidity of the negative reaction.

PATHOGENESIS.

i. Living Bacteria. —

(a) Psychrophilic Bacteria: When the organism will
only grow at or below 18° to 20° C.,

1. Prepare cultivations in nutrient broth and in-
cubate under optimum conditions.

2. After seven days' incubation inject that amount
of the culture corresponding to i per cent, of the
body- weight of a selected frog, into its dorsal lymph
sac.

3. Observe until death takes place, or, in the event
of a negative result, until the completion of twenty-
eight days.

4. If death .occurs, make a careful post-mortem
examination (vide page 287).

260 METHODS OF IDENTIFICATION.

(b) Mesophilic Bacteria: When the organism grows
at 35° to 37° C.,

1 . Prepare cultivations in nutrient broth and incu-
bate under optimum conditions for forty-eight hours.

2. Inoculate a selected white mouse, subcutaneously
at the root -of the tail, with an amount of cultivation
equivalent to i per cent, of its body-weight.

3. Inoculate a second mouse intraperitoneally with
a similar dose.

4. Observe carefully until death occurs, or until the
lapse of twenty-eight days.

5. If the inoculated animals succumb, make com-
plete post-mortem examination.

If death follows shortly after the injection of cul-
tivations of bacteria, the inoculation experiments
should be repeated two or three times. Then, if the
organism under observation invariably exhibits patho-
genic effects, steps should be taken to ascertain, if
possible, the minimal lethal dose (as described on
page 269) of the growth upon solid media for the frog
or white mouse respectively. Other experimental
animals — e. g., the white rat, guinea-pig, and rabbit —
should next be tested in a similar manner.

2. Toxins. — Prepare cultivations of the organism
under observation in glucose formate broth, and in-
cubate for fourteen days under optimum conditions.

(a) Intracellular or Insoluble Toxins:

1. Heat the fluid culture in a water-bath at 60° C.
for twenty minutes. (The resulting sterile, turbid
fluid is often spoken of as " killed " culture.)

2. Inject subcutaneously that amount of the culti-
vation corresponding to i per cent, of the body- weight
of the selected animal, usually one of the small rodents.

3. Inoculate a tube of sterile bouillon with a similar
quantity, and inoculate under optimum conditions.
This " control" then serves to demonstrate the freedom
of the toxin from living bacteria.

PATHOGENESIS.

26l

4. Observe during life or until the completion of
twenty-eight days, and in the event of death occurring
during that period, make a complete post-mortem ex-
amination.

5. Repeat the experiment at least once. In the
event of a positive result estimate the minimal lethal
dose of "killed" culture for each of the species of
animals experimented upon.

(b) Extracellular or Soluble Toxins:

i. Filter the cultivation through a porcelain filter
candle (Berkfeld) into a sterile filter flask, arranging
the apparatus as in the accompanying figure (Fig. 135).

Fig. 135. — Apparatus arranged for toxin filtration.

2. Inoculate mice, rats, guinea-pigs, and rabbits
subcutaneously with that quantity of toxin corre-
sponding to i per cent, of the body-weight of each
respectively, and observe, if necessary, until the com-
pletion of one month.

3. Inoculate a "control" tube of bouillon with a
similar quantity and incubate.

4. In the event of a fatal termination make com-
plete and careful post-mortem examinations.

5. Repeat the experiments and, if the results are
positive, ascertain the minimal lethal dose of toxin
for each of the susceptible animals.

XV. EXPERIMENTAL INOCULATION OF
ANIMALS.

THE animals generally employed in the study of
the pathogenic properties of the various micro-organ-
isms are:

Mouse.
Rat.

Guinea-pig.
Rabbit.
Pigeon.
Fowl.

Preparation. — Before inoculation, the experimental
animals should be carefully examined, to avoid the
risk of employing diseased animals; the weight should
be recorded and the rectal temperature taken.

Weighing. — The larger
animals are most conve-
niently weighed in a deci-
mal scale provided with a
metal cage for their re-
ception instead of the
ordinary pan (Fig. 136).

Mice and rats are weighed
in a modification of the
letter balance, weighing
to 250 grammes, which
has a conical wire cage
substituted for its pan

(Fig- 137).

The weight of inoculated animals should be observed
and recorded each day, at precisely the same hour,
during the entire period of observation, preferably
before the morning feeding.

262

Fig. ^6. — Rabbit scales.

CAGES.

263

Temperature. — To take the rectal temperature of
any of the laboratory animals, the animal should be
firmly held by an assistant and the bulb of an ordinary
clinical thermometer, well greased with vaseline, intro-
duced just within the sphincter ani. Allow it to remain
in this position for a few seconds, and then push it
on gently and steadily
until the entire bulb
and part of the stem,
as far as the constric-
tion, have passed into
the rectum. Three to
five minutes later, the
time varying, of course,
with the sensibility of
the thermometer used,
withdraw the instru-
ment and take the
reading.

Daily, if not more
frequently, observa-
tions should be made
of the temperature
of inoculated animals
during the entire pe-
riod they are under
observation.

Cages. — During the period which elapses between
inoculation and death, or complete recovery, the
experimental animals must be kept in suitable re-
ceptacles that can easily be kept clean and which can
be readily disinfected.

The mouse is usually stored in a glass jar (Fig. 138)
ii cm. high and n cm. in diameter, closed by a wire
gauze top which is weighted with lead or fastened to
the mouth of the jar by a bayonet catch. A matter
of great convenience is a small oblong label 5 cm. by

J37' — Mouse scales.

264 EXPERIMENTAL INOCULATION OF ANIMALS.

2.5 cm., sand-blasted on the side of the cylinder, as
marks made upon this with an ordinary lead pencil

show up well and only require
the use of a damp cloth to
remove them (Fig. 138).

The rat is kept under ob-
servation in a glass jar simi-
lar, but larger, to that used
for the mouse.

These jars are sterilised
after use either by chemical
reagents or by autoclaving.

The rabbit and the guinea-
pig are confined in cages of
suitable size, made entirely of
metal (Fig. 139). The sides
and top and bottom are of woven wire .work ; beneath
the cage is a movable metal tray filled with sawdust,
for the reception of the excreta. The cage as a whole
is raised from the ground on short legs. The sides,

Fig. 138. — Mouse jar.

Fig. 139. — Metal rabbit cage.

etc., are generally hinged so that the cage packs up

flat, for convenience of storing and also of sterilising.

The ordinary rat cage, a rectangular wire- work

APPARATUS REQUIRED. 265

box, 30 cm. from front to back, 20 cm. wide, and 14
cm. high, makes an excellent cage for guinea-pigs if
fitted with a shallow zinc tray, 35 by 24 cm., for it to
stand upon.

These cages are sterilised after use either by auto-
claving or spraying with formalin.

The tray which receives the dejecta should be
cleaned out and supplied with fresh sawdust each day,
and the soiled sawdust, remains of food, etc., should
be cremated.

As animal inoculation is purely a surgical operation,
the necessary instruments will be similar to those em-
ployed by the surgeon, and, like them, must be sterile.
In the performance of the inoculation strict attention
must be paid to asepsis and suitable precautions
adopted to guard against accidental contamination of
the material to be introduced into the animal. In
addition, the hands of the operator should be care-
fully disinfected.

The list of apparatus used in animal inocula-
tions given below comprises practically everything
needed for any inoculation. Needless to remark, all
the apparatus will never be required for any one
inoculation.

Apparatus Required for Animal Inoculation:

i. Water steriliser (vide page 38). It is also convenient
to have a second water steriliser, similar but smaller
(23 by 7 by 5 cm.), for the sterilisation of the syringes.

Fig. 140. — Hypodermic syringe with finger rests.

2. Injecting syringe. The best form is one of the ordi-
nary hypodermic pattern, fitted with finger rests,
but with the leather washers and the packing of the
piston replaced by those made of asbestos (Fig. 140).
The instrument must be easily taken to pieces, and

266 EXPERIMENTAL INOCULATION OF ANIMALS.

spare parts should be kept on hand to replace acci-
dental breakage or loss. A good supply of needles
must be kept on hand, both sharp-pointed and with
blunt ends. To sterilise the syringe, fill it with water,
loosen the packing of the piston and all the screw
joints, place it in the steriliser and boil for at least
five minutes. Disinfect the syringe after use, in a
similar manner. The needles, which are exceedingly
apt to rust after being boiled, should be stored in
a pot of absolute alcohol when not in use.

3. Surgical instruments, such as

Scissors, probe and sharp-pointed.

Dissecting forceps of various patterns.

Pressure forceps.

Aneurism needles, sharp and blunt.

Scalpels, )

Keratomes, >• with metal handles.

Trephines, )

Surgical needles.

Needle holder.

Sterilise these before use by boiling, and disinfect
them after use by the same means. Wipe perfectly
dry immediately the disinfection is completed.

4. Anaesthetic.

(a) General: The safest general anaesthetic for animals

is an A. C. E. mixture, containing by volume
alcohol i part, chloroform 2 parts, ether 6 parts,
and should be administered on a "cone" formed
by twisting up one corner of a towel and placing
a wad of cotton-wool inside it.

(b) Local:

Cocaine hydrochloride, 2 per cent, solution.
Eucaine, 2 per cent, solution.

5. Sterile capsules of various sizes.

10 c.c. (in tenths of a cu-

centimetre).

7. Flasks (75 c.c.) containing sterilised normal saline solu-

tion (or sterile bouillon).

8. Sterilised cotton- wool. Cotton- wool is packed loosely

in a copper cylinder similar to that used for storing
capsules, and sterilised in the hot-air oven.

9. Sterilised gauze. Gauze is sterilised in the same way

as cotton-wool.

10. Sterilised silk and catgut for sutures. These are
sterilised, as required, by boiling for some ten
minutes in the water steriliser.

THE PREPARATION OF THE INOCULUM. 267

11. Flexible collodion (or compound tincture of benzoin).

12. Grease pencil.

13. Tie-on celluloid labels, to affix to the cages.

14. Razor.

15. Small pot of warm water.

1 6. Liquid soap. Liquid soap is prepared as follows:

Measure out 100 grammes of soft soap and add to
500 c.c. of 2 per cent, lysol solution in a large glass
beaker; dissolve by heating in a water-bath at
about 90° C. Bottle and label " Liquid Soap."

Material Utilised for Inoculation. — The material in-
oculated may be either—

1 . Cultures of bacteria — grown in fluid media or on
solid media.

2. Metabolic products of bacterial activity — e. g.,
toxins in solution.

3. Pathological products (fluid secretions and excre-
tions, solid tissues).

The Preparation of the Inoculum.—
(a) Cultivations in Fluid Media.—

1. Flame the plug of the culture tube.

2. Remove the plug and flame the mouth of the
tube.

3. Slightly raise the lid of a sterile capsule, insert
the mouth of the culture tube into the aperture and
pour some of the cultivation into the capsule.

4. Remove the mouth of the culture tube from the
capsule, replace the lid of the latter, flame the mouth
of the tube, and replug.

5. Remove the syringe from the steriliser, squirt out
the water from its interior, and allow to cool.

6. Raise the lid of the capsule sufficiently to admit
the needle of the syringe and draw the required
amount of the cultivation into the barrel of the syringe.

(Or, remove a definite measured quantity of the cul-
tivation directly from the tube or flask by means of
a sterile graduated pipette, discharge the measured
amount into a sterile capsule, and fill into the syringe.)

268 EXPERIMENTAL, INOCULATION OF ANIMALS.

If it is necessary to introduce a large bulk of fluid
into the animal, the cultivation should be transferred
with aseptic precautions, to a sterile separatory funnel,
preferably of the shape shown in figure 141, and gradu-
ated if necessary. This is supported on a retort stand
and raised sufficiently above the level of the animal
to be injected, so as to secure a good "fall." A long
piece of sterile rubber tubing, fitted with an injection

Fig. 141. — Conical separatory funnel, fitted for injection of fluid cultivations.

needle and provided with a screw clamp, is now at-
tached to the nozzle of the funnel and the operation
completed according to the requirements of the par-
ticular case.

If the injection has to be made into the subcutaneous
tissue the "fall" may not be sufficient to force the
fluid in. In this case it will be necessary to transfer
the culture to a sterile wash-bottle and fasten a rubber

THE PREPARATION OF THE INOCULUM. 269

hand bellows to the air inlet tube (interposing an
air filter) and attach the tubing with the injection
needle to the outlet tube (Fig. 142). By careful use
sufficient force can be obtained to drive the injec-
tion in.

(6) Cultivations on Solid Media (e. g., Oblique A gar}. —

1 . By means of a sterile graduated pipette introduce
a suitable small quantity of sterile bouillon (or sterile
normal saline solution) into the culture tube.

2. With a sterile platinum loop or spatula scrape
the bacterial growth off the surface of the medium,

Fig. 142. — Arrangement of pressure injection apparatus.

and emulsify it with the bouillon. It then becomes
to all intents and purposes a fluid inoculum.

3. Pour the emulsion into a sterile capsule and fill
the syringe therefrom.

Minimal Lethal Dose : If the purpose of the inocula-
tion is to determine the minimal lethal dose, a slightly
different procedure is followed. For this purpose a
special platinum loop must be employed, some 2.5 mm.
by 0.75 mm., manufactured with parallel sides, and
calibrated by careful weighing. (One can determine
approximately by this method the amount of bacterial
growth the loop will hold when filled.)

2JO EXPERIMENTAL INOCULATION OF ANIMALS.

1. The cultivation must be prepared on a solid
medium of the optimum reaction, incubated at the
optimum temperature, and injected at the period of
greatest activity and vigour, of the particular organism
it is desired to test.

2. Arrange four sterile capsules in a row and label
them I, II, III, and IV. Into the first deliver 10 c.c.
sterile bouillon by means of a sterile graduated pipette ;
and into each of the remaining three, 9.9 c.c.

3. Remove one loopful of the bacterial growth from
the surface of the medium in the culture tube, observ-
ing the usual precautions against contamination, and
emulsify it evenly with the bouillon in the first capsule.
Each cubic centimetre of the emulsion will now con-
tain one-tenth of the organisms contained in the
original loopful (written shortly o. i loop).

4. Remove o.i c.c. of the emulsion in the first cap-
sule by means of a sterile graduated pipette and
transfer it to the second capsule and mix thoroughly.
Drop the infected pipette into a jar of lysol solution.
This makes up the bulk of the fluid in the second cap-
sule to 10 c.c., and therefore every cubic centimetre
of bouillon in capsule II contains o.ooi loop.

5. Similarly, o.i c.c. of the mixture is transferred
from capsule II to capsule III (i c.c. of bouillon in
capsule III contains o.ooooi loop), and then from
capsule III to capsule IV (i c.c. of bouillon in capsule
IV contains o.ooooooi loop).

6. With sterile graduated pipettes remove the neces-
sary quantity of bouillon corresponding to the various
divisors of ten of the loop from the respective capsules,
and transfer each "dose" to a separate sterile capsule
and label; and to such doses as do not amount to i c.c.
in bulk, add the necessary quantity of sterile bouillon.

7. Multiples of the loop are prepared by emulsifying
i, 2, 5, or 10 loops each with i c.c. sterile bouillon in
separate sterile capsules.

THE PREPARATION OF THE INOCULUM. 271

8. Inoculate a series of animals with these measured
doses, filling the syringe first from that capsule con-
taining the smallest dose, then from the capsule con-
taining the next smallest, and so on. If care is taken,
it will not be found necessary to sterilise the syringe
during the series of inoculations.

9. Plant tubes of gelatine or agar, liquefied by heat,
from each of the higher dilutions, say from o.ooooooi
loop to o.oi loop; pour plates and incubate. When
growth is visible enumerate the number of organisms
present in each, average up and calculate the number
of bacteria present in one loopful of the inoculum.

10. The smallest dose which causes the infection and
death of the animal inoculated is noted as the minimal
lethal dose (written shortly m. /. d.).

(c) Toxins. — Prepared by previously described
methods (vide page 260), are manipulated in a similar
manner to cultivations in fluid media.

(d) Pathological Products. — Fluid secretions, excre-
tions, etc., such as serous exudation, pus, blood, etc.,
are collected direct from the body in sterile capillary
pipettes (vide Fig. 10, page 21) in the following
manner:

1. Open the case containing the pipettes, grasp one
by the plugged end, remove it from the case, and
replace the lid of the latter.

2. Pass the entire length of the pipette twice or
thrice through the flame of the Bunsen burner.

3. Snap off the sealed end of the pipette with a pair
of sterile forceps.

4. Thrust the point of the pipette into the secretion,
apply the mouth to the plugged end, and fill the
pipette by suction.

5. Seal the point of the pipette in the flame. (If
using a pipette with a constriction below the plugged
mouthpiece, this portion of the pipette may also be
sealed in the flame.)

EXPERIMENTAL INOCULATION OF ANIMALS.

When about to perform the inoculation, snap the
resealed end of the pipette off with sterile forceps and
blow out the contents of the pipette into a sterile
capsule, from which the injecting syringe is filled.

If the material when discharged into the capsule is
very thick or viscous, a small quantity of sterile
bouillon or normal saline solution may be used to
dilute it, and thorough incorporation effected by the
help of a sterile platinum rod.

Solid tissues, such as spleen, lymph glands, etc.,
may be divided into small pieces by sterile instruments-
and rubbed up in a sterilised mortar with a small
quantity of sterile bouillon and the syringe filled from
the resulting emulsion.

Fig. 143. — Holding rabbits for shaving.

If it is desired to inoculate tissue en masse, remove
from the material a small cube of i or 2 mm. and
introduce it into a wound made by sterile instruments
in a suitable situation, and occlude the wounds by
means of a sealed dressing.

Method of Securing Animals During Inoculation.—
For the majority of inoculations, especially when no
anaesthetic is administered, it is customary to employ
an assistant to hold the animal (see Fig. 143); but
when the animal is anaesthetised, it is more convenient
to secure it firmly to some simple form of operating
table, such as Tatin's (Fig. 144), which will accommo-
date rabbits, guinea-pigs, and rats.

A useful piece of apparatus, too, is Voge's holder
for guinea-pigs, the method of using which is readily
seen from the accompanying figure (Fig. 145).

SECURING ANIMALS DURING INOCULATION. 273

The instrument itself consists of a hollow copper
cylinder, one end of which is turned over a ring of stout
copper wire, and from this open end a slot is cut

Fig. 144. — Operation table.

extending about half way along one side of the cylinder.
The opposite end is closed by a "pull-off" cap and is
perforated around its edge by a row of ventilating
holes, which correspond with holes cut in the rim of

the cap. In the event of the
animal resisting attempts to re-
move it from the holder, back-
wards, this cap is taken off and
the holder placed on the table
and the guinea-pig allowed to
walk out.

Fig. 145. — Taking guinea-
pig's temperature.

Fig. 146. — Voge' s holder.

To provide for different-sized animals, two sizes of
this holder will be found useful :

i. Length, 1 6 cm.; breadth, 6 cm.; size of slot,
8 cm. by 2.5 cm.

274 EXPERIMENTAL INOCULATION OF ANIMALS.

2. Length, 20 cm.; breadth, 8 cm.; size of slot,
10 cm. by 2.5 cm.

A convenient holder for mice and even small rats
is shown in figure 147, the tail being securely held by

Fig. 147. — Mouse holder.

the spring clip. Needless to say, the holder should
be entirely of metal, and the wire cage detachable and
easily renewed.

METHODS OF INOCULATION.1

The following methods of inoculation apply more
particularly to the rabbit, but from them it will readily
be seen what modifications in technique, if any, are
necessary in the case of the other experimental animals.

1. Cutaneous Inoculation. — (Anaesthetic, none.)

1. Have the animal firmly held by an assistant (or
secured to the operating table).

2. Apply the liquid soap to the fur, over the area
selected for inoculation, with a wad of cotton-wool,
and lather freely by the aid of warm water; shave
carefully and thoroughly.

3. Wash the shaved portion of skin thoroughly with
2 per cent, lysol solution.

4. Wash off the lysol with rectified spirit and allow
the alcohol to evaporate.

1 In the United Kingdom a special licence must be obtained from the Sec-
retary of State for the Home Department, granting permission to its holder to
perform inoculation upon the lower animals.

SUBCUTANEOUS INOCULATION. 275

5. Make numerous short, parallel, superficial inci-
sions with the point of a sterile scalpel.

6. When the oozing from the incisions has ceased,
rub the inoculum into the scarifications by means of
the flat of a scalpel blade, or a sterile platinum spatula.

7. Cover the inoculated area with a pad of sterile
gauze secured in situ by strips of adhesive plaster or
by sealing down the edges of the gauze with collodion.

8. Release the animal, place it in its cage, and affix
a label upon which is written:

(a) Distinctive name or number of the animal.

(b) Its weight.

(d) Date of inoculation.

2. Subcutaneous Inoculation. —

(a) Fluid Inoculum. — (Anesthetic, none.)
Steps 1-4. As for cutaneous inoculation.

5. Pinch up a fold of skin between the forefinger
and thumb of the left hand; take the charged hypo-
dermic syringe in the left hand, enter the needle into
the ridge of skin between the finger and thumb, and
push it steadily onwards until about 2 cm. of the needle
are lying in the subcutaneous tissue. Now release
the grasp of the left hand and slowly inject the fluid
contained in the syringe.

6. Withdraw the needle, and at the same moment
close the puncture with the left forefinger, to prevent
the escape of any of the inoculum. The infected
fluid, unless large in amount, will be absorbed within
a very short time.

7. Label, etc.

(b) Solid Inoculum. — (Anesthetic, none.)
Steps 1-4. As for cutaneous inoculation.

5. Raise a small fold of skin in a pair of forceps,
and make a small incision through the skin with a
pair of sharp-pointed scissors.

6. Insert a probe through the opening and push it

276 EXPERIMENTAL, INOCULATION OF ANIMALS.

steadily onwards in the subcutaneous tissue, and by
lateral movements separate the skin from the under-
lying muscles to form a funnel-shaped pocket with its
apex towards the point of entrance.

7. By means of a pair of fine-pointed forceps intro-
duce a small piece of the inoculum into this pocket
and deposit it as far as possible from the point of
entrance.

Or, improvise a syringe by sliding a piece of glass
rod (to serve as a piston) into the lumen of a slightly
shorter length of glass tubing and secure in position by
a band of rubber tubing. Sterilise by boiling. With-
draw the rod a few millimetres and deposit the piece of
tissue within the orifice of the tube, by means of sterile
forceps. Now pass the tube into the depths of the
''pocket," push on the glass rod till it projects beyond

Fig. 148. — Glass tube syringe for subcutaneous " solid" inoculation.

the end of the tube, and withdraw the apparatus, leav-
ing the tissue behind in the wound.

8. Close the wound in the skin with a dressing of
gauze sealed with collodion (or tinct. benzoin), having
previously inserted sutures, if necessary.

9. Label, etc.

3. Intramuscular. —

(a) Fluid Inoculum. — (Anesthetic, none.)

Steps 1-4. As for cutaneous inoculation.

5. Steady the skin over the selected muscle or
muscles with the slightly separated left forefinger
and thumb.

6. Thrust the needle of the injecting syringe boldly
into the muscular tissue and inject the inoculum
slowly.

7. Label, etc.

INTRAPERITONEAI,. 277

(b) Solid Inoculum. — (Anesthetic, A. C. E.}

1. Secure the animal to the operation table and
anaesthetise.

2. Shave and disinfect the skin at the seat of opera-
tion.

3. Surround the field of operation by strips of gauze
wrung out in 2 per cent, lysol solution.

4. Incise skin, aponeurosis, and muscle in turn.

5. Deposit the inoculum in the depths of the in-
cision.

6. Close the wound in the muscle with buried sutures
and the cutaneous wound with either continuous or
interrupted sutures.

Fig. 149. — Intraperitoneal inoculation — fluid.

7. Apply a sealed dressing of gauze and collodion.

8. Remove the animal from the operating table.

9. Label, etc.

4. Intraperitoneal. —

(a) Fluid Inoculum. — (Anaesthetic, none.)

Steps 1-4. As for cutaneous inoculation. Shave a

fairly broad transverse area, stretching from flank

to flank.

