THE ELEMENTS

OF

BACTERIOLOGICAL
TECHNIQUE

A LABORATORY GUIDE FOR
MEDICAL, DENTAL, AND TECHNICAL STUDENTS

BY

J. W. H. EYRE, M.D., M.S., F.R.S. (Eom.)

Director of the Bacteriological Department of Guy's Hospital, London, and Lecturer on

Bacteriology in the Medical and Dental Schools ; formerly Lecturer on Bacteriology

at Charing Cross Hospital Medical School, and Bacteriologist to Charing

Cross Hospital ; sometime Hunterian Professor, Royal College

of Surgeons, England

SECOND EDITION
REWRITTEN AND ENLARGED

PHILADELPHIA AND LONDON

W. B. SAUNDERS COMPANY

1915

Copyright, 1902, by W. B. Saunders and Company Revised, entirely
reset, reprinted, and recopyrighted July, 1913

Copyright, 1913, by W. B. Saunders Company

Registered at Stationers' Hall, London, England

Reprinted November, 1915

PRINTED IN AMERICA

PRESS OF

W. B. SAUNDERS COMPANY
PHILADELPHIA

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

PREFACE TO THE SECOND EDITION

BACTERIOLOGY is essentially a practical study, and
even the elements of its technique can only be taught
by personal instruction in the laboratory. This is a
self-evident proposition that needs no emphasis, yet
I venture to believe that the former collection of tried
and proved methods has already been of some utility,
not only to the student in the absence of his teacher,
but also to isolated workers in laboratories far removed
from centres of instruction, reminding them of for-
gotten details in methods already acquired. If this
assumption is based on fact no further apology is
needed for the present revised edition in which the
changes are chiefly in the nature of additions — ren-
dered necessary by the introduction of new methods
during recent years.

I take this opportunity of expressing my deep
sense of obligation to my confrere in the Physiolog-
ical Department of our medical school — Mr. J. H.
Ryffel, B. C., B. Sc. — who has revised those pages
dealing with the analysis of the metabolic products
of bacterial life; to successive colleagues in the
Bacteriological Department of Guy's Hospital, for
their ready co-operation in working out or in testing
new methods; and finally to my Chief Laboratory
Assistant, Mr. J. C. Turner whose assistance and ex-
perience have been of the utmost value to me in the
preparation of this volume. I have also to thank
Mrs. Constant Ponder for many of the new line
drawings and for redrawing a number of the original
cuts.

JOHN W. H. EYRE.

GUY'S HOSPITAL, S. E.

PREFACE TO THE FIRST EDITION

IN the following pages I have endeavoured to ar-
range 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 it only needs to be used to convince
one of its extreme utility, whilst its inclusion in an
elementary manual is calculated to induce in the
student habits of accurate observation and concise
description.

vii

viii PREFACE TO THE FIRST EDITION

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 inperfections 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 sec-
tion 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.

CONTENTS

PAGE
I. LABORATORY REGULATIONS i

II. GLASS APPARATUS IN COMMON USE ' . . 3

The Selection, Preparation, and Care of Glassware,
8 — Cleaning of Glass Apparatus, 18 — Plugging
Test-tubes and Flasks, 24.

III. METHODS OF STERILISATION 26

Sterilising Agents, 26 — Methods of Application, 27
— Electric Signal Timing Clock, 38.

IV. THE MICROSCOPE 49

Essentials, 49 — Accessories, 57 — Methods of Micro-
metry, 61.

V. MICROSCOPICAL EXAMINATION OF BACTERIA AND OTHER

MICRO-FUNGI 69

Apparatus and Reagents used in Ordinary Micro-
scopical Examination, 69 — Methods of Examina-
tion, 74.

VI. STAINING METHODS 90

Bacteria Stains, 90 — Contrast Stains, 93 — Tissue
Stains, 95 — Blood Stains, 97 — Methods of Demon-
strating Structure of Bacteria, 99 — Differential
Methods of Staining, 108.

VII. METHODS OF DEMONSTRATING BACTERIA IN TISSUES 114
Freezing Method, 115 — Paraffin Method, 117 —
Special Staining Methods for Sections, 121.

VIII. CLASSIFICATION OF FUNGI 126

Morphology of the Hypomycetes, 126 — Morphol-
ogy of the Blastomycetes, 129.

IX. SCHIZOMYCETES 13!

Anatomy, 134 — Physiology, 136 — Biochemistry,
144.

X. NUTRIENT MEDIA 146

Meat Extract, 148 — Standardisation of Media, 154
— The Filtration of Media, 156 — Storing Media in
Bulk, 159 — Tubing Nutrient Media, 160.

XI. ORDINARY OR STOCK CULTURE MEDIA 163

ix

X CONTENTS

PAGE
XII. SPECIAL MEDIA 182

XIII. INCUBATORS 216

XIV. METHODS OF CULTIVATION . . . • 221

Aerobic, 222 — Anaerobic, 236.

XV. METHODS OF ISOLATION 248

XVI. METHODS OF IDENTIFICATION AND STUDY 259

Scheme of Study, 259 — Macroscopical Examination
of Cultivations, 261 — Microscopical Methods, 272 —
Biochemical Methods, 276 — Physical Methods, 295
— Inoculation Methods, 315 — Immunisation, 321 —
Active Immunisation, 322 — The Preparation of
Haemolytic Serum, 327 — The Titrationof Haemolytic
Serum, 328 — Storage of Haemolysin, 331.