5. Place the left forefinger on one flank and the
thumb on the opposite, and pinch up the entire thick-
ness of the abdominal parietes in a triangular fold.
Now, by slipping the peritoneal surfaces (which are in

278 EXPERIMENT AI, INOCULATION OF ANIMALS.

apposition) one over the other, ascertain that no
coils of intestine are included in the fold.

6. Take the syringe in the right hand and with the
needle transfix the fold near its base (Fig. 149).

7. Now release the fold, but hold the syringe steady;
as the parietes flatten out, the point of the needle is
left free in the peritoneal cavity.

8. Inject the fluid from the syringe.

9. Label, etc.
Second Method:

Steps 1-4. As in the first method.

5. Heat platinum searing wire (0.5 mm. wire,
twisted to the shape indicated in figure 151, mounted
in an aluminium handle) to redness, and with it burn

Fig. 150. — Section of ab- Fig. 151. — Platinum wire for burning hole
dominal wall, etc., showing through parietes.

point of needle lying free above
the coils of intestine.

a hole through the skin and abdominal muscle down
to, but not through, the visceral peritoneum.

6. Fix a blunt-ended needle on to the charged
syringe, and by pressing the rounded end firmly against
the peritoneum it can easily be pushed through into the
peritoneal cavity.

7. Inject the fluid from the syringe.

8. Label, etc.

This method is especially useful when it is desired
to collect samples of the peritoneal fluid from time to
time during the period of observation, as fluid can be
removed from the peritoneal cavity, at intervals,
through this aperture in the abdominal parietes, by
means of a sterile capillary pipette.

INTRAPERITONEAL. 279

(b) Solid Inoculum (or the inoculation of gelatine
capsules x containing fluid cultivations). — (Anesthetic,
A. C. E.)

1. Anaesthetise the animal and secure it to the
operating table.

2. Shave a large area of the abdominal parietes.

3. Make an incision through the skin in the middle
line about 2 cm. in length, midway between the lower
end of the sternum and the pubes.

4. Divide the aponeuroses between the recti upon a
director.

5. Divide the peritoneum upon a director.

6. Introduce the inoculum into the peritoneal
cavity.

7. Close the peritoneal cavity with I,embert's su-
tures.

8. Close the skin and aponeurosis incisions together
with interrupted sutures, and apply a sealed dressing.

9. Release the animal from the operating table.

10. Label, etc.

1 Collodion sacs may be readily prepared by the following method :

1. Dip a small test-tube (5 by 0.5 cm.), bottom downwards, into a beaker
of collodion, and dry in the air ; repeat this process three or four times.

2. Dip the tube, with its coating of collodion, alternately into a beaker of
alcohol and one of water. This loosens the collodion and allows it to be
peeled off in the shape of a small test-tube.

3. Take a 20 cm. length of glass tubing, of about the diameter of the test-
tube used in forming the sac, and insert one end into the open mouth of the sac.

4. Suspend the glass tube with attached sac, inside a larger test-tube, by
packing cotton-wool in the mouth of the test-tube around the glass tubing,
and place in the incubator at 37° C. for twenty-four hours. When removed
from the incubator, the sac will be firmly adherent to the extremity of the
glass tubing.

5. Plug the open end of the glass tubing with cotton- wool, and sterilise the
test-tube and its contents in the hot-air oven.

To use the sac, remove the plug from the glass tubing, partly fill the sac
with cultivation to be inoculated, by means of a sterile capillary pipette, and
replug the tubing. When the abdominal cavity has been opened, remove
the tubing and attached sac from the protecting test-tube, close the sac by
tying a sterilised silk thread tightly around it a little below the end of the
glass tubing, and separate it from the tubing by cutting through the collodion
above the ligature, and the sac is ready for insertion in the peritoneal cavity.

280 EXPERIMENTAL INOCULATION OF ANIMALS.

5. Intracranial. — (Anesthetic, A. C. E.)

1. Anaesthetise the animal and secure it to the
operating table, dorsum uppermost.

2. Shave a portion of the scalp immediately in front
of the ears.

3. Mark out a crescentic flap of skin muscle, etc.,
convexity forwards, commencing 0.5 cm. in front of
the root of one ear and terminating at a similar spot

in front of the other ear. Reflect the
marked flap.

4. With a small trephine (diameter
0.5 cm.) remove a circular piece of bone
from the parietal segment. The centre
of the trephine hole should be at the
intersection of the median line and a
line joining the posterior canthi (Fig.

5. Introduce the inoculum by means
of a hypodermic syringe, perforating
the dura mater with the needle and de-
positing the material immediately below
this membrane.

6. Turn back the flap of skin and
r?ranil^ra"bbitra secure it in position with interrupted

sutures.

7. Dress with sterile gauze and wool and seal the
dressing with collodion.
6. Intraocular.—
(a) Fluid Inoculum. — (Anesthetic, cocaine.)

1. Instil a few drops of a sterile solution of cocaine,
2 per cent, (or B-eucaine, 2 per cent.), and repeat the
instillation in two minutes.

2. Five minutes later have the animal firmly held
by an assistant.

3. Steady the eye with fixation forceps; then pierce
the cornea with the needle of the syringe and make
the injection into the anterior chamber.

INTRAVENOUS. 2 8 1

4. Label, etc.

(b) Solid Inoculum. — (Anesthetic, A. C. E.)

1. Anaesthetise the animal and secure it firmly to
the operating table.

2. Irrigate the conjunctival sac thoroughly with
sterile saline solution.

3. Make an incision through the upper quadrant of
the cornea into the anterior chamber by means of a
triangular keratome.

4. Seize the solid inoculum in a pair of iris forceps,
introduce it through the corneal wound, and deposit
it on the anterior surface of the iris; withdraw the
forceps.

5. Again irrigate the sac and the surface of the cor-
nea.

6. Release the animal from the operating table.

7. Label, etc.

7. Intrapulmonary. —

Fluid Inoculum. — (Anaesthetic, none.)

1. Have the animal firmly held by an assistant.
(In this case the foreleg of the selected side is drawn
up by the assistant and held with the ear of that side.)

2. Shave carefully in the axillary line and disinfect
the denuded skin.

3. Thrust the needle of the syringe boldly through
the fifth or sixth intercostal space into the lung tissue.

4. Inject the contents of the syringe slowly.

5. Label, etc.

8. Intravenous.—

Fluid Inoculum,. — (Anesthetic, none.)

Vein selected, posterior auricular. Although this is
smaller than the median vein, it is firmly bound down
to the cartilage of the ear by dense connective tissue,
and is therefore more readily accessible. (In the
guinea-pig the jugular vein must be utilised, and in
order to perform the inoculation satisfactorily a gen-
eral anaesthetic must be administered to the animal.)

282 EXPERIMENTAL INOCULATION OF ANIMALS.

1. Have the animal firmly held by an assistant.
The selected ear is grasped at its root and stretched
forwards towards the operator.

2. Shave the posterior border of the dorsum of the
ear.

3. Disinfect the skin over the vein, rubbing it
vigourously with cotton- wool soaked in lysol. The
friction will make the vein more conspicuous. Wash
the lysol off with rectified spirit and allow the latter
to evaporate.

4. Direct the assistant to compress the vein at the
root of the ear. This will cause its peripheral portion
to swell up and increase in calibre.

Fig. 153. — Intravenous inoculation.

Care must be taken in preparing the inoculum, as
the injection of even small fragments may cause fatal
embolism. To obviate this risk the fluid should, if
possible, be filtered through sterile filter paper before
filling into the syringe.

Air bubbles, when injected into a vein, frequently
cause immediate death. To prevent this, the syringe
after being filled should be held in the vertical posi-
tion, needle uppermost. The needle should be thrust
through the centre of a piece of sterile filter paper and
the piston of the syringe pressed upwards until all the
air is expelled from the barrel and needle. Should any

INHALATION. 283

drops of the inoculum be forced out, they will fall
on the filter paper, which should be immediately
burned.

5. Hold the syringe as one would a pen and thrust
the point of the needle through the skin and the wall
of the vein till it enters the lumen of the vein (Fig. 1 53) .
Now press it onwards in the direction of the blood
stream — i. e.t towards the body of the animal.

6. Direct the assistant to cease compressing the
root of the ear, and slowly inject the inoculum. (If
the fluid is being forced into the subcutaneous tissue,
a condition which is at once indicated by the swelling
that occurs, the injection must be stopped and another
attempt made.)

7. Withdraw the needle.

8. Label, etc.

9. Inhalation. —

(a) Fluid Inoculum. — (Anaesthetic, none.)

1. Place the animal in a closed metal box.

2. Through a hole in one side introduce the nozzle
of some simple spraying apparatus, such as is used for
nasal medicaments.

3. Fill the reservoir of the instrument (previously
sterilised) with the fluid inoculum, and having at-
tached the bellows, spray the inoculum into the interior
of the box.

4. On the completion of the spraying, open the box,
spray the animal thoroughly with a 10 per cent, solution
of formaldehyde (to destroy any of the virus that may
be adhering to fur or feathers).

5. Transfer the animal to its cage.

6. Label, etc.

7. Thoroughly disinfect the inhalation chamber.

(b) Fluid or Powdered Inoculum. — (Ancesthetic, A. C.
E.)

i. Anaesthetise the animal and secure it firmly to
the operating table.

284 EXPERIMENTAL INOCULATION OF ANIMALS.

2. Pass a glass tube (previously sterilised) down the
larynx into the trachea.

3. Connect the straight portion of a Y-shaped piece
of tubing to the upper end of the sterilised tube and
couple one branch of the Y to a separatory funnel con-
taining the fluid inoculum, or insufflator containing the
powdered inoculum, and the other to a hand bellows.

4. Allow the fluid inoculum to run into the lungs by
gravity, or blow in the powdered inoculum by means
of a rubber-ball bellows.

5. Remove the intratracheal tube; release the animal
from the table.

6. Label, etc.

As an alternative method in the case of fairly large
animals, such as rabbits, etc., a sterile piece of glass
tubing of suitable diameter may be passed through
the larynx down the trachea almost to its bifurcation.
Fluid cultivations may then be literally poured into
the lungs, or cultivations, dried and powdered, may
be blown into the lung by the aid of a small hand
bellows.

One other method of inoculation remains to be
described, which does not require operative inter-
ference.

10. Feeding.—

1. Fluid Inoculum. — Small pieces of sterilised bread
or sop (sterilised in the steamer at 100° C.) are soaked
in the fluid inoculum and offered to the animals in a
sterile Petri dish or capsule.

2. Solid Inoculum. — Small pieces of tissue are placed
in sterile vessels and offered to the animals.

Raising the Virulence of an Organism. — If it is

desired to raise or " exalt" the virulence of a feebly
pathogenic organism, special methods of inoculation
are necessary, carefully adjusted to the exigencies of

ALTERING THE VIRULENCE. 285

each individual case. Among the most important
are the following:

1 . Passages of Virus. — The inoculation of pure culti-
vations of the organism into highly susceptible animals,
and passing it as rapidly as possible from animal to
animal, always selecting that method of inoculation —
e. q., intraperitoneal — which places the organism under
the most favorable conditions for its growth and
multiplication.

2. Virus Plus Virulent Organisms. — The inoculation
of pure cultivations of the organism together with pure
cultivations of some other microbe which in itself
is sufficiently virulent to ensure the death of the ex-
perimental animal, either into the same situation or
into some other part of the body. By this association
the organism of low virulence will frequently acquire
a higher degree of virulence, which may be still further
raised by means of "passages" (vide supra).

3. Virus Plus Toxins. — The inoculation of pure
cultivations of the organism into some selected situa-
tion, together with the subcutaneous, intraperitoneal,
or intravenous injection of a toxin, — e. g., one of those
elaborated by the proteus group, — either simultane-
ously with, before, or immediately after, the injection
of the organism. By this means the natural resistance
of the animal is lowered, and the organism inoculated
is enabled to multiply and produce its pathogenic
effect, its virulence being subsequently exalted by
means of " passages."

Attenuating the Virulence of an Organism. — Attenu-
ating or lowering the virulence of a pathogenic microbe
is usually attained with much less difficulty than the
exaltation of its virulence, and is generally effected
by influencing the environment of the cultivations,
as for example:

i. Cultivating in such media as are unsuitable by
reason of their (a) composition or (6) reaction.

286 EXPERIMENTAL, INOCULATION OF ANIMALS.

2. Cultivating in suitable media, but at an unsuitable
temperature.

3. Cultivating in suitable media, but in an unsuitable
atmosphere.

4. Cultivation in suitable media, but under unfavor-
able conditions as to light, motion, etc.

5. By a combination of two or more of the above
methods.

XVI. POST-MORTEM EXAMINATIONS
OF EXPERIMENTAL ANIMALS.

THE post-mortem examination should be carried out
as soon as possible after the death of the animal, for it
must be remembered that even in cold weather the
tissues are rapidly invaded by numerous bacteria
derived from the alimentary tract or the cavities of
the body, and from external sources.

The following outlines refer to a complete and ex-
haustive necropsy, and in routine work the examina-
tion will rarely need to be carried out in its entirety.
In all examinations, however, the searing irons must
be freely employed, and it must be recollected that one
instrument is only to be employed to seize or cut one
structure. This done, it must be regarded as con-
taminated and a fresh instrument taken for the next
step.

Apparatus Required :

Steriliser (vide page 38).

f Scissors.
Surgical instruments: -j Forceps.

( Scalpels.

Spear-headed platinum spatula (Fig. 156).
Searing irons (Fig. 154).
Platinum loop.

Tubes of media — bouillon and oblique agar.
Grease pencil.
Sterile capillary pipettes.
Sterile capsules.
Cover-slips.

Bottles of fixing fluid (for pieces of tissue intended for sec-
tioning) .

i. Place the various instruments, forceps, scissors,
scalpels, etc., needed for the autopsy inside the steril-
iser and sterilise by boiling for ten minutes; then raise

287

288

POST-MORTEM EXAMINATIONS.

the tray from the steriliser, close the lid of the latter,
and rest the tray on it.

2. Heat the searing irons to redness in a separate gas
stove.

3. Fasten the body of the animal, ventral surface
upwards (unless there is some special reason for having

Fig. 154- — Searing iron.

the dorsum exposed), out on a board by means of
copper nails driven through the extremities.

4. Drench the fur (or feathers) with lysol solution,
2 per cent. This serves the twofold purpose of pre-
venting the hairs from flying about and entering the
body cavities during the autopsy, and of rendering
innocuous any vermin that may be present on the
animal.

Fig. 155. — Apparatus for post-mortem examination, animal on board.

5. With sterile forceps and scalpel incise the skin in
the middle line from the top of the sternum to the
pubes. Make other incisions at right angles to the
first out to the axillae and groins, and reflect the skin
in two lateral flaps. (Place the now infected instru-
ments on the board by the side of the body or support
them on a porcelain knife rest.)

OF EXPERIMENTAL ANIMALS. 289

6. Inspect the seat of inoculation. If any local
lesion is visible, sear its exposed surface and remove
material to make cultivations and cover-slip prepara-
tions from the deeper parts by means of the platinum
loop. Collect specimens of pus or other exudation in
capillary pipettes for subsequent examination.

7. Sear the whole of the exposed surface of the
thorax with the searing irons.

8. Divide the ribs on either side of the sternum and
remove a rectangular portion of the anterior chest wall
with sterile scissors and a fresh pair of forceps, expos-
ing the heart. Place the infected instruments by the
side of the first set.

9. Raise the pericardial sac in a fresh pair of forceps
and burn through this structure with a searing iron.

10. Grasp the apex of the heart in the forceps and
sear the surface of the right ventricle.

11. Plunge the open point of a capillary pipette
through the seared area into the ventricle and fill
with blood.

Make cultivations and cover-slip preparations of the
heart blood.

12. Sear a broad track in the middle line of the ab-
dominal wall; open the peritoneal cavity by an incision
in the centre of the seared line.

13. Collect a specimen of the peritoneal fluid (or pus,
if present) in a capillary pipette. Make cultivations
and cover-slip preparations from this situation.

14. Collect a specimen of the urine from the dis-
tended bladder in a large pipette (in the manner indi-
cated for heart blood), for further examination, by
cultivations, microscopical preparations, and chemical
analysis.

15. Excise the spleen and place it in a sterile capsule.
(Sear the surface of this organ; plunge the spear-
headed spatula through the centre of the seared area,
twist it round between the finger and thumb, and re-

290 POST-MORTEM EXAMINATIONS

move it from the organ. Sufficient material will be
brought away in the eye in its head to make cultiva-
tions. A repetition of the process will afford material
for cover-slip preparations.)

1 6. In like manner examine the other organs — liver,
lungs, kidneys, lymphatic glands, etc. Prepare culti-
vations and cover-slip preparations.

17. Examine the other cavities of the body.

1 8. Remove small portions of various organs and
place in separate bottles of "fixing fluid" for future
sectioning. Affix to each bottle a label bearing all
necessary details as to its contents.

19. If necessary, remove portions of the organs for
preservation and display as museum specimens (vide
page 292).

20. Gather up all the infected instruments, return

Fig. .156. — Spear-headed spatula.

them to the steriliser, and disinfect by boiling for ten
minutes.

2 1 . Cover the exposed cavities of the body with blot-
ting or filter paper, moistened with 2 per cent, lysol
solution.

22. Cremate the cadaver together with the board
upon which it is fixed.

23. Stain the cover-slip preparations by suitable
methods and examine microscopically.

24. Incubate the cultivations and examine carefully
from day to day.

25. Make full notes of the condition of the various
body cavities and of the viscera immediately the
autopsy is completed ; and add the result of the micro-
scopical and cultural investigation when available.

26. Finally, the results of the action of the organism

OF EXPERIMENTAL, ANIMALS. 291

or organisms isolated may be summarised under the
following headings :

Tissue changes:1

1. Local — i. e., produced in the neighbourhood of

the bacteria.

Position: (a) At primary lesion.
(b) At secondary foci.
Character : (a) Vascular changes and . ,

tissue reactions.

(b) Degeneration and ch°ic
necrosis.

2. General (i. e., produced at a distance from the

bacteria, by absorption of toxins) :

(a) In special tissues — e. g., nerve cells and

fibres, secreting cells, vessel walls, etc.

(b) General effects of malnutrition, etc.
Symptoms :

(a) Associated with known tissue changes.

(b) Without known tissue changes.

Permanent Preparations — Museum Specimens.—

I. Tube Cultivations of Bacteria. — When showing
typical appearances these may be preserved, if not
permanently, at least for many years, as museum
specimens, by the following method:

1. Take a large glass jar 25 cm. high by 18 cm. diam-
eter, with a firm base and a broad flange, carefully
ground, around the mouth. The jar must be fitted
with a disc of plate glass ground on one side, to serve
as a lid.

2. Smear a layer of resin ointment (B. P.) on the
flange around the mouth of the jar.

3. Cover the bottom of the jar with a layer of cotton-
wool and saturate it with formalin.

4. Remove the cotton- wool plug from the culture
tubes and place them, mouth upwards, inside the jar.
(If water of condensation is present in any of the
culture tubes, it should be removed by means of a

1 This table is adapted from Muir and Ritchie's " Bacteriology."

292

POST-MORTEM EXAMINATIONS

capillary pipette before placing the tubes in the forma-
lin chamber.)

5. Adjust the glass disc, ground side downwards,
over the mouth of the jar and secure it by pressing it
firmly down into the ointment, with a rotary move-
ment.

6. Remove the tubes from the formalin chamber

after the lapse of a week, and dry the
exterior of each.

7. Seal the open mouth of each
tube in the blowpipe flame and label.

If the cultivations are intended for
museum purposes when they are first
planted, it is more convenient to em-
ploy Bulloch's tubes.

These are slightly longer than the
ordinary tubes, and are provided
with a constriction some 2 cm. below
the mouth (Fig. 157) — a feature
which renders sealing in the blow-
pipe flame an easy matter.

//. Tissues. — The naked-eye ap-
pearances of morbid tissues may be
preserved by the following method:

1 . Remove the tissue or organ from
the cadaver, using great care to avoid
distortion or injury.

2. Place it in a wide-mouthed stop-
pered jar, large enough to hold it con-
veniently, resting on a pad of cotton- wool, and arrange
it in the position it is intended to occupy (but if it
is intended to show a section of the tissue or organ,
do not incise it yet).

3. Cover with the Kaiserling solution, and stopper
the jar.

Kaiserling solution is prepared as follows :

Fig. 157. — Bulloch's
tubes.

OF EXPERIMENTAL ANIMALS. 293

Weigh out

Potassium acetate 30 grammes

Potassium nitrate 10 "

and dissolve in

Distilled water 750 c.c.

then add

Formalin 300 "

Filter.

4. After immersion in the formalin solution for
twenty-four to forty-eight hours (according to size),
transfer the tissue to a bath of methylated spirit for
ten minutes.

5. Remove to a fresh bath of spirit and watch care-
fully. Immediately the natural colours show in their
original tints, transfer to the mounting solution.

The mounting solution consists of

Glycerine , 500 c.c.

Distilled water 500 "

Formalin I "

6. After twenty-four hours in this solution transfer
to a museum jar, fill with fresh mounting solution,
and seal.

6a. Or transfer to museum jar and fill with lique-
fied gelatine, to which has been added i per cent,
formalin. Cover the jar and allow the gelatine to set.
When solid, seal the jar in the usual manner.

XVIL OUTLINES FOR THE STUDY OF
THE PATHOGENIC BACTERIA.

(THE outlines here given for the study of the patho-
genic bacteria are those in use in the author's elemen-
tary classes for medical and dental students, and for
those qualifying for the Public Health Service. They
represent the minimum of individual practical work
necessary for the acquisition of a sound knowledge
of the more important specific organisms of disease.
At the same time, it cannot be too strongly urged that
every student should, if possible, work out each organ-
ism completely and thoroughly on the lines suggested
in the Scheme for Study, on page 205.)

The student who has conscientiously worked out
the methods, etc., previously dealt with is in a position
to make accurate observations and to write precise
descriptions of the results of such observations. He
is, therefore, now entrusted with pure cultivations of
the various pathogenic bacteria, in order that he may
study the life-history of each and record the results
of his own observations — to be subsequently corrected
or amplified by the demonstrator. In this way he is
rendered independent of text-book descriptions, the
statements in which he is otherwise too liable to take
for granted, without personally attempting to verify
their accuracy. For this reason none of the bacteria
referred to in the following pages is described in detail,
nor are any photomicrographic reproductions inserted.

During the course of this work attention is also
directed, as occasion arises, to such other bacteria,
pathogenic or saprophytic, as are allied to the par-

294

THE ORGANISMS OF SUPPURATION. 295

ticular organisms under observation, or so resemble
them as to be possible sources of error, by working
them through on parallel lines. By this means a fund
of information is obtained with regard to the resem-
blances and differences, morphological and cultural, of
a large number of bacteria.

The Organisms of Suppuration.— Whilst nearly all
the pathogenic bacteria possess the power, under
certain conditions, of initiating purely pyogenic pro-
cesses in place of or in addition to their specific lesions
(e. g., the Bacillus tuberculosis, the pneumococcus,
etc.), there are a certain few organisms which commonly
express their pathogenicity in the formation of pus.
These are usually grouped together under the title
of "pyogenic bacteria," as distinct from those which
only occasionally exercise a pyogenic role.

The organisms included under this heading are:

1. Staphylococcus pyogenes albus.

2. Staphylococcus pyogenes aureus.

3. Staphylococcus pyogenes citreus.

4. Streptococcus pyogenes longus.

5. Micrococcus tetragenus.

6. Micrococcus gonorrhoeas.

7. Bacillus pyocyaneus.

296 OUTLINES FOR THE STUDY OF

Staphylococcus pyogenes Micrococcus candicans.

albus. Micrococcus agilis.

Staphylococcus pyogenes

aureus.
Staphylococcus pyogenes

citreus.

1. Prepare subcultivations from each:

Bouillon, ^

Agar streak, land incubate at 37° C.

Blood-serum, )

Agar streak, '

Gelatine stab, ., . , 0 ~

and incubate at 20° C.
Potato,

Litmus milk, t

Compare the naked-eye appearances of the cultures
from day to day. Note M. agilis refuses to grow
at 37° C.

2. Make hanging-drop preparations from the bouillon
and agar cultivations after twenty-four hours' incuba-
tion. Examine microscopically and compare.

3. Prepare cover-slip films from the agar cultures,
after twenty-four hours' incubation. Stain for flagella
by the modified Pitfield's method.

4. Make microscopical preparations of each from all
the various media after twenty-four and forty-eight
hours' and three days' incubation. Stain carbolic
methylene-blue, carbolic fuchsin, Gram's method.
Examine microscopically and compare.

5. Stain section of cardiac muscle provided (staphy-
lococcus in ulcerative endocarditis) by Gram's method,
and counterstain with eosin.

6. Stain film preparation of pus from an abscess
(containing Staphylococcus pyogenes aureus) with
carbolic methylene-blue and also by Gram's method,
counterstained with eosin.

7. Inoculate a white mouse subcutaneously with
three loopfuls of a forty-eight-hour agar cultivation

THE PATHOGENIC BACTERIA. 297

of the Staphylococcus aureus, emulsified with 0.2 c.c.
sterile broth.

Observe carefully during life, and if death occurs
make a careful post-mortem examination (page 287).

Gonococcus. Micrococcus tetragenus.

1. Prepare subcultivations of each:

Bouillon, V

Agar, ) and incubate at 37° C.

L,itmus milk,/

Gelatine streak,) ., . , 0 ~

5 \ and incubate at 20° C.
Potato, j

Observe the culture tubes from day to day.
Note that the gonococcus refuses to grow upon
these media either at 20° C. or at 37° C.

2. Prepare duplicate sets of subcultivations of the
gonococcus :

'incubate one set
Ascitic (or serum) bouillon, 0 ~

at 37° C. and
Serum agar, ,- ,,

the other at
Human blood agar. 0 ~

Examine the culture tubes from day to day.

Note that the gonococcus refuses to grow at 20° C.

3. Prepare cover-slip films of the Micrococcus tetra-
genus from all the media and of the gonococcus from
those media upon which it grows, after twenty-four
hours' and three days' incubation.

Stain carbolic methylene-blue, Gram's method.

4. Double stain the section of mouse's spleen (Micro-
coccus tetragenus) with picrocarmine and Gram's
method.

5. Stain cover-slip film preparations of urethral pus
(containing gonococci) with carbolic methylene-blue,
capsule stain, Gram's method, counterstained eosin.

6. Prepare three human serum agar plates (vide

298 OUTLINES FOR THE STUDY OF

page 198) in series from the specimen of urethral pus
and incubate at 37° C. for thirty-six hours.

Endeavour to isolate the M. gonorrhoese by subculti-
vating suspicious colonies in serum agar streak culture.

7. Inoculate a white mouse subcutaneously with
two loopfuls of a two-day agar cultivation of the
Micrococcus tetragenus.

Observe until death takes place, then make a com-
plete post-mortem examination.

Streptococcus pyo genes Ion- Streptococcus brems.

gus.
Streptococcus of bovine

mastitis.
Diplococcus pneumonia.

1. Prepare subcultivations from each:

Agar streak,

Blood agar streak,

Bouillon, and incubate at 37° C.

Litmus milk,

Potato,

Gelatine streak, 1

-T. . fand incubate at 20° C.

Potato, j

Compare the naked-eye appearances of the cultures
from day to day.

Note that the pneumococcus refuses to grow at 20° C.

2. Prepare cover-slip films from each culture tube
after twenty-four and forty-eight hours' incubation.

Stain carbolic methylene-blue, MacConkey's capsule
stain, Gram's method, and compare.

3. Examine agar cultivations at five and fourteen
days by means of stained preparations, for involution
forms, so-called arthrospores, etc.

4. Test the agar cultures for spores after fourteen
days' incubation. Result negative.

THE PATHOGENIC BACTERIA. 299

5. Stain film preparations from the specimen of pus
(containing streptococcus) carbolic methylene-blue,
Gram's method, counterstained eosin.

6. Double stain the sections of erysipelatous skin,
with picrocarmine and Gram's method.

7. Make cover-slip films from the rusty sputum
(from a case of pneumonia) and stain capsule stain,
and Gram counterstained eosin.

8. Stain the section of pneumonia lung by the Gram-
Weigert method, and counterstain with eosin.

9. Inoculate a rabbit intraperitoneally with a loopful
of the blood agar cultivation of the pneumococcus.

Observe until death occurs, recording the rectal
temperature at frequent intervals; then make a com-
plete post-mortem examination.

Observe the capsulated diplococci in the heart blood.

10. Inoculate a white mouse subcutaneously with
a loopful of the blood agar cultivation of the Strepto-
coccus longus.

Observe during life, and when death occurs, make
a complete post-mortem examination.

Bacillus of Friedldnder. B. of rhino scleroma.
(Pneumobacillus. )
i. Prepare subcultivations of each:

Bouillon,

Agar streak,

Blood-serum streak,
Potato,
Litmus milk.
Gelatine streak,
Gelatine stab,

Incubate at 37° C.

Incubate at 20° C.

Glucose gelatine shake.
Compare the naked-eye appearances of the cultures
from day to day.

300 OUTLINES FOR THE STUDY OF

2. Make hanging-drop preparations from the agar
cultures after twenty-four hours' incubation.

Examine microscopically, and compare.

3. Make film preparations of each, from all the
various media after twenty-four hours' and three days'
incubation.

Stain carbolic methylene-blue, carbolic fuchsin,
Gram's method. Examine microscopically and com-
pare.

Note the pleomorphism and involution forms.

4. Test the agar cultures for spores after seven
days' incubation. Result negative.

5. Stain the sections of mouse's kidney (containing
pneumobacilli) with carbolic methylene-blue.

6. Stain the sections of mouse's spleen (containing
pneumobacilli) with capsule stain.

7. Inoculate a mouse subcutaneously with a couple
of loopfuls of a forty-eight-hour-old potato cultivation,
emulsified with 0.2 c.c. sterile bouillon.

Observe during life, and after death make a com-
plete post-mortem examination.

Note the well-defined capsules around the bacilli in
the heart blood.

B. pyocyaneus. B. fluorescens liquefaciens.

B. fluorescens non-liquefa-

dens.

I. Prepare subcultivations of each:
Bouillon,

Agar streak, ^

* , - Incubate at 37° C.

Blood-serum streak,

Litmus milk.
Agar streak,
Gelatine stab,
Potato.

Incubate at 20° C.

THE PATHOGENIC BACTERIA. 301

Compare the naked-eye appearances of the cultures
from day to day.

Note the difference in the "optimum" temperatures.

2. Make hanging-drop preparations from the agar
and bouillon cultures after twenty -four hours' incuba-
tion.

Examine microscopically, and compare.

3. Prepare cultivations of each :

(a) Glucose formate bouillon, and incubate anae-
robically at 37° C. and 20° C.

Compare the culture tubes after three days' incuba-
tion

(b) Nitrate bouillon, and incubate at 37° C.
Compare the gas production from day to day.

fourteen days.

4. Endeavour to obtain solutions of the pigment,
formed upon agar and blood-serum in water, chloro-
form, alcohol.

5. Prepare cover-glass films from the agar cultiva-
tions at eighteen to twenty-four hours.

Stain by the modifield Pitfield method.

6. Make film preparations of each from the various
media after twenty-four hours' and three days' incu-
bation.

Stain carbolic methylene-blue, carbolic fuchsin,
Gram's method, and compare.

7. Stain the sections of guinea-pig's spleen (B.
pyocyaneus) with carbolic methylene-blue.

8. Make a complete post-mortem examination of
the body of the guinea-pig (septicaemia resulting from
pyocyaneus infection).

302 OUTLINES FOR THE STUDY OF

Vibrio cholerce. Vibrio metschnikom.

Vibrio of Finkler and Prior.
Spirillum rubrum.

i. Prepare subcultivations of each:
Bouillon,
Blood-serum streak,

Incubate at 37°
C.

Agar streak,

Peptone water,

Nitrate broth (i per cent.),

Litmus milk.

Gelatine stab, ) _ 0 ^

[ Incubate at 20° C.
Potato. J

Compare the naked-eye appearances of the cultures,
from day to day.

2. Make hanging-drop preparations from the bouillon
and agar cultures after eighteen hours' incubation.

Examine microscopically, and compare.

3. Prepare cover-glass films from the agar culti-
vations at eighteen to twenty-four hours.

Stain by the modified Pitfield method (for flagella).

4. Prepare cover-slip films from all the cultivations
after twenty-four and forty-eight hours' and five days'
incubation.

Stain carbolic methylene-blue, carbolic fuchsin
(diluted with water), by Gram's method. Examine
microscopically, and compare.

5. Test the agar cultivations for spores after four-
teen days' incubation at 37° C. Result negative.

6. Test the peptone water cultivation for indol
and nitrite after two, three, and five days' incubation.

7. Test the nitrate bouillon for nitrites after three
days' incubation.

8. Test a twenty -four-hour-old bouillon cultivation
against the serum of a guinea-pig that has been im-
munised against the Vibrio cholerae.

9. Make a careful post-mortem examination of the
guinea-pig (killed by intraperitoneal injection of the
V. cholerae).

THE PATHOGENIC BACTERIA. 303

Bacillus diphtheria. Bacillus of Hoffmann.

(Klebs-Loffler bacillus.) Bacillus of xerosis.
i. Prepare subcultivations of each:
Agar streak,
Blood-serum streak,

Potato,

Incubate at 37° C.

Bouillon,
Litmus milk.
Gelatine streak. Incubate at 20° C.
Compare the naked-eye appearance of the cultures
from day to day.

2. Make hanging-drop preparations from the bouil-
lon and agar cultivations after twenty-four hours'
incubation.

Examine microscopically, and compare.

3. Make cover-slip films from the blood-serum cul-
tures, after twelve to eighteen hours' incubation.

Stain Neisser's method.

4. Make film preparations of each from all the media
after twenty-four and forty-eight hours' and five
days' incubation.

Stain carbolic methylene-blue, carbolic fuchsin,
Gram's method, Neisser's method, and compare.

5. Test the agar cultivations for spores, after four-
teen days' incubation. Result negative.

6. Stain the sections of diphtheritic membrane (a)
with carbolic methylene-blue, and (6) by the Gram-
Weigert method — to demonstrate the fibrin.

Note the diphtheria bacilli in the superficial layers of
the membrane, often arranged in clusters and masses.

7. Inoculate a guinea-pig under the skin of the
abdomen with i c.c. of a forty-eight-hour-old bouillon
cultivation of the B. diphtherias.

Observe carefully until death occurs, then make
post-mortem examination.

Prepare cultivations on blood-serum from (a) local
lesion, (6) heart blood, and incubate at 37° C.

304 OUTLINES FOR THE STUDY OF

Note that a cultivation from a yields a growth of
the B. diphtheriae, while that from b does not.

Prepare cover-glass films from local lesion. Note
the bacilli present.

B. typhi abdominalis. B. coli communis.

B. enter itidis (of Gaertner) .
B. aquatilis sulcatus.
i. Prepare subcultivations of each:
Bouillon,

Litmus milk, ^

Incubate at 37° C.
Peptone water,

Agar streak.

Bouillon,

Potato,

Incubate at 20° C.

Gelatine streak.
Compare the naked-eye appearances of the cultures
from day to day.

Note that B. aquatilis sulcatus will not grow at 37° C.

2. Make hanging-drop preparations from the bouillon
and agar cultures after eighteen hours' incubation.

Examine microscopically, and compare.

3. Make cover-slip preparations from the agar culti-
vations after eighteen hours' incubation.

Stain modified Pitfield's method.

4. Make cover-slip preparations of each from all the
media after twenty-four hours' and five days' incuba-
tion.

Stain carbolic methylene-blue, Gram's method,
modified Pitfield's method, and compare.

5. Test agar cultivations at fourteen days for spores.
Result negative.

6. Test the peptone water cultivations at five days
for indol.

THE PATHOGENIC BACTERIA. 305

7. Prepare glucose-formate-gelatine "shake" culti-
vations and incubate at 20° C.

Compare from day to day.

8. Pour gelatine plate cultivations of each and in-
cubate at 20° C., for three or four days.

Compare the colonies, naked eye and under a i-inch
lens.

9. Stain the sections of typhoid ulcer with carbolic
methylene-blue.

10. Stain sections of guinea-pig spleen (B. typhi
abdominalis) with dilute carbolic fuchsin.

1 1 . Prepare a bouillon cultivation of each and incu-
bate at 20° C. for twenty-four hours.

Test each culture against blood-serum from a
typhoid patient.

Bacillus anthracis. Bacillus subtilis.

Bacillus mycoides.

1. Prepare subcultivations of each: •

Bouillon,

Agar streak,

T ? Incubate at 37° C.

Litmus milk,

Blood-serum streak.

Gelatine stab.)

Potato. '[ Incubate at 20° C.

Compare the naked-eye appearances of the cultures
from day to day.

2. Make hanging-drop preparations from the bouillon
and agar cultivations after twenty-four hours v incuba-
tion.

3. Make cover-slip preparations from the agar culti-
vations after twenty-four hours' incubation.

Stain modified Pitfield, examine microscopically,
and compare.

4. Make cover-slip preparations from the various

306 OUTLINES FOR THE STUDY OF

media after twenty-four and forty-eight hours' and
seven days' incubation.

Stain carbolic methylene-blue, carbolic fuchsin,
Gram's method, and compare.

5. Prepare cultivations of each, in nitrate broth,
incubate three days at 37° C., and test for nitrites.

6. Prepare cultivations of each, in peptone water,
incubate three days at 37° C., and test for indol.

7. Test the agar cultivations for spores, after seven
days' incubation.

8. Prepare cover-glass films from these agar cultiva-
tions and stain for spores by method 2 (vide page 91).

9. Double stain the sections of malignant pustule
(from man, containing anthrax bacilli) with picro-
carmine and Gram's method.

10. Stain the sections of guinea-pig's lung (contain-
ing B. anthracis) with carbolic methylene-blue.

IT. Inoculate a guinea-pig subcutaneously with two
loopfuls of a forty-eight-hour-old agar cultivation of the
B. anthracis, emulsified with i c.c. sterile bouillon.

Observe carefully during life.

When dead, make complete post-mortem examina-
tion.

Bacillus tuberculosis. Bacillus of aman tubercle.

Bacillus phlei.

(Timothy grass bacillus.)

i. Prepare subcultivations of th'e tubercle bacillus
and that of avian tubercle:

Blood-serum streak,
* Agar streak,

Glycerine agar streak, _

^/ ' .„ Incubate at 37° C.

Glycerine bouillon,

Litmus milk,
Glycerinated potato.

Glycerine agar streak, )

~J*\. [ Incubate at

Gelatine streak. J

THE PATHOGENIC BACTERIA.

307

Fig. 158.

308 OUTLINES FOR THE STUDY OF

Compare the culture tubes from day to day.
Note that no growth takes place at 20° C.

2. Prepare subcultivations of the B. phlei upon all
the ordinary media, and incubate at 37° C. and 20° C.

3. Make cover-slip preparations of each, from all
the various media after twenty-four hours' and seven
days' incubation.

Stain carbolic methylene-blue, carbolic fuchsin
(films of each organism should be stained in the usual
manner, and also by immersion in the stain for four
hours), Ziehl-Neelsen method, Gram's method. (Em-
ploy heat in staining with the aniline gentian violet,
as is done in the Ziehl-Neelsen method, also warm the
iodine solution.)

4. Double stain the sections of guinea-pig's spleen
(B. tuberculosis) with picrocarmine and Ziehl-Neelsen
method.

5. Stain the sections of lung (miliary tuberculosis)
by the Ziehl-Neelsen method, counter staining with
Bismarck brown.

6. Make a careful post-mortem examination of the
guinea-pig which has succumbed to general tubercu-
losis, as the result of subcutaneous inoculation at the
inner aspect of the bend of the left knee.

NOTE. — Every post-mortem examination of animals
infected with tuberculous material should include the
naked-eye and microscopical examination of the
popliteal, superficial, and deep inguinal, iliac, lumbar,
and axillary glands on each side of the body, also the
retrohepatic, bronchial, and sternal glands, the spleen,
liver, and lungs (see Fig. 158),

Bacillus lepra.

i. Stain the sections of (a) ulnar nerve and (6)
leprous nodule by the Ziehl-Neelsen method

This organism cannot yet be cultivated artificially.

THE PATHOGENIC BACTERIA. 309

Bacillus tetani.

Bacillus cedematis maligni.

Bacillus of symptomatic anthrax.

B. enteritidis sporogenes.

B. botulinus.

1. Prepare agar streak subcultivations from each and
incubate aerobically at 37° C.

Observe the culture tubes until the completion of
seven days* incubation. Result, no growth.

2. Prepare bouillon cultivations of —

Bacillus tetani and immediately inoculate the cul-
ture tube with B. prodigiosus.

Bacillus cedematis maligni and immediately inocu-
late the culture tube with Staphy loco ecus aureus.

Bacillus of symptomatic anthrax and immediately
inoculate the culture tube with B. pyocyaneus.

B. enteritidis sporogenes and immediately inoculate
the culture tube with B. subtilis.

Incubate these cultivations aerobically at 37° C.

Observe the symbiotic growth of the anaerobes.

3. Prepare subcultivations from each:

/Incubate anae-

Glucose formate agar streak, \ ro^lcaHy at

Blood-serum streak. ) £7 tl t fm

/ Bulloch's

\ apparatus).

Incubate anaero-
Glucose formate bouillon, «. ,, 0

bically at 37°

Glucose formate agar stab, ~ ,. -r, ;
T., M1 C. (in Buch-

Litmus milk. , L i_ \

ner s tubes).

Incubate an-
aerobically

Glucose formate gelatine stab.

Buchner's
tube).

4. Make hanging-drop preparations in the ordinary
way and examine microscopically.

at 37° C. (in

310 OUTLINES FOR THE STUDY OF

Make another set of hanging-drop preparations, but
before sealing the cell introduce a few crystals of
pyrogallic acid and a drop of caustic soda solution,
to absorb the oxygen and render the hanging drop
"anaerobic." Examine microscopically.

5. Prepare cover-slip films from the agar streak
cultivations at twenty-four hours.

Stain modified Pitfield (for flagella).

6. Prepare cover-slip films from each of the various
media after forty-eight hours' and three days' incu-
bation.

Stain carbolic methylene-blue and Gram's method.

7. Prepare cover-slip films from the agar cultiva-
tions after seven days' incubation.

Stain by Muller's method (for spores).

8. Make careful post-mortem examinations of the
guinea-pigs which have died as the result of infection
with the B. tetani, B. maligni, and B. enteritidis
sporogenes, respectively.

Bacillus pestis. B. septiccemia h&morrha-

gicce.
B. suipestifer.

(Bacillus of hog cholera.)
i. Prepare subcultivations from each:
Bouillon,

Agar streak,

and incubate at 37° C.

Litmus milk,
Gelatine streak,
Gelatine stab,

and incubate at 20° C.

•Potato,

Compare the naked-eye characters of the cultures
from day to day.

THE PATHOGENIC BACTERIA. 311

2. Add a loopful of sterile vaseline to a tube of
nutrient bouillon.

Prepare a subcultivation of the B. pestis in this and
incubate at 37° C.

Note the formation of stalactites depending from
the fat globules.

3. Make hanging-drop preparations from the bouillon
and agar cultures, after forty-eight hours' incubation.
Examine microscopically and compare.

4. Prepare cover-slip film preparations of each from
all the media after twenty-four, forty-eight hours'
and three days' incubation.

Stain carbolic methylene-blue, carbolic thionine blue,
Gram's method, and compare.

5. Prepare smear cultivations on the surface of very
dry agar and incubate at 37° C. for forty-eight hours.

Make cover-slip preparations from the growth and
stain with carbolic thionine blue.

Note the involution forms of B. pestis.

6. Examine the agar streak cultivations, after seven
days' incubation, for spores.

7. Sterilise some salt, in a glass capsule, in the hot-
air oven.

Add about 0.5 gramme to a tube of nutrient bouillon
and incubate at 37° C. for forty-eight hours, to deter-
mine its sterility.

8. Inoculate this bouillon with the B. pestis and
incubate at 37° C. for twenty-four hours.

Examine the resulting growth microscopically.
Note the plasmolysed bacilli.

9. Stain the sections of bubo (containing B. pestis)
with (a) Loftier 's methylene-blue and (b) carbolic
thionine blue.

10. Make a complete post-mortem examination of
the cadaver of the rat infected intraperitoneally with
the B. pestis.

312 OUTLINES FOR THE STUDY OF

Streptothrix actinomycotica. Cladothrix nivea.

1. Prepare subcultivations from each:

Bouillon, \

Glycerine bouillon,)

Agar, land incubate at 37° C.

Glycerine agar, \

Litmus milk, /

Gelatine streak,)

p Y and incubate at 20° C.

Compare the naked-eye appearances of the cultures
from day to day.

2. Prepare cover-slip film preparations of each from
all the media after twenty-four hours' and three days'
incubation.

Stain carbolic methylene-blue, carbolic fuchsin,
Gram's method. Examine microscopically and com-
pare.

3. Double stain sections of human liver (affected
with actinomycosis) with picrocarmine and the Gram-
Weigert method.

4. Stain the section of cow's tongue (infected with
actinomyces) by Gram's method and counterstain
with dilute aqueous solution of fuchsin.

5. Examine the pus from a breaking-down nodule
(from cow's tongue) with a hand lens, pick out some
of the minute yellowish granules with a sterile platinum
loop, and transfer to a sterile capsule.

6. Crush the selected granules with a sterile glass
rod. Make cover-slip preparations and stain by
Gram's method.

7. Inoculate four tubes of glycerine agar in series
with some of the material.

Observe the resulting growth macroscopically and
microscopically.

[ and incubate at 20° C.

THE PATHOGENIC BACTERIA. 313

B. influenzas. Bacillus cegyptiacus.

(Koch-Week's bacillus.)

1. Prepare subcultivations of each:

Bouillon, \

Serum bouillon,)

Agar, }and incubate at 37 C.

Blood agar,

Serum agar,
Observe from day to day.

Note that growth only occurs in serum and blood
media.

2. Prepare subcultivations from each:

Serum bouillon ^

Blood agar,
Observe from day to day for four days.
Note that no growth takes place.

3. Transfer the subcultivations (section 2) to the
incubator at 37° C.

Note that no growth takes place — the bacilli are
dead.

4. Prepare cover-slip films of each bacillus from all
the media upon which growth takes place, after six-
teen, twenty-four, and forty-eight hours' incubation.

Stain carbolic methylene-blue, very dilute aqueous
solution of fuchsin, Gram's method. Examine micro-
scopically and compare.

5. Prepare cover-slip preparations of the yellowish,
more solid masses of the specimen of sputum from a
case of influenza.

Stain in dilute fuchsin solution for twenty minutes.
Examine microscopically.

6. Wash some of these selected portions of sputum
in several changes of sterile distilled water.

7. Inoculate four blood agar tubes in series from the
washed sputum, and incubate at 37° C.

Observe the resulting growth macroscopically and
microscopically.

314 OUTLINES FOR THE STUDY OF

8. Prepare cover-slip films of pus from a case of
acute muco-purulent conjunctivitis.

Stain carbolic methylene-blue, dilute fuchsin solution.

9. Inoculate four blood agar tubes in series from the
pus from cases of acute conjunctivitis and incubate
at 37° C. Observe the resulting growth macro-
scopically and microscopically.

Bacillus mallei.

1. Prepare subcultivations :

Bouillon,

Agar smear,

Blood-serum, and lncubate at ^ C"

Litmus milk,

Gelatine streak,)

-PJ , \ and incubate at 20 C.

Potato, j

Observe the naked-eye appearances of the cultures
from day to day.

2. Make hanging-drop preparations from the bouillon
and agar cultivations.

3. Prepare cover-slip films from the agar and blood-
serum cultivations, after eighteen hours' and three days'
incubation.

Stain carbolic methylene-blue, Neisser's method,
and by Gram's method. Examine microscopically
and compare.

Also compare with corresponding preparations of
the B. diphtheriae.

4. Stain sections of the testicle of the guinea-pig
(containing B. mallei), by over staining with Nicolle's
gentian violet (vide page 83), and subsequently de-
colourising with i per cent, acetic acid.

5. Stain the sections of horse's lung (containing
glanders bacilli) with LofBer's methylene-blue.

THE PATHOGENIC BACTERIA. 315

6. Make a complete post-mortem examination of
the guinea-pig killed by infecting subcutaneously with
the B. mallei.

Micrococcus meiitensis.
i. Prepare subcultivations :

Bouillon, \

Glycerine agar smear,)

Brain agar smear, > Incubate at 37° C.

Blood-serum, \

Litmus milk. /

Agar streak,

Gelatine streak,

Incubate at 20° C.

Potato.

Observe the naked-eye appearances of the cultures
from day to day.

Note the extremely scanty growth on gelatine and
agar at 20° C.

2. Make hanging-drop preparations from the bouillon
and agar cultivations.

3. Prepare cover-slip films from the growth on each
of the media and stain carbolic methylene-blue,
dilute solution of fuchsin, by Gram's method; examine
microscopically and compare.

Note the bacillary forms on agar.

4. Examine the agar cultivations, after seven days'
incubation, for spores. Result negative.

5. Test a forty-eight-hour-old bouillon culture,
filtered through a sterile filter paper, against the serum
of a patient suffering from Malta fever.

XVHL BACTERIOLOGICAL ANALYSES.

EACH bacteriological or bacterioscopical analysis of
air, earth, sewage, various food-stuffs, etc., includes,
as a general rule, two distinct investigations:

1. Quantitative.

2. Qualitative.

The first aims simply at enumerating (approximately)
the total number of bacteria present in any given unit
of volume irrespective of the nature and character of
individual organisms.

The second seeks to classify the bacteria found,
and to accurately identify individual organisms. As a
subdivision of the qualitative examination, an esti-
mation is often made, and with a fair degree of accu-
racy, of the numbers of some particular organism
(e. g.y B. coli communis), present per unit, in the
sample under examination.

The general principles underlying the bacteriological
analyses of water, sewage, air and dust, soil, milk, ice
cream, meat, and other tinned stuffs, as exemplified
by the methods used by the author, are indicated
in the following pages, together with the methods of
testing filters and chemical germicides. It is hoped
that the methods given will be found to be capable
of expansion and adaptation to any circumstance or
set of circumstances which may confront the student.

BACTERIOLOGICAL EXAMINATION OF WATER.

1. Quantitative. —

Collection of the Sample. — As the quantity of water
actually used for this examination rarely exceeds 2 c.c.,
the most suitable vessels for the reception of the sample

316

BACTERIOLOGICAL EXAMINATION OF WATER. 317

are small glass bottles, 25 c.c. capacity, with narrow
necks and overhanging glass stoppers (to prevent
contamination of the bottle necks by falling dust).
These must be carefully sterilised in the hot-air steril-
iser (vide page 35).

If the sample is obtained from a tap or pipe, turn
on the water and allow it to run for a few minutes.
Remove the stopper from the bottle and retain it in
the hand whilst the water is allowed to run into the
bottle and three parts fill it. Re-
place the stopper and tie it down,
but do not seal it.

If the sample is obtained from a
stream, tank, or reservoir, fasten a
piece of stout wire around the neck
of the bottle, remove the stopper,
and retain it in the hand. Then,
using the wire as a handle, plunge
the bottle into the water, mouth
downwards, until it is well beneath
the surface; then reverse it, allow
it to fill, and withdraw it from the
water. Pour out a few cubic cen-
timetres of water from the bottle,
replace the stopper, and tie it down.

Or, use the apparatus designed
by v. Esmarch (Fig. 159), in which
the stopper can be removed, the bottle filled, and the
stopper replaced, whilst the bottle is below the surface
of the water, even at depths previously determined
upon. When the apparatus is taken out of the water,
the small bottles are filled from it, and packed in the
ice-box mentioned below.

To prevent the multiplication of the bacteria con-
tained in the water during transit from the place of
collection to the laboratory, enclose the bottles, rolled
in cotton- wool, in a double- walled metal box, pack the

Fig. 159. — Esmarch' s
collecting bottle for water
samples.

BACTERIOLOGICAL ANALYSES.

space between the walls with pounded ice, close the
metal box, and place it in a felt-lined wooden case
(Fig. 1 60). (It has been shown that the majority of
bacteria will survive exposure to the temperature of
melting ice for some days, while practically none will
multiply at this temperature.)

On reaching the laboratory, the method of examina-
tion consists in adding measured quantities of the

. Fig. 1 60. — Ice-box.

water sample to several tubes of nutrient media pre-
viously liquefied by heat, pouring plate cultivations
from each of these tubes, incubating at a suitable tem-
perature, and finally counting the colonies which
make their appearance on the plates.

The bacteria present in the water may comprise
not only varieties which have their normal habitat in
the water and will consequently be developed at 20°
C., but also varieties which have been derived from,

BACTERIOLOGICAL EXAMINATION OF WATER. 319

or are pathogenic for, the animal body, and which will
only develop well at a temperature of 37° C. In
order to demonstrate the presence of each of these
classes it will be necessary to plant both gelatine and
agar plates.

Further, the sample of water may contain moulds,
yeasts, or torulae, and the development of these will
be best secured by plating in wort gelatine and incu-
bating at 20° C.

Apparatus Required:

Plate-levelling stand.

Case of sterile plates.

Case of sterile pipettes, i c.c. (in tenths of a cubic centi-
metre) .

Case of sterile pipettes, 10 c.c. (in tenths of a cubic centi-
metre) .

Case of sterile capsules, 25 c.c. capacity.

Tubes of nutrient gelatine.

Tubes of nutrient agar.

Tubes of wort gelatine.

One 250 c.c. flask of sterile distilled water.

Tall cylinder containing 2 per cent, lysol solution.

Bunsen burner.

Grease pencil.

Water-bath regulated at 42° C.

METHOD. —

1. Arrange the plate-levelling platform with its
water compartment filled with water, at 45° C.

2. Number the agar tubes 1,2, and 3; the gelatine
tubes, consecutively, i to 6, and the wort tubes, 1,2,
and 3. Flame the plugs and see that they are not
adherent to the lips of the tubes.

3. Place the agar tubes in boiling water until the
medium is melted, then transfer them to the water-
bath regulated at 42° C. Liquefy the nutrient gelatine
and wort gelatine tubes by immersing them in the
same water-bath.

4. Remove the bottle containing the water sample
from the ice-box, distribute the bacterial contents

320 BACTERIOLOGICAL ANALYSES.

evenly throughout the water by shaking, cut the
string securing the stopper, and loosen the stopper,
but do not take it out.

5. Remove one of the i c.c. pipettes from the case,
holding it by the plain portion of the tube. Pass the
graduated portion twice through the Bunsen flame,
raise the bottle containing the water sample from the
bench in the other hand, grasp the stopper as if it
were a cotton-wool plug, and remove it from the bottle
with the hand holding the pipette; flame the mouth
of the bottle.

6. Pass the pipette into the mouth of the bottle,
holding its point well below the surface of the water.
Suck up rather more than i c.c. into the pipette; empty
the pipette by blowing. Now draw up exactly i c.c.
into the pipette. Withdraw the pipette from the
bottle, replace the stopper, and put the bottle down.

7. Take the first melted agar tube in the left hand,
remove the cotton-wool plug, and add to its contents
0.5 c.c. of the water sample from the pipette; replug
the tube and put it down. In a similar manner add
0.3 c.c. water to the contents of the second tube, and
0.2 c.c. to the contents of the third.

8. Drop the pipette into the cylinder containing
lysol solution.

9. Mix the water sample with the medium in each
tube in the manner described under plate cultivations ;
pour a plate from each tube. Label each plate with
(a) the distinctive name or number of the sample, (b)
the quantity of water sample it contains, and (c) the
date.

10. Allow the plates to set, and incubate at 37° C.

11. Empty the water chamber of the levelling
apparatus and refill it with ice- water.

12. By means of the sterile 10 c.c. pipette deliver
9.9 c.c. sterile distilled water into a sterile capsule.

13. Add o.i c.c. of the water sample to the 9.9 c.c.

BACTERIOLOGICAL EXAMINATION OF WATER. 321

sterile water in the capsule. This will give a dilution
of i in 100.

14. Plant the six tubes of nutrient gelatine in the
following manner: To the first tube add 0.5 c.c. of the
water sample direct from the bottle; to the second,
0.3 c.c.; and to the third, 0.2 c.c.; and pour a plate
of each tube. To the fourth tube add 0.5 c.c. of the
diluted water sample from the capsule; to the fifth,
0.3 c.c.; and to the sixth, 0.2 c.c.; and pour a plate
from each.

15. Label each plate with the quantity of the water
sample it contains — that is, 0.5 c.c., 0.3 c.c. ,0.2 c.c.,
0.005 c-c-> 0.003 c-c-> and 0.002 c.c.

1 6. Allow the plates to set, and incubate at 20° C.

17. To the first tube of liquefied wort gelatine add
0.5 c.c. water sample; to the second, 0.3 c.c.; and to
the third, 2 c.c.

1 8. Label the plates, allow them to set, and incubate
at 20° C.

19. Count the number of colonies that have de-
veloped upon the agar at 37° C. after twenty-four
hours' incubation.

20. Note the number of colonies present on each of
the gelatine and wort gelatine plates after forty-eight
hours' incubation.

21. Replace in the incubator, count again at three
days, four days, and five days.

22. Calculate the number of organisms present per
cubic centimetre of the original water from the average
of the six gelatine plates.

Method of Counting.— The most accurate method
of counting the colonies on each of the plates is by
means of either Jeffer's or Fakes' counting disc. Each
of these discs consists of a piece of paper, upon which
is printed a dead black disc, subdivided by concentric
circles and radii, printed in white. In Jeffer's counter,
each subdivision has an area of i square centimetre;

322 BACTERIOLOGICAL ANALYSES.

Fig. 161. — Jeffer's disc.

Fig. 162. — Fakes' disc.

BACTERIOLOGICAL EXAMINATION OF WATER. 323

in Fakes' counter, radii divide the circle into sixteen
equal sectors, and counting is facilitated by equidistant
concentric circles.

(a) In the final counting of each plate, place the
plate over the counting disc, and centre it, if possible,
making its periphery coincide with one or other of the
concentric circles.

(b) Remove the cover of the plate, and by means of
a hand lens count the colonies appearing in each of the
sectors in turn. Make a note of the number present
in each.

(c) If the colonies present are fewer than 500, the
entire plate should be counted. If, however, they
exceed this number, enumerate one-half, or one-
quarter of the plate, or count a sector here and there,
and from these figures estimate the number of colonies
present on the entire plate.

It will be noted that the quantities of water selected
for addition to each set of tubes of nutrient media
total i c.c.; consequently the plates in a measure
control each other; that is, the second and third
plates of a series should together contain as many
colonies as the first, and the second should contain
about half as many more than the third.

2. Qualitative Examination.—

Collection of the Sample. — The quantity of water
required for this examination is about 2000 c.c., and
the vessel usually chosen for its reception is an ordinary
blue glass Winchester quart bottle, sterilised in the
hot-air oven, the stopper covered by a piece of sterile
cotton- wool, and over this a paper or parchment cap
fastened with string. The bottle may be packed in
a wooden box or in an ordinary wicker case. The
method of collecting the sample is identical with that
described under the heading of Quantitative Examina-
tion; there is, however, not the same imperative

324 BACTERIOLOGICAL ANALYSES.

necessity to pack the sample in ice for transmission
to the laboratory.

Examination. — The qualitative bacteriological ex-
amination of water is usually directed to the deter-
mination of the presence or absence of certain patho-
genic organisms, usually one or more, but very rarely
all, of those comprised in the following list :

I. B. coli communis.
II. B. typhi abdominalis.

III. B. enteritidis of Gaertner.

IV. B. enteritidis sporogenes.
V. Streptococci.

VI. Vibrio cholerae.
VII. B. anthracis.
VIII. B. tetani.

When these are present they are usually very highly
diluted, and it is necessary, before commencing the
examination, to adopt some means by which —

1. All the bacteria present in the sample of water,
pathogenic or otherwise, may be concentrated in a
small space.

2. The harmless non-pathogenic bacteria may be
destroyed or their growth inhibited.

The first of these objects is usually effected by
filtration of the water sample through a porcelain
filter candle, and the subsequent emulsion of the
bacterial residue of the original water with a small
measured quantity of sterile bouillon.

The second is attained by so arranging the en-
vironment (i. e., media, incubation temperature, and
atmosphere) as to favor the growth of the pathogenic
organisms at the expense of the harmless saprophytes.

Apparatus Required:

Sterile Berkfeld porcelain filter candle, fitted with rubber
washer.

Rubber cork to fit the mouth of the filter candle, perfo-
rated with one hole.

Kitasato serum flask, 2000 c.c. capacity.

BACTERIOLOGICAL EXAMINATION OF WATER. 325

Fleuss air pump or water force pump.

Wulff's bottle, fitted as wash-bottle, and containing sul-
phuric acid (to act as a safety valve between filter and
pump).

Pressure tubing, clamps, pinch-cock.

Retort stand, with ring and clamp.

Fig. 163. — Water filtering apparatus. That portion of the figure to the left
of the vertical line is drawn to a larger scale than that on the right, in order
to show details of the force pump.

Rubber cork for the neck of Winchester quart, perforated
with two holes and fitted with one 6 cm. length of
straight glass tubing, and one V-shaped piece of glass
tubing, one arm 32 cm. in length, the other 52 cm., the
short arm being plugged with cotton-wool. The rub-
ber stopper must be sterilised by boiling and the glass
tubing by hot air, before use.

326

BACTERIOLOGICAL ANALYSES.

Flask containing 250 c.c. sterile broth.

Test-tube brush to fit the lumen of the candle, enclosed in
a sterile test-tube (and previously sterilised by dry heat
or by boiling).

Case of sterile pipettes, 10 c.c. in tenths.

Case of sterile pipettes, i c.c. in tenths.

Case of sterile pipettes, i c.c. in hundredths.

Tubes of various nutrient media (according to require-
ments).

Twelve Buchner's tubes with rubber stoppers.

Pyrogallic acid, 10 per cent, aqueous solution.

Dekanormal caustic soda solution.

METHOD.—

i. Fit up the filtering apparatus as in the accom-
panying diagram (Fig. 163), interposing the wash-
bottle with sulphuric acid between the
filter flask and the force-pump (in the
position occupied in the diagram by
the central vertical line), and placing
another screw clamp on the rubber
tubing connecting the lateral arm of
the filter flask with the wash-bottle.

2. Filter the entire 2000 c.c. of water
through the filter candle.

3. When the filtration is completed,
screw up the clamps and so occlude
the two pieces of pressure tubing.

4. Reverse the position of the glass
tubes in the WulfTs bottle so that the
one nearest the air pump now dips into
the sulphuric acid.

5. Slowly open the metal clamps and
allow air to gradually enter filter flask,
having first passed through the acid,
and restore the pressure.

6. Unship the apparatus, remove the cork from the
mouth of the candle.

7. Pipette 10 c.c. of sterile broth into the interior
of the candle, and by means of the sterile test-tube

Fig. 164. — Sterile
test-tube brush.

THE COU AND TYPHOID GROUPS. 327

brush (Fig. 164) emulsify the slimy residue which
lines the candle, with the broth.

The entire bacterial contents of the original 2000 c.c.
of water are now suspended in 10 c.c. of broth, so that
i c.c. of the suspension is equivalent, so far as the
contained organisms are concerned, to 200 c.c. of the
original water.

Up to this point the method is identical, irrespective
of the particular organism whose presence it is desired
to demonstrate; but from this point onwards the
methods must be specially adapted to the isolation
of definite groups of organisms or of individual bacteria.

The Coli and Typhoid Groups.—

1 . Number ten tubes of bile salt broth (vide page
1 69) , consecutively from i to 10.

2. To each of the tubes of bile salt broth add varying
quantities of the suspension by means of suitably
graduated sterile pipettes, as follows :

No. 2 ... o.oi c.c. (equivalent to 2 c.c. of the original water sample).

No. 3 ... 0.02 " " " 4 " " " " " "

No. 4 . . .0.05 " " " 10 " " " " " "

No. 5 ... o.i " " "20 " " " " " "

No. 6 ... 0.2 " " " 40 " " " " " . "

No. 7 ... 0.3 " " " 60 " " "

No. 8 ... 0.5 " " «< 100 " " " "

No. 9 . . . i.o " " <« 200 c« '« " " " "

No. 10 ... 2.5 " " " 500 4< " " " " "

(To No. i should be added i c.c. of the original
water sample before the filtration is commenced.1)

3. Put up each tube anaerobically in a Buchner's
tube and incubate at 42° C.

1 If a positive result is obtained when using this method, it only needs a
simple calculation to determine the smallest quantity (down to I c.c.) of the
sample that contains at least one of the germs. For instance, if growth occurs
in all the tubes from 4 to IO, and that growth is subsequently proved to be due
to the presence of the B. coli, then it follows that at least one colon bacillus is
present in every 10 c.c. of the water sample, but not in every 4 c.c. If, on the
other hand, the presence of the B. coli can only be proved in tube No. 10, then
the average number of colon bacilli present in the sample is two per litre.

328 BACTERIOLOGICAL ANALYSES.

4. Examine after twenty-four hours' incubation.
Note in each culture tube:

(a) The presence or absence of visible growth.

(6) The reaction of the medium in each tube as in-
dicated by the colour change (if any) the litmus has
undergone.

(c) The presence or absence of gas formation as in-
dicated by froth on the surface of the medium and
the collection of gas in the fermentation tube.

5. Replace those tubes which show no signs of growth
in the incubator. Examine after another period of
twenty-four hours with reference to the same points.

6. Remove the culture tubes which show visible
growth from the Buchner's tubes and make gelatine
plate cultivations whether gas production and acid
production are present or not. Incubate at 22° C.
for forty-eight or seventy- two hours.

7. Pick off coliform or typhiform colonies and sub-
cultivate in:

1. Nutrient bouillon.

2. Glucose formate bouillon.

3. Glycerine bouillon.

4. Lactose litmus bouillon.

5. Peptone water.

6. Litmus milk.

7. Glucose gelatine stab.

8. Lactose gelatine stab.

9. Maltose gelatine stab.

10. Neutral red-agar stab.

11. Nutrient gelatine streak.

12. Potato.

8. Differentiate by means of the characters of the
resulting cultural reactions into members of the coli
group, members of the Gaertner group, and members
of the typhoid group.

9. Confirm these results by testing the organisms
isolated with serum obtained from animals experi-

THE COLI AND TYPHOID GROUPS.

329

mentally protected against each of these groups of
bacteria.

ANALYSIS OF MEMBERS OF THE COLI AND TYPHOID GROUPS.

Lactose litmus bouillon.

Gas.

B. coli communis and its allies.

Gas in glucose gelatine.
Acid and coagulation in milk.
General turbidity and indol in bouillon.
Fermentation in saccharose media.

No gas

Gaertner and typhoid
groups.

Glucose gelatine.

Gas.

Gaertner.

No gas.

Typhoid.

Litrm

Acid
Alkal
Nocc

is milk.

at first,
ine later,
•agulation.

Bot

Get
No
• Ser

illon.

eral turbidity,
indol.
um reaction.

Litmus

Acid.
No co

milk. Bou

Gen
agulation. No
Sen

illon.

eral turbidity,
indol.
im reaction.

Alternative Methods. —
(A) The Carbolic Method:

1. Take ten tubes of carbolised bouillon (vide page
144) and number them consecutively from i to 10.

2. Inoculate each tube with a different amount of the
suspension, as in the previous method.

3. Incubate aerobically at 37° C.

4. Examine the culture tubes after twenty-four
hours' incubation.

5. From those tubes which show signs of growth,

330 BACTERIOLOGICAL ANALYSES.

pour plates in the usual manner, using carbolised
gelatine (vide page 149) in place of the ordinary gela-
tine, and incubate at 20° C. for three, four, or five days
as may be necessary.

6. Subcultivate from any colonies that make their
appearance, and determine their identity on the
lines laid down in the previous method.

(B) Parietti's Method:

1. Take nine tubes of Parietti's bouillon (vide
page 144) — i. e., three each of those containing o.i
c.c., 0.2 c.c., and 0.5 c.c. of Parietti's solution respec-
tively. Mark plainly on the outside of each tube the
quantity of Parietti's solution it contains.

2. To each tube add a different amount of the original
water, or of the suspension, and incubate at 37° C.

3. Examine the culture tubes after twenty-four and
forty-eight hours' incubation, and plate in gelatine
from such as have grown.

4. Pick off suspicious colonies, if any such appear
on the plates, subcultivate them upon the various
media, and identify them.

(C) Eisner's Method: This method simply consists
in substituting Eisner's potato gelatine (vide page 164)
for ordinary nutrient gelatine in any of the previously
mentioned methods.

(D) The Candle Method:

1. Remove the rubber stopper from the mouth of
the filter candle, introduce 10 c.c. sterile bouillon into
its interior, and emulsify the bacterial sediment; re-
plug the mouth of the candle with a wad of sterile
cotton- wool.

2. Remove the filter candle from the filter flask
and insert it into the mouth of a flask or a glass cylinder
containing sterile bouillon sufficient to reach nearly up
to the rubber washer on the candle.

3. Incubate for twenty-four to thirty-six hours at
37° C.

BACILLUS ENTERITIDIS SPOROGENES. 331

4. From the now turbid bouillon in the glass cylinder
pour gelatine plates and incubate at 20° C.

5. Subcultivate and identify any suspicious colonies
that appear.

(The method depends upon the assumption that
members of the typhoid and coli groups find their way
through the porcelain filter from the interior to the
surrounding bouillon at a quicker rate than the asso-
ciated bacteria.)

B. Enteritidis Sporogenes.—

1. Transfer 5 c.c. of the emulsion from the filter
candle to a sterile test-tube and plug carefully.

2. Place the test-tube in the interior of the benzole
bath employed in separating out spore-bearing organ-
isms (vide page 202), and expose to a temperature of
80° C. for twenty minutes.

3. Number ten tubes of litmus milk consecutively
from i to 10.

4. Remove the test-tube from the benzole bath and
shake well to distribute the spores evenly through
the fluid.

5. To each tube of litmus milk add a measured quan-
tity of the suspension corresponding to the amounts
employed in isolating the coli group (vide page 327).

6. Put up each tube anaerobically in a Buchner's
tube and incubate at 37° C.

7. Examine after twenty-four hours' incubation.
Note (if the B. enteritidis sporogenes is present)—

(a) Acid reaction of the medium as indicated by
the colour of the litmus or its complete decolourisation.

(b) Presence of clotting, and the separation of clear
whey.

(c) Presence of gas, as indicated by fissures and
bubbles in the coagulum, and possibly masses of
coagulum driven up the tube almost to the plug.

8. Replace the tubes which show no signs of growth
in the incubator for a further period of twenty-four

332 BACTERIOLOGICAL ANALYSES.

hours and again examine with reference to the same
points.

9. Remove those tubes which give evidence of growth
from the Buchner's tubes and carefully pipette off
the whey; examine the whey microscopically.

10. Inoculate two guinea-pigs subcutaneously with
0.5 c.c. of the whey each and observe the result.

Streptococci.—

1. Melt ten tubes of nutrient agar in boiling water
and cool to 42° C.

2. Number the tubes consecutively from i to 10.

3. To each of the tubes of liquefied agar add a
measured quantity of the emulsion, corresponding to
those amounts employed in isolating members of the
coli group (vide page 327).

4 Pour plates in the usual manner and incubate
aerobically at 37° C. for twenty-four hours.

5. Examine the plates carefully, pick off suspicious-
looking colonies, and subcultivate in broth.

6. If the resulting growth appears, microscopically,
to be composed of streptococci, subcultivate on the
various media and identify.

Vibrio Cholerse.—

1 . Number ten tubes of peptone water consecutively
from i to 10.

2. To each of the tubes of peptone water add a mea-
sured quantity of the suspension, corresponding to
those amounts employed in isolating the members of
the coli group (vide page 327).

3. Incubate aerobically at 37° C. for twenty-four
hours. Examine the tubes carefully for visible growth,
especially delicate pellicle formation, which if present
should be examined microscopically for vibrios.

4. Inoculate fresh tubes of peptone water from such
of the tubes as exhibit pellicle formation, — from the
pellicle itself, — and incubate at 37° C. for twenty-
four hours.

BACILLUS ANTHRACIS — BACILLUS TETANI. 333

5. Prepare gelatine and agar plates in the usual way
from such of these tubes as show pellicle formation.

6. Pick off from the plates any colonies resembling
those of the Vibrio cholerae and subcultivate upon all
the ordinary laboratory media.

7. Test the vibrio isolated against the serum of an
animal immunised to the Vibrio cholerae.

B. Anthracis.—

1. Transfer 5 c.c. of the emulsion from the filter
candle to a sterile test-tube and plug carefully.

2. Place the test-tube in the interior of the benzole
bath employed in separating out spore-bearing organ-
isms (vide page 202), and expose to a temperature
of 80° C. for twenty minutes.

3. Melt three tubes of nutrient agar in boiling water
and cool to 42° C.

4. Number the tubes i, 2, and 3. To No. i add
0.2 c.c., to No. 2 add 0.3 c.c., and to No. 3 add 0.5 c.c.
of the suspension, and pour plates therefrom.

5. Incubate at 37° C. for twenty-four or forty-
eight hours.

6. Pick off any colonies resembling those of anthrax
and subcultivate on all the ordinary laboratory media.

7. Inoculate a young white rat subcutaneously (on
the inner aspect of one of the hind legs) with two
loopfuls of the cultivation on agar, emulsified with
i c.c. sterile bouillon. Observe during life, and, if
the animal succumbs, make a complete post-mortem
examination.

B. Tetani.-

1. Proceed as detailed above in steps i and 2 for
the isolation of the B. anthracis.

2. Add i c.c. of the suspension to each of three tubes
of glucose formate broth, and incubate anaerobically
in Buchner's tubes at 37° C.

3. From such of the tubes as show visible growth
(with or without the production of gas) after twenty-

334 BACTERIOLOGICAL ANALYSES.

four hours' incubation inoculate guinea-pigs, subcu-
taneously (under the skin of the abdomen), using o.i
c.c. of the bouillon cultivation as a dose. Observe
carefully during life, arid, if death occurs, make a
complete post-mortem examination.

4. From the same tubes pour agar plates and in-
cubate anaerobically in Bulloch's apparatus, at 37° C.

5. Subcultivate suspicious colonies on the various
media, incubate anaerobically, making control cultiva-
tions on glucose formate agar, stab and streak, to
incubate aerobically.

EXAMINATION OF SEWAGE AND SEWAGE EFFLUENTS.

Quantitative. —

Collection of the Sample. — As only small quantities
of material are needed, the samples should be collected
in a manner similar to that described under water for
quantitative examination and transmitted in the ice
apparatus used in packing those samples.

Apparatus Required. — As for water (vide page 319),
substituting gelatine tubes for the agars and the
wort gelatines.

METHOD. —

1. Arrange four sterile capsules in a row and number
them I, II, III, IV.

2. Pipette 9 c.c. sterile bouillon into capsule No. I.

3. Pipette 9.9 c.c. sterile bouillon into capsules II,
III, and IV.

4. Add i c.c. of the sewage to capsule No. I by means
of a sterile pipette, and mix thoroughly.

5. Take a fresh sterile pipette and transfer o.i c.c.
of the mixture from No. I to No. II and mix thor-
oughly.

6. In like manner transfer o.i c.c. from No. II to
No. Ill, and then o.i c.c. from No. Ill to No. IV.

EXAMINATION OF AIR. 335

Now I c.c. of dilution No. I contains O. I c.c. of the original sewage.

I " " « " II " O.OOI " " " "

I " " *< «« HI " 0.00001 " " " "

! " « « « IV " 0.0000001 " " " "

7. Pour a set of gelatine plates from the contents
of each capsule, three plates in a set, and containing
respectively 0.2, 0.3, and 0.5 c.c. of the dilution.
Label carefully; incubate at 20° C. for three, four, or
five days.

8. Enumerate the organisms present in those sets
of plates which have not liquefied, probably those
from dilution III or IV, and calculate therefrom the
number present per cubic centimetre of the original
sample of sewage.

Qualitative. — The qualitative examination of sewage
is but rarely required. When necessary, however, it
is conducted on lines similar to those indicated under
the corresponding section of water examination.

EXAMINATION OF AIR.

Quantitative. —

Apparatus Required:

Aspirator bottle, 10 litres capacity, fitted with a delivery
tube, and having its mouth closed by a perforated rubber
stopper, through which passes a short length of glass
tubing.

Brlenmeyer flask, 250 c.c. capacity (having a wide mouth
properly plugged with wool), containing 50 c.c. sterile
bouillon.

Rubber stopper to fit the mouth of the flask, perforated
with two holes, and fitted as follows :

Take a 15 cm. length of glass tubing and bend up 3
cm. at either end at right angles to the main length of
tubing. Pass one of the bent ends through one of the
perforations in the stopper ; plug the opposite end with
cotton- wool.

Take a glass funnel 5 or 6 cm. diameter with a stem
12 cm. in length and bend the stem close up to the apex
of the funnel, in a gentle curve through a quarter of a
circle ; pass the long stem through the other perforation
in the rubber stopper.

336

BACTERIOLOGICAL ANALYSES.

Rubber tubing.

Screw clamps and spring clips, for tubing.
Water steriliser.
Retort stand and clamps.

Apparatus for plating (as for enumeration of water organ-
isms, -vide page 319).

METHOD.—

i. Fill 10 litres of water into the aspirating bottle
and attach a piece of rubber tubing with a screw
clamp to the delivery tube. Regulate the screw clamp,

Fig. 165. — Arrangement of apparatus for air analysis.

by actual experiment, so that the tube delivers i c.c.
of water every second.

At this rate the aspirator bottle will empty itself
in just under three hours. Occlude the rubber tube
below the screw clamp by means of a spring clip,
and make up the contents of the aspirator bottle to
10 litres again.

2. Sterilise the fitted rubber cork, with its funnel

EXAMINATION OX AIR. 337

and tubing, by boiling in the water steriliser for ten
minutes.

3. Remove the cotton- wool plug from the flask, and
replace it by the rubber stopper with its fittings. Make
sure that the end of the stem of the funnel is immersed
in the bouillon.

4. Place the flask in a glass or metal vessel and pack
it round with pounded ice. Arrange the flask with its
ice casing just above the neck of the aspirator bottle.

5. Connect up the free end of the glass tube from the
flask — after removing the cotton- wool plug — with the
air-entry tube in the mouth of the aspirating bottle.

6. Remove the spring clip from the rubber tube, and
allow the water to run.

Replenish the ice from time to time if necessary.
(In emptying itself the aspirator bottle will aspirate
10 litres of air slowly through the broth in the Brlen-
meyer flask.)

7. When the aspiration is completed, disconnect the
flask and remove it from its ice packing.

8. Liquefy three tubes of nutrient gelatine and add
to them 0.5 c.c., 0.3 c.c., and 0.2 c.c., respectively, of
the broth from the flask, by means of a sterile gradu-
ated pipette, as in the quantitative examination of
water. Pour plates.

9. Pour a second similar set of gelatine plates.

10. Incubate both sets of plates at 20° C.

11. Enumerate the colonies present in the two sets
of gelatine plates after three, four, or five days and
average the results from the numbers so obtained;
estimate the number of micro-organisms present in
i c.c., and then in the 50 c.c. of broth in the flask.

12. The result of air examination is usually expressed
as the number of bacteria present per cubic metre
(i. e.t kilolitre) of air; and as the number of organisms
present in the 50 c.c. bouillon only represent those

338 BACTERIOLOGICAL ANALYSES.

contained in 10 litres of air, the resulting figure must
be multiplied by 1000.
Qualitative. —

1. Proceed exactly as in the quantitative examina-
tion of air (vide supra), steps i to 10.

2. Pour plates of wort agar with similar quantities
of the air-infected bouillon, and incubate at 37° C.

3. Pour plates of nutrient agar with similar quan-
tities of the bouillon and incubate at 37° C.

4. Pour similar plates of wort gelatine and incubate
at 20° C.

5. Pick off the individual colonies that appear in
the several plates, subcultivate them on the various
media, and identify them.

EXAMINATION OF SOIL.

Collection of Sample. — A small copper capsule 6 cm.
high by 6 cm. diameter, with "pull-off" cap secured
by a bayonet catch, previously sterilised in the hot-
air oven, is the most convenient receptacle for samples
of soil.

The instrument used for the actual removal of the
soil from its natural position will vary according to

Fig. 1 66. — Soil scoop.

whether we require surface samples or soil from vary-
ing depths. In the first case, use an iron scoop, shaped
like a shoe horn, but provided with a sharp spine
(Fig. 1 66). This is wrapped in asbestos cloth and
sterilised in the hot-air oven. When removed from
the oven, wrap in a piece of oiled paper, silk, or gutta-

EXAMINATION OF SOIL.

339

percha tissue, secured with string, as a further protec-
tion against contamination.

On reaching the spot whence the samples are to be
taken, the coverings of the scoop are removed, and the
asbestos cloth employed to brush away loose stones
and debris from the selected area. The surface soil
is then broken up with the point of the scoop, scraped
up and collected in the body of the scoop, and trans-
ferred to the sterile cap-
sule for transmission.

If it is desired to ob-
tain samples of the earth
from varying depths,
some form of borer, such
as that designed by
Fraenkel (sterilised in a
manner similar to that
adopted for the scoop),
must be employed for
the purpose (Fig. 167).

Quantitative. — Four
distinct investigations
are included in the com-
plete quantitative bac-
teriological examination
of the soil:

1 . The enumeration of
the aerobic organisms.

2. The enumeration of
the spores of aerobes.

3. The enumeration of the anaerobic organisms (in-
cluding the facultative anaerobes).

4. The enumeration of the spores of anaerobes.
Further, by a combination of the results of the first

and second, and of the third and fourth of these,
the ratio of spores to vegetative forms is obtained.

Fig. 167. — Fraenkel' s borer.

340 BACTERIOLOGICAL ANALYSES.

Apparatus Required:

Case of sterile capsules (25 c.c. capacity).

Case of sterile graduated pipettes, 10 c.c. (in tenths of a

cubic centimetre).
Case of sterile graduated pipettes, i c.c. (in tenths of a

cubic centimetre).

Flask containing 250 c.c. sterile bouillon.
Tall cylinder containing 2 per cent, lysol solution.
Plate-levelling stand.
12 sterile plates.
Tubes of nutrient gelatine.
Tubes of wort gelatine.
Tubes of nutrient agar.
Tubes of glucose formate gelatine.
Tubes of glucose formate agar.
Water-bath regulated at 42° C.
Bunsen burner.
Grease pencil.

Sterile mortar and pestle (agate).

Sterile wide-mouthed Erlenmeyer flask (500 c.c. capacity).
Sterile metal funnel with short wide bore delivery tube to

just fit mouth of flask.

Solid rubber stopper to fit the flask (sterilised by boiling).
Pair of scales.
Counterpoise (Fig. 88).
Sterile metal (nickel) spoon or spatula.
Fractional steriliser (Fig. 113).

METHOD. —

1. Arrange four sterile capsules numbered I, II, III,
and IV; pipette 9 c.c. sterile bouillon into the first
capsule, and 9.9 c.c. into each of the remaining three.

2. Pipette 100 c.c. sterile bouillon into the Krlen-
meyer flask.

3. Remove the cotton- wool plug from the flask and
replace it by the sterile funnel.

4. Place flask and funnel on one pan of the scales,
and counterpoise accurately.

5. Empty the sample of soil into the mortar and
triturate thoroughly.

6. By means of the sterile spatula add 10 grammes
of the earth sample to the bouillon in the flask.

The final results will be more reliable if steps 2, 3, 4,

EXAMINATION OF SOIL. 341

and 5 are performed under a hood — to protect from
falling dust, etc.

7. Remove the funnel from the mouth of the flask;
replace it by the rubber stopper and shake vigourously ;
then allow the solid particles to settle for about a
minute.

8. Pipette off i c.c. of the supernatant bouillon,
termed the "soil water," and add it to the contents
of capsule I ; mix thoroughly.

9. Remove o. i c.c. of the infected bouillon from
capsule I and add it to capsule II, and mix.

10. In like manner add o.i c.c. of the contents of
capsule II to capsule III and then o.i c.c. of the
contents of capsule III to capsule IV.

Then I c.c. fluid from capsule I contains soil water from .01 gm. earth.

I « " " « II " " " " .0001 "

I « « «« " III " " " " .oooooi " "
I « « " « IV " " " " .oooooooi " "

(A) Aerobes (Vegetative Forms and Spores). —

11. Pour a set of gelatine plates from the contents
of each capsule — two plates in a set, and containing
respectively o.i c.c. and 0.3 c.c. of the diluted soil
water. Label and incubate.

12. Pour similar sets of wort gelatine plates from the
contents of capsule II and III, label, and incubate at
20° C.

13. Pour similar sets of agar plates from the contents
of capsules II and III; label and incubate at 37° C.

14. "Count" the plates after incubation for three,
four, or five days, and from the figures thus obtained
estimate —

(a) The number of aerobic micro-organisms present
per gramme of the soil.

(b) The number of yeasts and moulds present per
gramme of the soil.

342 BACTERIOLOGICAL ANALYSES.

(B) Anaerobes (Vegetative Forms and Spores). —

15. Pour similar sets of plates in glucose formate
gelatine and agar and incubate in Bulloch's anaerobic
apparatus.

1 6. Pipette 5 c.c. soil water into a sterile tube.

17. Place in the differential steriliser at 80° C. for
ten minutes.

1 8. Pour two sets of four gelatine plates containing
o.i, 0.2, 0.5, and i c.c. respectively of the soil water;
label and incubate at 20° C., one set aerobically, the
other anaerobically in Bulloch's apparatus.

19. "Count" the plates (delay the enumeration as
long as possible) and estimate the number of spores
of aerobes and anaerobes respectively present per
gramme of the soil.

20. Calculate the ratio existing between spores
alone and the total number of organisms.

Qualitative Examination. — The qualitative examina-
tion of soil is usually directed to the detection of one
or more of the following :

I. Members of the coli or typhoid group.
II. Bacillus enteritidis sporogenes.

III. Streptococci.

IV. Bacillus anthracis.
V. Bacillus tetani.

VI. Bacillus oedematis maligni.
VII. The nitrous organisms.
VIII. The nitric organisms.

1. Transfer the remainder of the soil water (88
c.c.) to a sterile Erlenmeyer flask by means of a sterile
syphon.

2. Fix up the filtering apparatus as for the qualita-
tive examination of water, and filter the soil water.

3. Suspend the bacterial residue in 5 c.c. sterile
bouillon (technique similar to that described for the
water sample).

EXAMINATION OF SOU,. 343

Every cubic centimetre of suspension now contains
the soil water from nearly i gramme of earth.

The methods up to this point are identical no matter
which organism or group of organisms it is desired
to isolate; but from this stage onwards the process is
varied slightly for each particular bacterium.

The Coli Group.—

Bacillus Enteritidis Sporogenes. —

Bacillus Anthracis.—

Bacillus Tetani.—

The methods adopted for the isolation of these
organisms are identical with those already described
under water (page 327 et seq.).

Bacillus (Edematis Maligni. — Method precisely sim-
ilar to that employed for the B. tetani.

The Nitrous Organisms.—

The Nitric Organisms.—

i. Take six tubes of Warrington's solution (vide
page 172) and number them consecutively from i to 6.

2. To tube No. i add o. i c.c. of the suspension.

« « « 2 c 02 « « « i

" " " 3
« « « 4

" " '" 5

" " « 6

0-5
i.o

2.5

Label and incubate at 30° C.

3. Examine after twenty-four and forty-eight hours'
incubation. From those tubes that show signs of
growth make subcultivations in fresh tubes of the
same medium and incubate at 30° C.

4. Make further subcultivations from such of those
tubes as show growth, and again incubate.

5. If growth occurs in these subcultures, make sur-
face smears on plates of Winogradsky's silicate jelly
(vide page 172).

6. Pick off such colonies as make their appearance
and subcultivate in each of these two media.

344 BACTERIOLOGICAL ANALYSES.

EXAMINATION OF MILK.

Quantitative. —

Collection of Sample. — " One-cow" milk, if taken
from the apparently healthy animal (that is, an animal
without any obvious lesion of the udder or teats)
with ordinary precautions as to cleanliness, avoidance
of dust, etc., contains but few organisms, and may
be received directly into small sterile bottles (similar
to those referred to under the collection of water for
quantitative examination), packed in the ice-box for
transmission, and dealt with in precisely the same
manner as an ordinary water sample. In dealing with
one-cow milk, from a suspected, or an obviously dis-
eased animal, a complete analysis should include the
examination (both qualitative and quantitative) of
samples of (a) fore-milk, (b) mid-milk, (c) strippings,
and, if possible, from each quarter of the udder, and
the specimen should then be collected as described
for mixed milk.

"Mixed" milk, on the other hand, by the time it
leaves the retailer's hands, usually contains as many
micro-organisms as an equal volume of sewage, and it
becomes necessary to adopt special methods of collec-
tion, and, when collected, to estimate the number of
its contained bacteria by the methods employed in
the examination of sewage.

The apparatus used for the collection of a retail
mixed milk sample consists of a cylindrical copper case,
1 6 cm. high and 9 cm. in diameter, provided with a
"pull-off" lid, containing a milk dipper, also made of
copper; and inside this, again, a wide-mouthed, stop-
pered glass bottle of 200 c.c. capacity (about 14 cm.
high by 7 cm. diameter), having a tablet for notes,
sand-blasted on the side. The copper cylinder and its
contents, secured from shaking by packing with cot-
ton-wool, are sterilised in the hot-air oven.

EXAMINATION OF MILK.

345

When collecting a sample,

1. Remove the cap from the cylinder.

2. Draw out the cotton-wool.

3. Lift out the bottle and dipper together.

4. Receive the milk in the sterile dipper, and pour it
directly into the sterile bottle.

5. Enter the particulars necessary for the identi-
fication of the specimen, on the tablet, with a lead
pencil, or pen and ink.

6. Repack the apparatus.

Fig. 1 68. — Milk-collecting bottle and dipper.

Four such bottles should be filled, so as to give a
total of about 800 c.c. milk for examination. The four
cases may be packed in an ice-box similar to, but
larger than, that used for water specimens.

Apparatus Required:

Case of sterile capsules (25 c.c. capacity).
Case of sterile graduated pipettes, 10 c.c. (in tenths of a
cubic centimetre).

346 BACTERIOLOGICAL ANALYSES.

Case of sterile graduated pipettes, i c.c. (in tenths of a

cubic centimetre).

Flask containing 250 c.c. sterile bouillon.
Tall cylinder containing 2 per cent, lysol solution.
Plate-levelling stand.
Case of sterile plates.

Tubes nutrient gelatine agar (-J- 10 reaction).
Tubes of wort gelatine.
Tubes of nutrient agar (+10 reaction).
Water-bath regulated at 42° C.
Bunsen burner.
Grease pencil.

METHOD.—

1. Arrange four sterile capsules in a row; number
them I, II, III, and IV.

2. Fill 9 c.c. sterile bouillon into the first, and 9.9
c.c. bouillon into each of the three remaining capsules.

3. Remove i c.c. milk from one of the bottles by
means of a sterile, pipette and dilute it exactly as
described for sewage (vide page 334).

Then I c.c. of dilution I contains O.I c.c. milk sample.

I " « •« II " o.ooi " "

i " " " III " o.ooooi " " "

i « «« " IV " o.ooooooi " " "

4. Melt the gelatine agar and the agar tubes in
boiling water; then transfer to the water-bath and
cool them down to 42° C.

5. Number the gelatine agar tubes consecutively i
to 8.

6. .To the first three tubes add 0.2 c.c., 0.3 c.c., and
0.5 c.c. respectively of the diluted milk from capsule
IV.

7. To the second set of three tubes add similar
quantities of the diluted milk from capsule III.

8. To the two remaining tubes add o.i c.c. and 0.2
c.c. of the diluted milk from capsule II.

9. Pour plates from the eight gelatine agar tubes;
label, and incubate at 28° C. (or 30° C.).

10. Liquefy three wort gelatine tubes and to them

EXAMINATION OF MILK. 347

add o.i c.c. of the diluted milk from capsules I, II,
and III respectively.

11. Pour plates from the wort gelatine; label, and
incubate at 20° C.

12. Add to each of three agar tubes o.i c.c. of the,
diluted milk from capsule II, III, and IV.

13. Pour plates from the agar tubes; label, and in-
cubate at 37° C.

14. After twenty-four and forty-eight hours' incu-
bation, " count" the agar plates and estimate the
number of "organisms growing at 37° C." present
per cubic centimetre of the sample of milk.

15. After three, four, or five days' incubation,
"count" the gelatine agar plates and estimate there-
from the total number of organisms present per cubic
centimetre of the sample of milk.

1 6. After a similar interval "count" the wort
gelatine plates and estimate the number of moulds
and yeasts present per cubic centimetre of the sample
of milk.

Qualitative. — The qualitative bacteriological exam-
ination of milk is chiefly directed to the detection of the
presence of one or more of the following pathogenic
bacteria :

I. Members of the typhi and coli groups.
II. Bacillus enteritidis of Gaertner.

III. Bacillus enteritidis sporogenes.

IV. Vibrio cholerae. *«,
V. Bacillus diphtheriae.

~ VI. Bacillus tuberculosis.

VII. Streptococcus pyogenes longus.

VIII. Staphylococcus pyogenes aureus.

Of these, the first six occur as accidental contamina-
tions (the vehicle of transmission in the case of the
first five usually being water), while the last three are
usually derived directly from the cow.

In milk, as in water, the first essential is the con-

34-8 BACTERIOLOGICAL ANALYSES.

centration of the bacterial contents of a large volume
of the sample into a small compass. In this process,
however, thorough centrifugalisation is substituted for
nitration.

Apparatus Required:

, A centrifugal machine. This machine, to be of real ser-
vice in the bacteriological examination of milk, must
conform to the following requirements :

1. The centrifugal machine must be of such size, and

should carry tubes or bottles of such capacity, as
to enable from 250 to 500 c.c. of milk to be
manipulated at one time.

2. The rate of centrifugalisation should be from 2500

to 3000 revolutions per minute.

3. The portion of the machine destined to carry the

tubes should be a metal disc, of sufficient weight
to ensure good "flank" movement, continuing
over a considerable period of time. In other
words, the machine should run down very
gradually and slowly after the motive power is
removed, thus obviating any disturbance of the
relative positions of particulate matter in the
solution that is being centrifugalised.

4. The machine should preferably be driven by elec-

tricity, or by power, but in the case of hand-
driven machines —

(a) The gearing should be so arranged that

the requisite speed is obtained by not
more than forty or fifty revolutions of
the crank handle per minute, so that it
may be maintained for periods of twenty
or thirty minutes without undue exer-
tion.

(b) The handle employed should be provided

with a special fastening (e. g., a clutch
similar to that employed for the free
wheel of a bicycle), or should be readily
detachable so that, on ceasing to turn,
the handle should not, by its weight and
air resistance, act as a brake and stop
the machine too suddenly.
One of the few satisfactory machines of this class is

shown in figure 169.

y Sterile centrifugal tubes, of some 60-70 c.c. capacity,
tapering to a point at the closed end, plugged with
cotton- wool.

EXAMINATION OF MII^K. 349

u Sterilised cork borer.

t/ Case of sterile pipettes, 10 c.c. (in tenths of a cubic centi-
metre) .

Case of sterile pipettes, i c.c. (in tenths of a cubic centi-
metre).
Flask of sterile bouillon.

Fig. 169. — Electrically driven centrifugal machine.

METHOD.—

1. Fill the milk sample into the tubes, and replace
the cotton-wool plugs by solid rubber stoppers (steril-
ised by boiling), and fit the tubes in the centrifugal
machine.

2. Centrifugalise the milk sample for twenty to
thirty minutes at a speed of 2500 revolutions per
minute.

3. Remove the motive power and allow the machine
to slow down gradually.

350

BACTERIOLOGICAL ANALYSES.

4. Remove the tubes of milk from the centrifuge.
Each tube will now show (Fig. 170):

(a) A superficial layer of cream (varying in thickness
with different samples) churned into a semi-solid mass,
which can be shown to contain some organisms and a
few leucocytes.

(b) A central layer of separated milk, thin, watery,
and opalescent, and containing extremely few bacteria.

majority of the contained bacteria and
leucocytes, together with adventitious
matter, such as dirt, hair, epithelial
cells, etc.

•f .5. Withdraw the rubber stopper and
remove a central plug of cream from
each tube by means of a sterile cork
borer; place these masses of cream in
sterile capsules.

,/ 6. Remove all but the last 3 or 4 c.c.
of separated milk from each tube, by
means of sterile pipettes.

7. Mix the deposits thoroughly with
the residual milk, pipette the mixture
from each tube into a fresh sterile tube,
and mix together; then fill with sterile
bouillon (or normal saline solution).

8. Place the mixed deposits in the
centrifuge, counterpoise with another tube containing
an equal volume of water, and centrifugalise, as before.

9. Pipette off all the supernatant fluid and invert
the tube to drain on to a pad of sterilised cotton- wool,
contained in a beaker. (This wool is subsequently
cremated.)

10. Examine both cream and deposit microscopi-
cally—

(a) In hanging drops.

(b) In film preparations stained carbolic methylene-

Fig. 170. — Milk in
centrifuge tube.

EXAMINATION OF MILK. 351

blue, Gram's method, Neisser's method, Ziehl-Neelsen's
method.

ii. Adapt the final stages of the investigation to
the special requirements of each individual sample,
as follows :

Members of the Typhoid and Colon Groups. —

Bacillus Enteritidis of Qaertner. —

Bacillus Enteritidis Sporogenes. —

Vibrio Cholerse.—

When searching for any or either of these organisms,
emulsify the deposit with 10 c.c. sterile bouillon and
proceed with the examination as described under
water.

NOTE. — The B. coli communis, derived from the
alvine discharges of the cow, is almost universally
present in large or small numbers, in retail milk. Its
detection, therefore, unless in enormous numbers,
when it indicates want of cleanliness, is of little value.

B. Diphtherias.—

(A) i. Plant three sets of serial cultivations from
(a) cream (twelve tubes in each set) upon oblique
inspissated blood-serum, (6) deposit (twelve tubes in
each set) upon oblique inspissated blood-serum, and
incubate at 37° C.

2. Pick off any suspicious colonies which may have
made their appearance nine hours after incubation,
and subcultivate upon blood-serum; return the
original tubes to the incubator.

3. Repeat this after eighteen hours' incubation.

4. From the resulting growths make cover-slip
preparations and stain carbolic methylene-blue, Neis-
ser's method, Gram's method.

5. Inoculate guinea-pigs subcutaneously with forty-
eight-hour-old glucose bouillon cultivation derived
from the first subcultivation, and observe the result.

6. Inoculate guinea-pigs subcutaneously with filtered

352 BACTERIOLOGICAL ANALYSES.

glucose bouillon cultivations (toxins ?) and observe
the result.

(B) i. Emulsify the remainder of the deposit with
3 c.c. sterile bouillon and inoculate two guinea-pigs,
thus : guinea-pig a, subcutaneously with i c.c. emulsion;
guinea-pig 6, subcutaneously with 2 c.c. emulsion; and
observe the result.

2. If either or both of the inoculated animals suc-
cumb, make complete post-mortem examination and
endeavour to isolate the pathogenic organisms from
the local lesion.

Bacillus Tuberculosis. — Add 5 c.c. sterile bouillon
to the deposit in the tube and emulsify thoroughly.

(A) i. Inoculate each of three guinea-pigs (previ-
ously tested with tuberculin, to prove their freedom
from spontaneous tuberculosis) subcutaneously at the
inner aspect of the bend of the left knee, with i c.c.
of the emulsion.

2. Introduce a small quantity of the cream into a
subcutaneous pocket prepared at the inner aspect of
the bend of the right knee of each of these three animals.
Place a sealed dressing on the wound.

3. Observe carefully, and weigh accurately each
day.

4. Kill one guinea-pig at the end of the second
week and make a complete post-mortem examination.
(Compare Fig. 158, page 307.)

5. If the result of the examination is negative or
inconclusive, kill a second guinea-pig at the end of the
third week and examine carefully.

6. If still negative or inconclusive, kill the third
guinea-pig at the end of the sixth week. Make a careful
post-mortem examination.

(B) i. Place the tube containing the remainder of
the emulsion in a water-bath at 56° C. for ten minutes.

2. Remove the tube from the water-bath and cool
rapidly.

EXAMINATION OF MILK. 353

3. Inoculate each of two guinea-pigs, intraperi-
toneally, with i c.c. of the emulsion.

4. Kill one of the guinea-pigs at the end of the
first week and examine carefully.

5. Kill the second guinea-pig at the end of the second
week and examine carefully.

NOTE. — No value whatever attaches to the result
of a microscopical examination for the presence of the
B. tuberculosis unless confirmed by the result of
inoculation experiments.

Streptococcus Pyogenes Longus. —

(A) i. Plant serial cultivations from the deposit
upon (a) oblique inspissated blood-serum (six tubes
in series) and (6) oblique nutrient agar (six tubes in
series).

2. If the resulting growth shows colonies which
resemble those of the streptococcus, make subcultiva-
tions upon agar and in bouillon in the first instance
and study carefully.

(B) i. Plant a large loopful of the deposit into each
of three tubes of glucose formate bouillon, and incu-
bate anaerobically (in Buchner's tubes) for twenty-
four hours at 37° C.

2. If the resulting growth resembles that of the
streptococcus, make subcultivations upon nutrient
agar.

3. Prepare subcultivations of any suspicious colonies
that appear, upon all the ordinary media, and study
carefully.

If the streptococcus is successfully isolated, inocu-
late serum bouillon cultivations into the mouse,
guinea-pig, and rabbit, to determine its pathogenicity
and virulence.

Staphylococcus Pyogenes Aureus. —

i. Prepare serial cultivations upon oblique nutrient
agar (eight tubes in series).
23

354 BACTERIOLOGICAL ANALYSES.

2. Incubate at 37° C. for twenty-four hours.

3. Pick off any suspicious colonies, plant on oblique
agar, and incubate at 20° C. Observe pigment forma-
tion.

4. Prepare subcultivations from any suspicious
growths upon all the ordinary media and study care-
fully.

ICE CREAM.

Collection of the Sample. —

1. Remove the sample from the drum in the ladle
or spoon with which the vendor retails the ice cream,
and place it at once in a sterile copper capsule, similar
to that employed for earth samples.

2. Pack for transmission in the ice-box.

3. On arrival at the laboratory place the copper
capsules containing the ice cream in the incubator at
20° C. for fifteen minutes — that is, until at least some
of the ice cream has become liquid.

Qualitative and Quantitative Examination. — Treat
the fluid ice cream as milk and conduct the examina-
tion in precisely the same manner as described for
milk (vide page 346).

EXAMINATION OF CREAM AND BUTTER.

Collection of the Sample. — Collect, store, and trans-
mit samples to the laboratory, precisely as is done in
the case of ice cream.

Quantitative. —

Apparatus Required:
Sterile test-tube.
Sterilised spatula.
Water-bath regulated at 42° C.
Case of sterile plates.

Case of sterile graduated pipettes, i c.c. (inhundredths).
Tubes of gelatine-agar (-J- 10 reaction).
Plate-levelling stand, with its water chamber filled with
water at 42° C.

EXAMINATION OF CREAM AND BUTTER. 355

METHOD. —

1 . Transfer a few grammes of the sample to a sterile
test-tube by means of the sterilised spatula.

2. Piace the tube in the water-bath at 42° C. until
the contents are liquid.

3. Liquefy eight tubes of gelatine- agar and place
them in the water-bath at 42° C., and cool down to
that temperature.

4. Inoculate the gelatine-agar tubes with the fol-
lowing quantities of the sample by the help of a sterile
pipette graduated to hundredths of a cubic centimetre

—viz., o.i, 0.2, 0.3, 0.5, o.oi, 0.02, 0.03, and 0.05 c.c.

5. Pour a plate cultivation from each of the gelatine-
agar tubes and incubate at 28° C.

6. "Count" the plates after three days' incubation,
and from the figures thus obtained estimate the number
of organisms present per cubic centimetre of the sample.

Qualitative. —

Apparatus Required:

Sterile beaker, its mouth plugged with sterile cotton- wool.

Scales and weights.

Sterilised spatula.

Water-bath regulated at 42° C.

Separatory funnel, 250 c.c. capacity, its delivery tube
protected against contamination by passing it through
a cotton-wool plug into the interior of a small Erlen-
meyer flask which serves to support the funnel. This
piece of apparatus is sterilised en masse in the hot-air
oven.

Centrifugal machine.

Sterile tubes (for the centrifuge) closed with solid rubber
stoppers.

Case of sterile pipettes.

METHOD.—

1. Weigh out 100 grammes of the sample in a sterile
beaker.

2. Plug the mouth of the beaker with sterile cotton-
wool and immerse the beaker in a water-bath at 42° C.
until the contents are completely liquefied.

356 BACTERIOLOGICAL ANALYSES.

3. Fill the liquefied butter into the sterile separatory
funnel.

4. Transfer the funnel to the incubator at 37° C.
and allow it to remain there for four days.

At the end of this time the contents of the funnel
will have separated into two distinct strata.

(a) A superficial oily layer, practically free from
bacteria.

(b) A deep watery layer, turbid and cloudy from
the growth of bacteria.

5. Draw off the subnatant turbid layer into sterile
centrifugal tubes, previously warmed to about 42° C.,
and centrifugalise at once.

6. Pipette off the supernatant fluid and fill the tubes
with sterile i per cent, sodium carbonate solution
previously warmed slightly; stopper the tubes and
shake vigour ously for a few minutes.

7. Centrifugalise again.

8. Pipette off the supernatant fluid; filling the tubes
with sterile bouillon, shake well, and again centrif-
ugalise, to wash the deposit.

9. Pipette off the supernatant fluid.

10. Prepare cover-slip preparations, fix and clear
as for milk preparations, stain carbolic methylene-blue,
Gram's method, Ziehl-Neelsen's method, and examine
microscopically with a y^-inch oil-immersion lens.

11. Proceed with the examination of the deposit
as in the case of milk.

EXAMINATION OF UNSOUND MEATS.

(INCLUDING TINNED OR POTTED MEATS, FISH, ETC.)

Qualitative. —

Apparatus Required:

Erlenmeyer flask (500 c.c. capacity) containing 250 c.c.

sterile bouillon and fitted with solid rubber stopper.
Scissors and forceps.

EXAMINATION OF UNSOUND MEATS. 357

Water steriliser.

Hypodermic syringe.

Case of sterile capsules.

Filtering apparatus as for water analysis.

Case of sterile plates.

Case of sterile graduated pipettes, 10 c.c. (in tenths of a

cubic centimetre).
Case of sterile graduated pipettes, i c.c. (in tenths of a

cubic centimetre).
Plate-levelling stand.
Tubes of nutrient gelatine.
Tubes of nutrient agar.
Water-bath regulated at 42° C.
Bulloch's apparatus.

METHOD.—

(A) i. Mince a portion of the sample by the aid of
sterile scissors and forceps, and add the minced sample
to the bouillon in the flask.

2. Make an extract by standing the flask in the
water-bath at 42° C. for half an hour, shaking its
contents from time to time.

3. Pipette off 10 c.c. of the extract into a sterile
test-tube and remove for use under section C.

4. Filter the extract through a sterile Berkfeld
filter.

5. Emulsify the bacterial residue with 10 c.c. sterile
bouillon.

6. Pour a set of gelatine and a set of agar plates from
tubes containing 0.2, 0.3, and 0.5 c.c. of the extract,
and incubate aerobically, the gelatine set at 20° C.,
the agar at 37° C.

7. Pour duplicate sets of gelatine and agar plates
and incubate anaerobically in Bulloch's apparatus,
at similar temperatures.

8. Subcultivate from the colonies that make their
appearance and identify the various organisms.

9. Continue the investigations with reference to the
detection of pathogenic organisms as described under
water (page 327 et seq.).

BACTERIOLOGICAL ANALYSES.

(B) i. Feed rats and mice on portions of the sample
and observe the result.

2. If any of the animals die, make complete post-
mortem examinations and endeavour to isolate the
pathogenic organisms.

(C) i. Inoculate rats, mice, and guinea-pigs sub-
cutaneously and intraperitoneally with various quan-
tities of the bouillon extract, and observe the result.

2. If any of the animals succumb to the inocula-
tion, make careful post-mortem examinations and en-
deavour to isolate the pathogenic organisms.

EXAMINATION OF FILTERS.

Porcelain filter candles are examined with reference
to their power of holding back all the micro-organisms
suspended in the fluids which are filtered through
them, and permitting the passage of only germ-free
filtrates. The examination is conducted as follows:

Apparatus Required:

Filtering apparatus — one or other of those described under
Examination of Water. The actual filter candle that is
used must be the one it is intended to test ; the arrange-
ment of the apparatus will therefore need to be varied
with each different form of filter (see also pages 47, 48).

Plate-levelling stand.

Case of sterile plates.

Case of sterile pipettes, 10 c.c. (in tenths).

Case of sterile pipettes, i c.c. (in tenths).

Tubes of nutrient gelatine.

Flask containing sterile normal saline solution.

Sterile measuring flask, 1000 c.c. capacity.

METHOD. —

1. Prepare surface cultivations, on nutrient agar in
a culture bottle, of the Bacillus prodigiosus, and incu-
bate at 20° C., for forty-eight hours.

2. Pipette 5 c.c. sterile normal saline into the culture
bottle and emulsify the entire surface growth in it.

3. Pipette the emulsion into the sterile measuring

EXAMINATION OF DISINFECTANTS. 359

flask and dilute up to 1000 c.c. by the addition of
sterile water.

4. Pour the emulsion into the filter reservoir and
start the filtration.

5. When the filtration is completed, pour six gela-
tine plates each containing i c.c. of the filtrate.

6. Incubate at 20° C. until, if necessary, the comple-
tion of seven days.

EXAMINATION OF DISINFECTANTS.

Disinfectants or Germicides are examined with
reference to three points:

(A) Inhibition coefficient — i. e.,that percentage of
the disinfectant present in the nutrient medium
which is sufficient to prevent the growth and multi-
plication of bacteria therein.

(B) Inferior lethal coefficient — i. e., the time ex-
posure necessary to kill vegetative forms suspended
in water at 20° to 25° C., in which the disinfectant is
present in medium concentration (concentration in-
sufficient to cause plasmolysis).

(C) Superior lethal coefficient — i. e.y the time ex-
posure necessary to kill spores under conditions similar
to those obtaining in B.

The methods here detailed only specifically refer
to those disinfectants mentioned under Germicides in
the Scheme for the Study of Bacteria (page 252), but
the technique is practically similar for all other chemi-
cal disinfectants.

Inhibition Coefficient. —

Apparatus Required:

Case of sterile pipettes, 10 c.c. (in tenths).

Case of sterile pipettes, i c.c. (in tenths).

Sterile tubes or capsules for dilutions.

Tubes of nutrient bouillon.
Materials Required:

i . Five per cent, aqueous solution of carbolic acid.

360 BACTERIOLOGICAL ANALYSES.

2. One per cent, aqueous solution of perchloride of mer-

cury.

3. One-tenth per cent, aqueous solution of formaldehyde.

METHOD.—

1. Prepare a series of six tube cultivations, in
bouillon (each tube containing 10 c.c. of medium),
of each organism employed in the test and add 2 c.c.
of the 5 per cent, carbolic acid solution (i : 100) to
the first, i c.c. (i : 200) to the second, 0.6 c.c.
(i : 300) to the third, 0.5 c.c. (i : 400), to the fourth,
0.4 c.c. (i : 500) to the fifth, and 0.2 c.c. (i : 1000) to
the sixth.

2. Prepare a similar series of tube cultivations and
add o.i c.c. (i : 1000), 0.05 c.c. (i : 2000), 0.03 c.c.
(i : 3000), 0.025 c.c. (i : 4500), 0.02 c.c. (i : 5000), and
o.oi c.c. (i : 10,000) of the i per cent, perchloride of
mercury solution.

3. Prepare a similar series of tube cultivations and
add i c.c. (i : 1000), 0.4 c.c. (i : 2500), 0.2 c.c. (i : 5000),
o.i c.c. (i : 10,000), 0.075 c.c. (i : 15,000), and 0.05
c.c. (i : 20,000) of the o.i per cent, formaldehyde
solution.

4. Incubate all three sets of cultivations under opti-
mum conditions as to temperature and atmosphere.

5. Examine each of the culture tubes from day to
day, until the completion of seven days, and note
those tubes, if any, in which growth takes place.

Inferior Lethal Coefficient. —

Apparatus Required:

Highly concentrated solutions of the disinfectants.

Sterile test-tubes in which to make dilutions from the con-
centrated solutions of the disinfectants.

Hanging-drop slides.

Cover-slips.

Krlenmeyer flask containing 100 c.c. sterile distilled water.

Case of sterile pipettes, 10 c.c. (in tenths of a cubic centi-
metre).

Case of sterile pipettes, i c.c. (in tenths of a cubic centi-
metre).

EXAMINATION OF DISINFECTANTS. 361

METHOD.—

1. Prepare a surface cultivation of each of the
"test" organisms upon nutrient agar in a culture
bottle and incubate under optimum conditions for
forty-eight hours; then examine the cultivation micro-
scopically to determine the absence of spores.

2. Prepare solutions of different percentages of each
disinfectant.

3. Make a series of hanging-drop preparations from
the agar culture, using a loopful of disinfectant solu-
tion of the different percentages to prepare the emulsion
on each cover-slip.

4. Examine microscopically and note the strongest
solution which does not cause plasmolysis and the
weakest solution which does plasmolyse the organism.

5. Make control preparations of these two solutions
and determine the percentage to be tested. .

6. Pipette 10 c.c. sterile water into the culture
bottle and suspend the entire surface growth in it.

7. Transfer the suspension to the Erlenmeyer flask
and mix it with the 90 c.c. of sterile water remaining
in the flask.

8. Pipette 10 c.c. of the diluted suspension into each
of ten sterile test-tubes.

9. Label one of the tubes "Control" and place it
in the incubator at 20° C.

10. Add to each of the remaining tubes a sufficient
quantity of a concentrated solution of the disinfectant
to produce the percentage previously determined upon
(vide step 5).

11. Incubate the tubes at 20° C.

12. At hourly intervals remove the control tube
and one of the tubes with added disinfectant from the
incubator.

13. Make a subcultivation from both the control and
the test suspension, upon the surface of nutrient agar;
incubate under optimum conditions.

362 BACTERIOLOGICAL, ANALYSES.

14. Observe these culture tubes from day to day
until the completion of seven days, and determine
the shortest exposure necessary to cause the death of
vegetative forms.

Superior Lethal Coefficient. —

1. Prepare surface cultivations of the " test " organ-
isms upon nutrient agar in a culture bottle, and incu-
bate under optimum conditions, previously determined,
for the formation of their spores.

2. Employ that percentage solution of the disinfec-
tant determined in the previous experiment, and com-
plete the investigations as detailed therein, steps 6 to
14, increasing the interval between planting the sub-
cultivations to two, three, or five hours if considered
advisable.

NOTE. — Where it is necessary to leave the organisms
in contact with a strong solution of the disinfectant
for lengthy periods, some means must be adopted to
remove every trace of the disinfectant from the bac-
teria before transferring them to fresh culture media;
otherwise, although not actually killed, the presence
of the disinfectant may prevent their development,
and so give rise to an erroneous conclusion. In such
cases proceed as follows :

1. Transfer the suspension of bacteria to sterile
centrifugal tubes; add the required amount of dis-
infectant, and allow it to remain in contact with the
bacteria for the necessary period.

2. Centrifugalise thoroughly, pipette off the super-
natant fluid; fill the tube with sterile water and dis-
tribute the deposit evenly throughout the fluid.

3. Centrifugalise again, pipette off the supernatant
fluid; fill the tube with sterile water; distribute the
deposit evenly throughout the fluid, and transfer the
suspension to a litre flask.

4. Make up to a litre by the addition of sterile
water; filter the suspension through a sterile porcelain
candle.

5. Emulsify the bacterial residue with 5 c.c. sterile
bouillon.

6. Prepare the necessary subcultivations from this
emulsion.

INDEX.

ABBA'S condenser, 56
Aberration, chromatic, 57

spherical, 56
Absolute alcohol as a fixative, 75

as an antiseptic, 33
Absorbent paper for drying cover-
slips, 65

A. C. E. mixture, 266
Acetic acid for clearing films, 76
Acid-fast bacilli, to stain, 95, 106
Acid production, analysis table,

227, 228
by bacteria, 224
qualitative examination, 225
quantitative examination,

225

Actinomyces bo vis, 312
Action of different gases on bac-
teria, 240

Aerobic cultures, 177
Aerogenic bacteria, 111
Agar expansion table, 135
gelatine, 153

method of preparation, 149
rapid method of preparing, 150
Agglutination reaction, 252

macroscopical observation of,

259
microscopical observation of,

256
Air, analysis of, qualitative, 338

quantitative, 335
filter, 43
pump, 45

Albumin solution, Mayer's, 103
Alkaline serum agar, 157
Ammonia production, 229
Amphitrichous bacteria, 115
Anaerobic cultures, 186
Anaesthetics, 266
Analysis of air, apparatus for, 335
method of, 336
qualitative bacteriological,

338
quantitative bacteriological,

335

of butter, qualitative bacterio-
logical, 355

Analysis of butter, quantitative

bacteriological, 354
of cream, qualitative bacterio-
logical, 355
quantitative bacteriological,

354

of ice-cream, qualitative bac-
teriological, 354
of meat, apparatus for, 356
method of, 357
qualitative bacteriological,

375

of milk, apparatus for, 345
collection of samples, 344
method of, 346
qualitative bacteriological,

347
quantitative bacteriological

344

of sewage, qualitative bacterio-
logical, 335
quantitative bacteriological,

334

of soil, apparatus for, 339
collection of samples, 338
method of, 340
qualitative bacteriological,

342
quantitative bacteriological,

339

of water, apparatus for, 324
method of, 326
qualitative bacteriological,

323
quantitative bacteriological,

316

Anatomy of bacteria, 113
Aniline dyes, 77

gentian violet, 85
Anthrax, bacillus of, 305
Antiseptics, 33
action of, 359

Arnold's steam sterilizer, 39
Ascitic bouillon, 156
Ascomycetae, 109
Ascospores, 110
Asparagin media, 171
Aspergillus, 108

363

364

INDEX.

Atmospheric conditions, 239

Attenuating the virulence of or-
ganisms, 285

Autoclave, 42

Autopsy, method of conducting,
287

BACILLUS, 112
aegyptiacus, 313
anthracis, 305

in water, 333
aquatilis sulcatus, 304
botulinus, 309
chauvei, 309
coli communis, 304

in water, 327
diphtherias, 302

in milk, 351
enteritidis of Gartner, 304

in water, 327
sporogenes, 309
in water, 331
fluorescens liquefaciens, 300

non-liquefaciens, 300
influenzas, 313
leprae, 308
mallei, 314
mycoides, 305
oedematis maligni, 309

in soil, 343

of avian tubercle, 306
of Friedlander, 299
of Hoffmann, 302
of rhinoscleroma, 299
of symptomatic anthrax, 309
pestis, 310
phlei, 306
pyocyaneus, 300
septicaemiae haemorrhagicae, 310
subtilis, 305
suipestifer, 310
tetani, 309

in water, 333
tuberculosis, 306

in milk, 352
typhi abdominalis, 304

in water, 327
typhosus, 304
xerosis, 302

Bacteria, classification of, 111
microscopical examination of,

stained, 80
unstained, 72
Bacterial enzymes, 222

food-stuffs, 121
Base of microscope, 52
Beer wort, 165
Beet-root medium, 164
Beggiotoa, 1 1 3

Benzole bath, 202
Berkfeld filter, 45
Bile-salt agar, 169

broth, 169

Biochemistry of bacteria, 221
Bismarck brown, 84
Blastomycetes, morphology of,

109

Blood agar, 158
pipettes, 22

serum, collection of, 153
inspissated, 155, 156
to inspissate, 155
Body tube of microscope, 52
Botkin's anaerobic method, 192
Bouillon, preparation of, 141
Brain agar, 150
Bread paste, 167
Brownian movement, 74
Buchner's anaerobic method, 189
Bulloch's anaerobic method, 194

tubes, 292
Butter, analysis of, apparatus for,

355

method of, 355
qualitative, 355
quantitative, 354

CAGES, 263

for guinea-pigs, 264

for mice, 263

for rabbits, 264

for rats, 264

Camera lucida, Abbe, 60
Capillary pipettes, 20

graduated, 23
Capsule of bacteria, 113

thermo-regulator, 175

to stain the, 86, 105
Capsules, collodion, inoculation of,

279
preparation of, 279

glass, 20

to clean infected, 26

to clean new, 25

to sterilise, 36
Carbolic acid, 33
Carbolised agar, 152

bouillon, 144

gelatine, 149
Carbon dioxide, 233
Carrot medium, 164
Cell wall of bacteria, 114
Centrifugalised milk, 350
Centrifugal machine, 349
Chemical products of bacteria, 221
Chloroform, 33
Cholera, 302
Chromatic aberration, 57

INDEX.

365

Chromogenic bacteria, 111
Cladothrix, 113

nivea, 312

Classification of bacteria, 111
Clearing films with acetic acid, 76
Coarse adjustment, 53
Cocaine, 266
Coccus, 110
Coefficient, inferior lethal, 360

of inhibition, 359

superior lethal, 359
Cohn's solution, 171
Collection of pus, 271

of water samples, 316
Collodion capsules, 279
Coloured light, action of, 252
Columella, 108
Compensation eyepiece, 56
Conidia, 109

Continuous sterilisation, 41
Corrosive sublimate, Lang, 76
Cotton-wool filter, 43
Counterstaining, 78
Cover-slip films, 75
Cover-slips, 27

to clean new, 28

used, 28

Crates for test-tubes, 36
Cream, analysis of, apparatus for,

355

method of, 355
qualitative, 355
quantitative, 354
Crenothrix, 113
Culture bottles, 19

flasks, 19
Cutaneous inoculation, 274

DAUGHTER cells, 110
Daylight, diffuse, action of, 250
Decolourising agents, 78
Definition of objective, 57
Description of plate culture, 207
Descriptive terms, 208
Desiccation, effect of, 249
Desiccator, Miiller's, 249
Diaphragm, iris, 55
Diastatic enzymes, 223
Differential steriliser, 202
Diluting chamber, 196
Diphtheria, bacillus of, 302
Diplobacillus, 112
Diplococcus, 111

pneumonise, 292
Discontinuous sterilisation, 40
Discs of plaster-of -Paris, 173
Disinfectants, 33

action of, 359

testing power of, 359

Dosage of inoculum, 269
Double nosepiece, 58
Dropping bottles, 68
Dry heat, 34
Dunham's solution, 168
Dyes, aniline, 77

EDGE of individual colonies, char-
acters of, 213
Egg-albumen media, 160
Egg to clear nutrient media with,

145

Eisenberg's milk-rice medium, 167
Elevation of colonies, description

of, 209

Eisner's gelatine, 164
Endogenous spores, varieties of,

118

English proof agar, 1 70
Enumerating discs, Jeffer's, 322

Fakes', 322
Enumeration of micro-organisms,

321
Environmental conditions, 121,

238
Enzyme production by bacteria,

222

Eosin, 83

Esmarch's anaerobic culture
method, 187

roll culture, 199

water-bottle, 317
Estimation of reaction of media,

128

Ether, 33
Eucaine, 266
Exalting virulence of organisms,

284
Expansion table for agar, 135

for gelatine, 134
Experimental animals, 261

inoculation of animals, 261
Extracellular toxins, testing of/

261

Eyepieces, 56
Eye-screen, 59

FEEDING experiments, 284

Fermentation tubes, 24

Field of objective, 57

Filar micrometer, 63

Filling tubes, etc., with medium,

. 138

Film preparations, fixing, 75

making, 75

staining, 76

Filter candles, testing efficiency
of, 358

to disinfect, 34

366

INDEX.

Filter candles, to sterilise, 34

flasks, 19

papers, 136

to fold, 136
Filtering agar, 137

gelatine, 137
Filters, 43

Berkfeld, 45

Chamberland, 44

cotton-wool, 43
Filtration by aspiration, 45

of media, 137

under pressure, 47
Fine adjustment, 54
Fish, bacteriological analysis of,
356

bouillon, 162

gelatine, 162

agar, 163

Fission, multiplication by, 116
Fixation by heat, 75

of tissues, 98
Fixing fluids, 75
Flagella, 115

to stain, 87
Flasks, Bohemian, 18

Erlenmeyer's, 18

filter, 19

Kolle's culture, 19

to clean infected, 28

to clean new, 25

to plug, 29

to sterilise, 36

Fluid media, description of, 217
Foot of microscope, 52
Formaldehyde, 33
Formalin method of preserving

cultures, 291
tissues, 292

Fractional sterilisation, 37, 38
Frankel and Voges' solution, 171
Freezing method of sectioning, 98
French proof agar, 170
Fresh preparations of bacteria, 69
Fuchsin, 82

GAS analysis, qualitative, 235

quantitative, 235
collecting apparatus, 236
production by bacteria, 233
tubes for media, 139

Gasperini's solution, 167

Gelatine agar, 153
expansion table, 134
method of preparation, 145
rapid method of preparation.
145

General anaesthetics, 266

Gentian violet, 83

German lined paper, 65
Germicides, 33

testing power of, 359
Geryk air-pump, 45
Glanders, bacillus of, 314
Glucose formate agar, 152
bouillon, 142
gelatine, 148
Glycerinated potato, 164
Glycerine agar, 151

blood-serum, 155

bouillon, 142

potato broth, 164
Gpadby's gelatine, 165
Gonidium, 108
Goniodophore, 109
Gonococcus, 297
Graduated pipettes, 20
Gram's differential staining

method, 93
Gram-Weigert staining method,

94, 104

Grease pencils, 68
Griiber's reaction, 252
Guinea-pig cages, 264
Gulland's solution, 76
Gum solution, 99

H^MATOCYTOMETER Cell, 196

Haematoxylin, 85
Hanging-drop cultures, 184

preparation, 72
examination of, 72
staining of, 74

slides, 65

Hardening tissues, 98
Hay infusion, 165
Hearson's water-bath, 242
Heat, effect of, 242
Heiman's serum agar, 157
Hesse's anaerobic culture method,

187

Hog cholera bacillus', 310
Holder for guinea-pigs, 273

for mice, 274
Hot air, 35
Hot-air oven, 35

to use the, 36
Hot-water funnel, 138
Human blood agar plates, 198
Huyghenian eyepiece, 56
Hydrogen, detection of, 233

generating apparatus, 191
Hypha, 107
Hyphomycetes, 107

morphology of, 107

reproduction of, 107

ICE-BOX for water samples, 317

INDEX.

367

Ice-cream, analysis of, qualitative,

354

quantitative, 354
Impression films, 79
Incubators, 174
Indol production, 230
Inferior lethal coefficient, 360
Influence of environment on bac-
terial growth, 121
Inhalation experiments, 283
Inhibition coefficient, 359
Inoculating syringe, 265
Inoculation, cutaneous, 274

intracranial, 280

intramuscular, 276

intraocular, 280

intraperitoneal, 277

intrapulmonary, 281

intravenous, 281

of bacteria, effects of, 291

of collodion capsules, 279

subcutaneous, 275
Inoculum, character of, 267

preparation of, 267
Inosite-free bouillon, 141
Insoluble toxins, testing of, 260
Intermittent sterilisation, 40
Intracellular toxins, testing of,

260

Intracranial inoculation, 280
Intramuscular inoculation, 276
Intraocular inoculation, 280
Intraperitoneal inoculation, 277
Intrapulmonary inoculation, 281
Intravenous inoculation, 281
Invertin enzymes, 223
Involution forms, 116
Iodine solution, 94
Iron bouillon, 143

peptone solution, 168
Isolation by animal experiment,
204

by differential atmosphere, 203
incubation, 201
media, 200
sterilisation, 201

by dilution, 196

by plate cultures, 197

of sporing bacteria, 201

JEFFER'S enumerating discs, 322

KAISERLING solution, 292
Kanthack's serum agar, 157
Killed cultivations, 260
Kipp's hydrogen apparatus, 191
Kitasato's serum flasks, 19
Klebs-Loffler bacillus, 302
Koch's steam steriliser, 39

Koch-Week's bacillus, 313
Kolle's culture flasks, 19

LAB. enzymes, 224
Lactose litmus agar, 152

bouillon, 141

gelatine, 148
Lakmus molke, 144
Lang's solution, 76
Lead bouillon, 143
Leptothrix, 112, 113
Lethal dose, minimal, 269
Light, action of, 250
Liquid soap, 267
Lithium carmine, 84
Litmus bouillon, 141
gelatine, 148
milk, 161

cultivations, descriptions of,

217

whey, 161

Local anaesthetics, 266
Loffier's serum, 156
Lophotrichous bacteria, 116
Lorrain Smith's serum, 156
Lugol's solution, 94
Lysol, 33

MAcCoNKEv's capsule stain, 86

media, 169

MacCrorrie's flagella stain, 89
Macroscopical examination of cul-
tures, 207
Malta fever, 315

Margin of individual colonies, 213
Material for inoculation, 267
Mayer's albumin, 103
Measuring bacteria, 60
Meat, bacteriological analysis of,

356
extract, 127

reaction of, 128
Mechanical stage, 54

tube length, 53
Media, culture:

agar gelatine (Guarnieri), 152

ascitic bouillon, 156

asparagin medium (Frankel and

Voges), 171
(Uschinsky), 171
beer wort, 165
beet-root, 164
bile-salt agar (MacConkey), 169

broth (MacConkey), 169
blood-agar (Washbourn), 158
blood-serum, 153
(Loffler), 156
(Lorrain Smith), 156
brain agar, 150

368

INDEX.

Media, culture:
bread paste, 167
carbolised agar, 152
carrot, 164
Cohn's solution, 171
egg-albumen, 160

(Tarchanoff and Kolesnikoff),

160

English proof agar (Blaxall) , 1 70
fish bouillon, 162
gelatine, 162

agar, 163
French proof agar (Sabouraud),

170

gelatine agar, 152
glucose formate agar, 152
bouillon (Kitasato), 142
gelatine (Kitasato), 148
glycerinated potato, 164

broth, 164
glycerine agar, 151
blood -serum, 155
bouillon, 142
hay infusion, 165
inosite-free media bouillon

(Durham), 141
iron bouillon, 143

peptone solution (Pakes), 168
lactose litmus agar (Wurtz), 152
bouillon, 144
gelatine (Wurtz), 148
Lakmus molke, 144
lead bouillon, 143
litmus bouillon, 143
gelatine, 148
milk, 161
whey, 161
milk, 160

rice (Eisenberg), 167

(Soyka), 167
Naegeli's solution, 171
nitrate bouillon, 143

water (Pakes), 168
nutrient agar-agar, 149
bouillon, 141
gelatine, 145

rapid method of preparing,

145

parsnip, 164
Pasteur's solution, 170
peptone water (Dunham), 168

rosolic acid water, 168
plaster-of -Paris discs, 173
potato, 163

gelatine (Eisner), 164

(Goadby), 165
serum agar (Heiman), 157

(Kanthack and Stevens),
157

Media, culture:

serum agar (Wertheimer), 156

bouillon, 156

silicate jelly (Winogradsky), 172
spleen agar, 150
sugar agar, 151
bouillon, 142
gelatine, 148

sulphindigotate agar, 152
bouillon (Weyl), 143
gelatine (Weyl), 148
turnip, 164
urine agar, 159
gelatine, 158

(Heller), 159

wheat broth (Gasperini), 167
whey agar, 162
gelatine, 161
wine must, 167

Winogradsky's solution (for
nitric organisms), 172
(for nitrous organisms),

172

wort agar, 166
gelatine, 166

yeast water (Pasteur), 170
Media, filtration of, 137
Media, tubing, 138
Medium store boxes, 140
Merismopedium, 111
Mesophilic bacteria, 122

pathogenic effects, 260
Metabolic products of bacteria,

123

Metachromatic granules, 1 1 5
Metal instruments, to sterilise, 37,

Methods of identification of bac-
teria, 205
of inoculation, 274
Methylene-blue, 81
Meyer's carmine, 85
Micrococcus, 110
agilis, 296
candicans, 296
melitensis, 315
tetragenus, 297
Micrometer, 59
filar, 63
net, 62
ocular, 61
stage, 60

Micrometry, methods of, 60
Micron, 60

Microscope for bacteriology, 51
Microscopical examination of bac-
teria, 218
stained, 80
unstained, 72

INDEX.

369

Milieu d'epreuve, 170
Milk, analysis of, qualitative, 347
quantitative, 345

medium, 160

rice media, 167
Minimal lethal dose, 269
Mirrors for microscope, 56
Moist heat, 37
Molecular movement, 74
Monotrichous bacteria, 115
Motility, examination for, 72
Moulds, examination of, 107

for paraffin imbedding, 102
Mounting film preparations, 78

paraffin sections, 102
Mouse cages, 263

holder, 274

scales, 263
Mucorinae, 107
Mucor mucedo, 108
Muffle furnace, 34
Miiller's desiccator, 249
Museum specimens, preparation

of cultures for, 291
of tissues for, 292
Mycelium, 107
Mycoprotein, 115

NAEGEU'S solution, 171
Naked flame, 34
Neisser's staining method, 95
Net micrometer, 62
Nitrate bouillon, 143

water, 169

Nitric organisms in soil, 343
Nitroso-indol reaction, 230
Nitrous organisms in soil, 343
Nosepiece, double, 58

triple, 58
Novy's anaerobic method, 193

jars, 193

Numerical aperture, 57
Nutrient media, 125

OBJECTIVES, 56
Ocular micrometer, 61
Oculars, 56
Oese, .66
Oidium, 109
Oil of garlic, 33

of mustard, 33

Operating table for animals, 272
Optical characters of colonies 213

tube length, 53

Optimum reaction of medium, de-
termination of, 248

temperature, determination of,
241

Orsat-Lunge gas analysis appara-
tus, 235
Orth's carmine, 84

FAKES' counting disc, 322

filter reservoir, 49
Papier Chardin, 137
Paraffin sections, mounting, 102

staining, 103
Parietti's bouillon, 144
Parsnip medium, 164
Passages of virus, 285
Pasteur-Chamberland filter, 44
Pasteur pipettes, 21

solutions, 170
Pathogenesis, methods of testing,

259

Pathogenic bacteria, 111
Pediococcus, 111
Penicillium, 108
Peptone rosolic acid water, 168

water, 168

Perchloride of mercury, 33
Perisporiaceae, 108
Peritrichous bacteria, 1 16
Permanent preparations of bac-
teria, 291
of tissues, 292
Petri's dishes, 19
Phenol production, 231
Photogenic bacteria, 111
Picric acid solution, 104
Picrocarmine, 84
Pigment production, 232
Pipettes, 20, 21, 22, 23

to clean infected, 27
new, 25

to sterilise, 36
Plasmolysis, 114
Plaster-of -Paris discs, 173
Plate box, 20

cultures, 181

levelling stand, 182
Plates, 19

to clean infected, 26
new, 25

to sterilise, 36
Platinum needles, 66

method of mounting, 67
Pneumobacillus, 299
Pneumococcus, 298
Polar granules, 115
Porcelain filter, 44
Post-mortem examinations, 287
Potato gelatine, 164, 165

medium, 163

Potted meat, bacteriological anal-
ysis of, 356
Pouring plates, 182, 183

370

INDEX.

Primary colours, action of, 252
Proteolytic enzymes, 222
Psychrophilic bacteria, 122
pathogenic effects, 259
Pus, collection of, 271
Pyrogallic'acid solution, 189

RABBIT cages, 264

scales, 261
Raising virulence of organisms,

284

Ramsden's micrometer, 63
Range of temperature, 241
Rat cages, 264

Reaction of medium, effect of, 248
optimum, 248
range of, 248

Reducing agent production, 232
Reduction of nitrates, 232
Reichert's thermo-regulator, 175
Relation of bacteria to environ-
ment, 238
Removal of material from culture

tubes, 69

Rennet enzymes, 224
Reproduction of bacteria, 116
Resistance to lethal agents, 249
Roll cultures, 199
Roux's anaerobic culture method,
188

SABOURAUD'S medium, 170
Saccharomyces, morphology of,

110

Safranine, 84
Saprogenic bacteria, 111
Sarcina, 111
Scales, decimal, 261

trap, 146

Scalpels, to sterilise, 37
Scheme of study for bacteria, 205
Schizomycetes, morphology of, 111
Scissors, to sterilise, 37
Searing irons, 288
Sedimentation tubes, 23
Serial cultivations, 199
Serum agar, 156, 157
plate cultures, 198

bouillon, 156

inspissator, 155
Sewage, analysis of, qualitative,

335

quantitative, 334
Shake cultivations, 181
description of, 217
Shape of individual colonies, 208
Silicate jelly, 1 72
Size of individual colonies, 208
Slides, 27

Slides, to clean new, 27

used, 28
Smear cultures, 179, 180

description of, 214
Soap, liquid, 267
Soil, analysis of, qualitative, 342

quantitative, 339
Soluble toxins, testing of, 261
Soyka's milk-rice medium, 167
Spear-headed spatula, 290
Specific serum, collection of, 253

dilution of, 255
Spherical aberration, 56
Spirillum, 112
rubrum, 302
Spirochseta, 113
Spleen agar, 150
Sporangium, 108
Spore formation, arthrogenous, 11?
endogenous, 117
method of, 116
observation of, 219
germination, method of, 119

observation of, 219
to stain, 91

Spores, characters of, 118
Stab cultures, 180

description of, 215
Stage micrometer, 60

of microscope, 54
Staining paraffin sections, 103

reactions of bacteria, 221
Stains, 77
Standardising bouillon, 133

media, 132

Standard soda solution, 132
Staphylococcus, 112
pyogenes albus, 296
aureus, 296

in milk, 353
citreus, 296
Steam steriliser, Arnold's, 39

Koch's, 39
streaming, 39
Sterigma, 108

Sterilisation by chemicals, 33
by dry heat, 34
by filters, 43
by moist heat, 37
by streaming steam, 39
by superheated steam, 40
of gases, 43
of liquids, 44
Sterilising agents, 32
Store boxes for media, 140
Streak cultures, 179, 180

description of, 214
Streaming movement, 74-
Streptobacillus, 112

INDEX.

371

Streptococci in water, 332
Streptococcus, 111

brevis, 298

of bovine mastitis, 298

pyogenes longus, 298

in milk, 353
Streptothrix, 113

actinomycotica, 312
Structure of individual colonies,

211

Subcutaneous inoculation, 275
Substage condenser, 55
Sugar agar, 151

bouillon, 142

gelatine, 148
Sulphindigotate agar, 152

bouillon, 143

gelatine, 148

Sulphuretted hydrogen, 234
Sunlight, direct, action of, 251
Superheated steam, 41
Superior lethal coefficient, 359
Suppuration, organisms of, 295
Surface of individual colonies, 210
Swarm spores, 108
Syringe for subcutaneous inocula-
tion of solid material, 276

hypodermic, 265

TATIN'S operating table, 272
Taxonomy, 263, 273
Temperature, action of, 241
optimum, 241
range, 241
Testing filters, 358
Test objects for objectives, 58
Test-tubes, 17

to clean infected, 25
to clean new, 24
to plug, 29
to sterilise, 36
Tetrad, 111

Thermal death - point, J_22
determination of, 242
of spores, 244
of vegetative forms, 242
Thermophilic bacteria, 122
Thermo-regulators, capsule, 175

Reichert's, 175
Thionine blue, 83
Thiothrix, 113
Timothy grass bacillus, 306
Tinned meat, analysis of, 356
Tissues for sectioning, fixing, 98
freezing, 100
hardening, 98
imbedding, 101
washing, 99
Titration of media, 129

Torulae, 110
Toxins, testing of, 260
Triple nosepiece, 58
True motility, 74
Tube cultures, 177

inoculating, 178

preparation of, 178
Tubercle bacillus, 306

to stain, 95, 106
Tubing media, 138
Turnip medium, 164

URINE agar, 159

gelatine, 158, 159

media, 158
Uschinsky's solution, 171

VAN ERMENGEM'S flagella stain,

Vesuvin, 84
Vibrio, 113

cholerae, 302
in water, 332

Metschnikovi, 302

of Kinkier and Prior, 302
Virulence, attenuating, 285

raising, 284

Voges' guinea-pig holder, 272
Volatile oils as disinfectants, 33

WARM stage, 58

Washing tissues, 99

Water, analysis of, qualitative,

323
quantitative, 316

steriliser, 38
Weighing animals, 261
Wertheimer's serum agar, 157
Wheat broth, 167
Whey agar, 162

gelatine, 161
Widal's reaction, 253
Wine must, 167
Winogradsky's jelly, 172

solutions, 172

Wire baskets for test-tubes, 36
Wort agar, 166

gelatine, 166
Wright's anaerobic method, 190

XEROSIS bacillus, 302

YEASTS, examination of, 110
Yeast water, 170

ZiEHL-Neelsen staining method,

95, 106
Zooglcea, 114
Zymogenic bacteria, 1 1 1

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Church and Peterson's
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Nervous and Mental Diseases. By ARCHIBALD CHURCH, M. D.,
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Northwestern University Medical School, Chicago; and FREDERICK
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THE NEW (6th) EDITION

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OPINIONS OF THE MEDICAL PRESS

American Journal of the Medical Sciences

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DISEASES OF CHILDREN.

KerrV Diagnostics qf w
Diseases qf Children ?

Diagnostics oi the Diseases of Children. By LEGRAND KERR,
M. D., Professor of Diseases of Children, Brooklyn Postgraduate Med-
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FOR THE PRACTITIONER

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New York State Journal of Medicine

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Kerley's Treatment qf
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Treatment of the Diseases of Children. By CHARLES GILMORE
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School and Hospital. Octavo of 628 pages, illustrated. Cloth, $5.00
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THE NEW (2d) EDITION

This work has been prepared for the physician engaged in general practice.
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NURSING.

Nursing in Diseases of the
Eye, Ear, Nose, and Throat

Nursing in Diseases of the Eye, Ear, Nose, and Throat. By the

Committee on Nurses of the Manhattan Eye, Ear, and Throat Hospital:
J. EDWARD GILES, M. D., Surgeon in the Eye Department ; ARTHUR B.
DUEL, M. D. (Chairman), Surgeon in the Ear Department ; HARMON
SMITH, M. D., Surgeon in the Throat Department. Assisted by JOHN
R. SHANNON, M. D., Assistant Surgeon in the Eye Department ; and
JOHN R. PAGE, M. D., Assistant Surgeon in the Ear Department. With
chapters by HERBERT B. WILCOX, M. D., Attending Physician to the
Hospital; and Miss EUGENIA D. AYERS, Superintendent of Nurses.
I2mo of 260 pages, illustrated. Cloth, $1.50 net.

A VALUABLE BOOK

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style throughout is simple, plain, and definite.

New York Medical Journal

" Every side of the question has been fully taken into consideration."

Stoney's
Materia Medica for Nurses

Practical Materia Medica for Nurses, with an Appendix containing
Poisons and their Antidotes, with Poison-Emergencies ; Mineral Waters ;
Weights and Measures ; Dose-List, and a Glossary of the Terms used
in Materia Medica and Therapeutics. By EMILY A. M. STONEY, of the
Carney Hospital, South Boston. I2mo of 3OOpages. Cloth, $1.50 net.

THE NEW (3d) EDITION

In making the revision for this new third edition, all the newer drugs have
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Stoney's Nursing

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EMILY A. M. STONEY, Superintendent of the Training School for Nurses
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THE NEW (4th) EDITION

In this volume the author explains the entire range of private nursing as dis-
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Stoney's Technic for Nurses

Bacteriology and Surgical Technic for Nurses. By EMILY A. M.
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Trained Nurse and Hospital Review

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Spratling on Epilepsy

Epilepsy and Its Treatment. By WILLIAM P. SPRATLING, M. D.,
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The Lancet. London

" Dr. Sp ratling's work is written throughout in a clear and readable style. . . . The work
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NURSING.

Aikens' Primary Studies for Nurses illustrated

PRIMARY STUDIES FOR NURSES: A Text-Book for First-year Pupil
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Aikens' Clinical Studies for Nurses fSS"

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up in a concise, forceful way.

Dietetic and Hygienic Gazette

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numerous and well selected."

Aikens' Training-School Methods

HOSPITAL TRAINING-SCHOOL METHODS AND THE HEAD NURSE. By
CHARLOTTE A. AIKENS, formerly Director of Sibley Memorial Hospital,
Washington, D. C. 12 mo of 267 pages. Cloth, $1.50 net.

Trained Nurse and Hospital Review

" There is not a chapter in the book that does not contain valuable suggestions."

Aikens9 Hospital Management J"*t Ready

HOSPITAL MANAGEMENT. BY CHARLOTTE A. AIKENS, formerly Direc-
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Miss Aikens' long experience as hospital director has well fitted her to
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Hoxie's Medicine for Nurses

Practice of Medicine for Nurses. A Text-Book for Nurses and Students,
of Domestic Science, and a Hand-Book for All Those Who Care for the Sicfc^
By GEORGE HOWARD HOXIE, M. D., Professor of Internal Medicine, U»i--
versity of Kansas. With a Chapter on Technic of Nursing by PEARL L..
LAPTAD, Principal of the Training School for Nurses, University of Kansas,.
I2mo of 284 pages, illustrated. Cloth, $1.50 net,

This work is truly a practice of medicine for the nurse, enabling her to recognize any
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most acceptable, particularly emphasizing the nurse's part.

Trained Nurse and Hospital Review

" This book has our unqualified approval."

McCombs' Diseases of Children for Nurses New

Diseases of Children for Nurses. By ROBERT S. McCoMBS, M. D.,,
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Dr. McCombs' experience in lecturing to nurses has enabled him to emphasize just those-
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National Hospital Record

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Wilson's Obstetric Nursing

A Reference Hand-Book of Obstetric Nursing. By W. REYNOLDS
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32mo of 258 pages, illustrated. Flexible leather, $1.25 net.

Dr. Wilson's work discusses the subject of obstetrics entirely from the nurse's point o|
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agement. The text is copiously illustrated.

American Journal of Obstetrics

" Every page emphasizes the nurse's relation to the case."

Friihwald and Westcott on Children

Diseases of Children. A Practical Reference Book for Students and
Practitioners. By PROFESSOR DR. FERDINAND FRUHWALD, of Vienna.
Edited, with additions, by THOMPSON S. WESTCOTT, M. D., University of
Pennsylvania. Octavo, 533 pages, 176 illustrations. Qoth, $4.50 net.

Boyd's State Registration for Nurses

State Registration for Nurses. By LOUIE CROFT BOYD* R, N., Graduate
Colorado Training-school for Nurses. Octavo of 42 pages* 50 cents net.

NURSING.

Macfarlane's Gynecology for Nurses illustrated

A REFERENCE HAND-BOOK OF GYNECOLOGY FOR NURSES. By CATH-
ARINE MACFARLANE, M. D., Gynecologist to the Woman's Hospital of
Philadelphia. 321110 of 150 pages, with 70 illustrations. Flexible
leather, $1.25 net.

A. M. Seabrook, M. D., Woman's Medical College of Philadelphia.

" It is a most admirable little book, covering in a concise but attractive way the subject from
the nurse's standpoint."

Galbraith's Personal Hygiene and Physical Training

for Women Recently Issued

PERSONAL HYGIENE AND PHYSICAL TRAINING FOR WOMEN. By
ANNA M. GALBRAITH, M.D., Fellow New York Academy of Medicine,,
i2mo of 371 pages, with original illustrations. Cloth, $2.00 net.

Dr. Galbraith's book is just what has long been needed — a simple manual
of hygiene and physical training along scientific lines.

De Lee's Obstetrics for Nurses New (3d) Editfon

OBSTETRICS FOR NURSES. By JOSEPH B. DELEE, M. D., Professor of
Obstetrics in the Northwestern University Medical School. 12010 vol-
ume of 512 pages, fully illustrated. Cloth, $2.50 net.

J. Clifton Edgar, M. D.,

Professor of Obstetrics and Clinical Midwifery, Cornell Medical School y N. Y.

" It is far-and-away the best that has come to my notice, and I shall take great pleasure in recom-
mending it to my nurses and students as well."

Davis' Obstetric Nursing New (3d)

OBSTETRIC AND GYNECOLOGIC NURSING. By EDWARD P. DAVIS, A. M.,
M. D., Professor of Obstetrics, Jefferson Medical College and Philadel-
phia Polyclinic. i2mo of 436 pages, illustrated. Buckram, $1.75 net.

The Lancet, London

" Not only nurses, but even newly qualified medical men, would learn a great deal by a perusal of
this book. It is written in a clear and pleasant style, and is a work we can recommend."

Beck's Hand-Book for Nurses New (2d) Editioll

A REFERENCE HAND-BOOK FOR NURSES. By AMANDA K. BECK, of
Chicago, 111. 32mo of 200 pages. Flexible leather, $1.25 net.

This little book contains information upon every question that comes to a
nurse in her daily work, and embraces all the information that she requires
to carry out any directions given by the physician.

Boston Medical and Surgical Journal

" Muflt be regarded as an extremely useful book, not only for nurses, but for physicians."

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A TEXT-BOOK ON PRACTICAL FEVER NURSING. By EDWARD C.
B.EGISTER, M. D., Professor of the Practice of Medicine in the North
Carolina Medical College. i2mo of 352 pages. Cloth, $2.50 net.

The work completely covers the field of practical fever nursing. The illustrations shov»
the nurse how to perform those measures that come within her province.

Trained Nurse and Hospital Review

" Nurses will find this book of great value in this practical branch of their work."

Hecker, Trumpp, and Abt on Children

ATLAS AND EPITOME OF DISEASES OF CHILDREN. By Dr. R. HECKER
and Dr. J. TRUMPP, of Munich. Edited, with additions, by ISAAC A.
ABT, M.D., Assistant Professor of Diseases of Children, Rush Medical
College, Chicago. With 48 colored plates, 144 text-cuts, and 453 pages
of text. Cloth, 15.00 net.

The many excellent lithographic plates represent cases seen in the authors' clinics, and
have been selected with great care, keeping constantly in mind the practical needs of the
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equivalent to actual clinical observation. The editor, Dr. Isaac A. Abt, has added all new
methods of treatment.

Johns Hopkins Hospital Bulletin

" The entire field has been covered. With the excellent plates, it will be found of real
value to both students and practitioners."

Lewis* Anatomy and Physiology The New (id) Edition

ANATOMY AND PHYSIOLOGY FOR NURSES. By LEROY LEWIS, M.D.,
Surgeon to and Lecturer on Anatomy and Physiology for Nurses at the
Lewis Hospital, Bay City, Michigan. i2mo of 375 pages, with 150
illustrations. Cloth, $1.75 net.

A demand for such a work as this, treating the subjects from the nurses point of view,
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employed by him in teaching these branches, making the text unusually simple and clear.

The Nurses Journal of the Pacific Coast

" It is not in any sense rudimentary, but comprehensive in its treatment of the subjects
in hand. The application of the knowledge of anatomy in the care of the patient is
emphasized."

Friedenwald and Ruhrah's Dietetics New (id) Edition

DIETETICS FOR NURSES. By JULIUS FRIEDENWALD, M. D., Professor
of Diseases of the Stomach, and JOHN RUHRAH, M. D., Professor of
Diseases of Children, College of Physicians and Surgeons, Baltimore.
i2mo volume of 395 pages. Cloth, $1.50 net.

This work has been prepared to meet the needs of the nurse, both in the training
school and after graduation. It aims to give the essentials of dietetics, considering briefly
the physiology of digestion and the various classes of foods and the part they play in
nutrition.

American Journal of Nursing

" It is exactly the book for which nurses and others have long and vainly sought. A
simple manual of -dietetics, which does not turn into a cook-book at the end of the first
or second chapter.

NURSING AND CHILDREN. II

T» i» T> mr •

Pauls Fever Nursing New (2d) Edition

NURSING IN THE ACUTE INFECTIOUS FEVERS. By GEORGE P. PAUL,
M.D., Assistant Visiting Physician to the Samaritan Hospital, Troy, N. Y.
i2mo of 246 pages. Cloth, $1.00 net.

Dr. Paul has taken great pains in the presentation of the care and management of each
fever. The book treats of fevers in general, then each fever is discussed individually, and
the latter part of the book deals with practical procedures and valuable information.

The London Lancet

" The book is an excellent one and will be of value to those for whom it is intended.
It is well arranged, the text is clear and full, and the illustrations are good."

Paul's Materia Medica for Nurses

MATERIA MEDICA FOR NURSES. By GEORGE P. PAUL, M.D., Assistant
Visiting Physician to the Samaritan Hospital, Troy. i2mo of 240 pages,
Cloth, #1.50 net.

Dr. Paul arranges the physiologic actions of the drugs according to the action of the
drug and not the organ acted upon. An important section is that on pretoxic signs,
giving the warnings of the full action or the beginning toxic effects of the drug, which,
if heeded, may prevent many cases of drug poisoning.

The Medical Record, New York

"This volume will be of real help to nurses; the material is well selected and well
arranged, and the book is as readable as it is useful."

Pyle's Personal Hygiene The New (4th) Edition

A MANUAL OF PERSONAL HYGIENE : Proper Living upon a Physiologic
Basis. By Eminent Specialists. Edited by WALTER L. PYLE, A.M.,
M.D., Assistant Surgeon to Wills Eye Hospital, Philadelphia. Octavo
volume of 472 pages, fully illustrated. Cloth, £1.50 net.

To this new edition there have been added, and fully illustrated, chapters on Domestic
Hygiene and Home Gymnastics, besides an appendix containing methods of Hydro-
therapy, Mechanotherapy, and First Aid Measures. There is also a Glossary of the
medical terms used.

Boston Medical and Surgical Journal

" The work has been excellently done, there is no undue repetition, and the writers
have succeeded unusually well in presenting facts of practical significance based on sound
knowledge."

Galbraith's Four Epochs of Woman's Life second Edition

THE FOUR EPOCHS OF WOMAN'S LIFE. By ANNA M. GALBRAITH,
M.D. With an Introductory Note by JOHN H. MUSSER, M.D., Univer-
sity of Pennsylvania. i2mo of 247 pages. Cloth, $1.50 net.
Birmingham Medical Review

" We do not as a rule care for medical books written for the instruction of the public ;
but we must admit that the advice in Dr. Galbraith's work is in the main wise and whole-

Starr on Children second Edition

AMERICAN TEXT-BOOK OF DISEASES OF CHILDREN. Edited by Louis
STARR, M.D., assisted by THOMPSON S. WESTCOTT, M.D. Octavo,, 1244
pages, illustrated. Cloth, $7.00 net; Half Morocco, #8.50 net.

12 SAUNDERS* BOOKS ON

Brower and Bannister
on Insanity

A Practical Manual of Insanity. For the Student and General
Practitioner. By DANIEL R. BROWER, A.M., M.D., LL. D., Professor
of Nervous and Mental Diseases in Rush Medical College, in affiliation
with the University of Chicago ; and HENRY M. BANNISTER, A. M.,
M. D., formerly Senior Assistant Physician, Illinois Eastern Hospital
for the Insane. 'Handsome octavo of 426 pages, with a number of
full-page inserts. Cloth, $3.00 net.

FOR STUDENT AND PRACTITIONER

This work, intended for the student and general practitioner, is an intelligible,
up-to-date exposition of the leading facts of psychiatry, and will be found of in-
valuable service, especially to the busy practitioner unable to yield the time for a
more exhaustive study. The work has been rendered more practical by omitting
elaborate case records and pathologic details, as well as discussions of speculative
and controversial questions.

American Medicine

" Commends itself for lucid expression in clear-cut English, so essential to the student in
any department of medicine. . . . Treatment is one of the best features of the book, and for
this aspect is especially commended to general practitioners."

Bergey's Hygiene

The Principles of Hygiene: A Practical Manual for Students,
Physicians, and Health Officers. By D. H. BERGEY, A. M., M. D.,
Assistant Professor of Bacteriology in the University of Pennsylvania.
Octavo volume of 555 pages, illustrated. Cloth, $3.00 net.

THE NEW (3d) EDITION

This book is intended to meet the needs of students of medicine in the
acquirement of a knowledge of those principles upon which modern hygienic
practises are based, and to aid physicians and health officers in familiarizing
themselves with the advances made in hygiene and sanitation in recent years.
This new third edition has been very carefully revised, and much new matter
added, so as to include the most recent advancements.

Buffalo Medical Journal

" It will be found of value to the practitioner of medicine and the practical sanitarian ; and
students of architecture, who need to consider problems of heating, lighting, ventilation, water
supply, and sewage disposal, may consult it with profit."

CHILDREN AND HYGIENE. 13

Griffith's Care of the Baby

The Care of the Baby. By J. P. CROZER GRIFFITH, M. D., Clinical
Professor of Diseases of Children, University of Penn. ; Physician to the
Children's Hospital, Phila. I2mo, 455 pp. Illustrated. Cloth, $1.50 net.

THE NEW (5th) EDITION

The author has endeavored to furnish a reliable guide for mothers. He has
made his statements plain and easily understood, in the hope that the volume
may be of service not only to mothers and nurses, but also to students and practi-
tioners whose opportunities for observing children have been limited.

New York Medical Journal

" We are confident if this little work could find its way into the hands of every trained
nurse and of every mother, infant mortality would be lessened by at least fifty per cent."

Crothers* Morphinism

Morphinism and Narcomania from Opium, Cocain, Ether, Chloral,
Chloroform, and other Narcotic Drugs ; also the Etiology, Treatment,
and Medicolegal Relations. By T. D. CROTHERS, M. D., Superintendent
of Walnut Lodge Hospital, Hartford, Conn. Handsome I2mo of 351
pages. Cloth, $2.00 net.

The Lancet, London

"An excellent account of the various causes, symptoms, and stages of morphinism, the
discussion being throughout illuminated by an abundance of facts of clinical, psychological, and
social interest."

Ruhrah's Diseases of Children

A Manual of Diseases of Children. By JOHN RUHRAH, M. D.,
Professor of Diseases of Children, College of Physicians and Surgeons,
Baltimore. I2mo of 534 pages, fully illustrated. Flexible leather]
$2.50 net.

THE NEW (3d) EDITION

In revising this work for the second edition Dr. Ruhrah has carefully in-
corporated all the latest knowledge on the subject. All the important facts are
given concisely and explicitly, the therapeutics of infancy and childhood being
outlined very carefully and clearly. There are also directions for dosage and
prescribing, and many useful prescriptions are included.

American Journal of the Medical Sciences

"Treatment has been satisfactorily covered, being quite in accord with the best teaching,
yet withal broadly general and free from stock prescriptions."

14 SAUNDERS' BOOKS ON

Peterson and Haines'
Legal Medicine & Toxicology

A Text-Book of Legal Medicine and Toxicology. Edited by
FREDERICK PETERSON, M. D., Professor of Psychiatry in the College
of Physicians and Surgeons, New York; and WALTER S. HAINES,
M. D., Professor of Chemistry, Pharmacy, and Toxicology, Rush
Medical College, in affiliation with the University of Chicago. Two-
imperial octavo volumes of about 750 pages each, fully illustrated.
Per volume: Cloth, $5.00 net; Sheep or Half Morocco, $6.50 net
Sold by Subscription.

IN TWO VOLUMES

The object of the present work is to give to the medical and legal professions
a comprehensive survey of forensic medicine and toxicology in moderate compass.
This, it is believed, has not been done in any other recent work in English. Under
" Expert Evidence" not only is advice given to medical experts, but suggestions
are also made to attorneys as to the best methods of obtaining the desired infor-
mation from the witness. An interesting and important chapter is that on "The
Destruction and Attempted Destruction of the Human Body by Fire and Chemi-
cals." A chapter not usually found in works on legal medicine is that on " The
Medicolegal Relations of the X-Rays."
Columbia Law Review

" For practitioners in criminal law and for those in medicine who are called upon to give
court testimony in all its various forms ... it is extremely valuable."

Fiske's Human Body

Structure and Functions of the Body. By ANNETTE FISKE, A.M.,.
Graduate of the Waltham Training School for Nurses. I2mo of 221
pages, illustrated. Cloth, $1.25 net.

JUST READY

The way in which this book presents anatomy and physiology is a departure
from the usual method — a departure, however, of a very practical kind. Miss
Fiske has woven the physiology in with the anatomy, thus making her work a
most readable one. It is an extremely practical book — one that can be readily
understood.

LEGAL MEDICINE.

Draper's Legal Medicine

A Text-Book of Legal Medicine. By FRANK WINTHROP DRAPER, A. M.,
M. D., Late Professor of Legal Medicine in Harvard University, Boston.
Octavo of 573 pages, illustrated. Cloth, $4.00 net ; Half Morocco, $5.50 net.

Hon. Olin Bryan, LL. B., Baltimore Medical College.

" A careful reading of Draper's Legal Medicine convinces me of the excellent character
of the work. It is comprehensive, thorough, and must, of a necessity, prove a splendid
acquisition to the libraries of those who arc interested in medical jurisprudence."

Chapman's Medical Jurisprudence Third Edition

Medical Jurisprudence, Insanity, and Toxicology. By HENRY C.
CHAPMAN, M. D., late Professor of Institutes of Medicine and Medical Juris-
prudence in Jefferson Medical College, Philadelphia. I2mo of 329 pages,
illustrated. Cloth, $1.75 net.

Golebiewski and Bailey's Accident Diseases

Atlas and Epitome of Diseases Caused by Accidents. By DR. ED.

GOLEBIEWSKI, of Berlin. Edited, with additions, by PEARCE BAILEY, M. D.,
Consulting Neurologist to St. Luke's Hospital, New York. With 71 colored
illustrations on 40 plates, 143 text illustrations, and 549 pages of text. Cloth,
$4.00 net. In Saunders1 Hand-Atlas Series.

Hofmann and Peterson's Legal Medicine

Atlas of Legal Medicine. By DR. E. VON HOFMANN, of Vienna.
Edited by FREDERICK PETERSON, M. D., Professor of Psychiatry in the
College of Physicians and Surgeons, New York. With 120 colored figures
on 56 plates and 193 half-tone illustrations. Cloth, $3.50 net.

Jakob and Fisher's Nervous System

and itS Diseases In Saunders* Hand-Atlases

Atlas and Epitome of the Nervous System and its Diseases. By

PROFESSOR DR. CHR. JAKOB, of Erlangen. From the Second Revised
German Edition. Edited, with additions, by EDWARD D. FISHER, M. D.,.
Professor of Diseases of the Nervous System, University and Bellevue
Hospital Medical College, New York. With 83 plates and copious text.
Cloth, $3. 50 net.

Abbott's Transmissible Diseases second Edition

The Hygiene of Transmissible Diseases : Their Causes, Modes of Dis-
semination, and Methods of Prevention. By A. C. ABBOTT, M. D., Pro-
fessor of Hygiene and Bacteriology, University of Pennsylvania. Octavo of
351 pages, illustrated. Cloth, 12.50 net

16 SAWDERS' BOOKS ON CHILDREN.

American Pocket Dictionary just Ready-New (?th) Edition

AMERICAN POCKET MEDICAL DICTIONARY. Edited by W. A. NEW-
MAN BORLAND, M. D., Editor "American Illustrated Medical Dic-
tionary." Containing the pronunciation and definition of the principal
words used in medicine and kindred sciences, with 64 extensive tables.
With 610 pages. Flexible leather, with gold edges, $1.00 net; with
patent thumb index, $1.25 net.

" I can recommend it to our students without reserve." — J. H. HOLLAND, M. D., Dean
9f the Jefferson Medical College, Philadelphia.

Morrow's Immediate Care of Injured

IMMEDIATE CARE OF THE INJURED. By ALBERT S. MORROW, M. D.,
Attending Surgeon to the New York City Hospital for the Aged and
Infirm. Octavo of 340 pages, with 238 illustrations. Cloth, $2.50 net.

Dr. Morrow's book on emergency procedures is written in a definite and decisive style,
the reader being told just what to do in every emergency. It is a practical book for every
day use, and the large number of excellent illustrations can not but make the treatment to
be pursued in any case clear and intelligible. Physicians and nurses will find it indispensible.

Powell's Diseases of Children Third Edition,

ESSENTIALS OF THE DISEASES OF CHILDREN. By WILLIAM M. POWELL,
M. D. Revised by ALFRED HAND, JR., A. B., M. D., Dispensary
Physician and Pathologist to the Children's Hospital, Philadelphia.
i2mo volume of 259 pages. Cloth, $1.00 net. In Saunders9
Question- Compend Series.

Shaw on Nervous Diseases and Insanity Fourth Edition

ESSENTIALS OF NERVOUS DISEASES AND INSANITY : Their Symptoms
and Treatment. A Manual for Students and Practitioners. By the late
JOHN C. SHAW, M. D., Clinical Professor of Diseases of the Mind and
Nervous System, Long Island College Hospital, New York. i2mo of
204 pages, illustrated. Cloth, $1.00 net. In Saunders' Question- Com-
pend Series.

*' Clearly and intelligently written ; we have noted few inaccuracies and several sug-
gestive points. Some affections unmentioned in many of the large text-books are noted."
— Boston Medical and Surgical Journal.

Starr's Diets for Infants and Children

DIETS FOR INFANTS AND CHILDREN IN HEALTH AND IN DISEASE. By
Louis STARR, M. D., Consulting Pediatrist to the Maternity Hospital,
Philadelphia. 230 blanks (pocket-book size). Bound in flexible leather,
$ i. 25 net.

Grafstrom's Mechano-Therapy second Revised Edition

A TEXT-BOOK OF MECHANO-THERAPY (Massage and Medical Gymnas-
tics). By AXEL V. GRAFSTROM, B. Sc., M. D., Attending Physician to
the Gustavus Adolphus Orphange, Jamestown, New York. i2mo, 200
pages, illustrated. Cloth, £1.25 net.

THIS BOOK IS DUE ON THE LAST DATE
STAMPED BELOW

AN INITIAL FINE OF 25 CENTS

WILL BE ASSESSED FOR FAILURE TO RETURN
THIS BOOK ON THE DATE DUE. THE PENALTY
WILL INCREASE TO SO CENTS ON THE FOURTH
DAY AND TO $1.OO ON THE SEVENTH DAY
OVERDUE.

BIOLOGY LIBRARY

OCT 1 6 1941

/" A X

1954

APR 2 6 1963

LD 21-5m-7,'37

T.C 88540

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