XVII. EXPERIMENTAL INOCULATION OF ANIMALS ...... 332

Selection and Care of Animals, 335 — Methods of
Inoculation, 352.

XVIII. THE STUDY OF EXPERIMENTAL INFECTIONS DURING LIFE 370

General Observations, 371 — Blood Examinations,
373 — Serological Investigations, 378 — Agglutinin,
381 — Opsonin, 387 — Immune Body, 393.

XIX. POST-MORTEM EXAMINATION OF EXPERIMENTAL ANIMALS 396
XX. THE STUDY OF THE PATHOGENIC BACTERIA 408

XXI. BACTERIOLOGICAL ANALYSES 415

Bacteriological Examination of Water, 416 — Ex-
amination of Milk, 441 — Ice Cream, 457 — Ex-
amination of Cream and Butter, 457 — Examination
of Unsound Meats, 460 — Examination of Oysters
and Other Shellfish, 463 — Examination of Sewage
and Sewage Effluents, 466 — Examination of Air, 468
— Examination of Soil, 470 — Testing Filters, 478 —
Testing of Disinfectants, 480.

APPENDIX 492

INDEX .505

[N THE VEGETABLE KINGDOM

OGAMS

•PHYTA

ngi

«3

0)
O

g

g

o
a

g

aJ

omycetes Gyrr

8
<

M

a
-M

c

0)

1

CD
O

Mucorinae Penici
Asper

1

w

0)

8

{>,

THE POSITION OF BACTERIA ]

CRYPT

THALLC

fe
tt

o
1

CD

o

a

o
a

w

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a

to

a

, .2

_o

' 6
.2

'a

i

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t/3 '

1)

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3

L>
^

.2
•9

1

pq t

BACTERIOLOGICAL TECHNIQUE.

I. LABORATORY REGULATIONS.

The following regulations are laid down for observ-
ance in the Bacteriological Laboratories under the
direction of the author. Similar regulations should
be enforced in all laboratories where pathogenic
bacteria are studied.

BACTERIOLOGICAL DEPARTMENT.

HANDLING OF INFECTIVE MATERIALS.

The following Regulations have been drawn up in the interest
of those working in the Laboratory as well as the public at large,
and will be strictly enforced.

Their object is to avoid the dangers of infection which may
arise from neglect of necessary precautions or from carelessness.

Everyone must note that by neglecting the general rules laid
down he not only runs grave risk himself, but is a danger to others.

I REGULATIONS.

1. Each worker must wear a gown or overall, provided at his
own expense, which must be kept in the Laboratory.

2. The hands must be disinfected with lysol 2 per cent, solu-
tion, carbolic acid 5 per cent, solution, or corrosive sublimate
i per mille solution, after dealing with infectious material, and
before using towels.

3. On no account must Laboratory towels or dusters be used
for wiping up infectious material, and if such towels or dusters do
become soiled, they must be immediately sterilised by boiling.

4. Special pails containing disinfectant are provided to receive
any waste material, and nothing must be thrown on the floor.

I

2 LABORATORY REGULATIONS

5. All instruments must be flamed, boiled, or otherwise dis-
infected immediately after use.

6. Labels must be moistened with water, and not by the mouth.

7. All disused cover-glasses, slides, and pipettes after use in
handling infectious material, etc., must be placed in 2 per cent,
lysol solution. A vessel is supplied on each bench for this
purpose.

8. All plate and tube cultures of pathogenic organisms when
done with, must be placed for immediate disinfection in the boxes
provided for the purpose.

9. No fluids are to be discharged into sinks or drains unless
previously disinfected.

10. Animals are to be dissected only after being nailed out on
the wooden boards, and their skin thoroughly washed with
disinfectant solution.

11. Immediately the post-mortem examination is completed
each cadaver must be placed in the zinc animal-box — without
removing the carcase from, the post-mortem board — and the cover
of the box replaced, ready for carriage to the destructor.

12. Dead animals, when done with, are cremated in the destruc-
tor, and the laboratory attendant must be notified when the bodies
are ready for cremation.

13. None of the workers in the laboratory are allowed to enter
the animal houses unless accompanied by the special attendant
in charge, who must scrupulously observe the same directions
regarding personal disinfection as the workers in the laboratories.

14. No cultures are to be taken out of the laboratory without
the permission of the head of the Department.

15. All accidents, such as spilling infected material, cutting
or pricking the fingers, must be at once reported to the bacteri-
ologist in charge.

II. 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. 18X1.5 cm., to contain media for ordinary tube
cultivations.

2. 18X1.3 cm., to contain media used for pouring
plate cultivations, and also for holding sterile " swabs."

3. 18X2 cm., to contain wedges of potato, beet-
root, or other vegetable media.

4. 13X1.5 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 $1.50, or 6 shillings per gross.) Such
tubes are expensive it is true, but they are sufficiently
stout to resist rough handling, do not usually break
if accidentally allowed to drop (a point of some moment
when dealing with cultures of pathogenic bacteria),
can be cleaned, sterilised, and used over and over again,
and by their length of life fully justify their initial
expense.

A point be noted is that the manufacturers rarely
turn out such tubes as these absolutely uniform in

3

4 GLASS APPARATUS IN COMMON USE

calibre, and a batch of 1 8 by 1.5 cm. tubes usually con-
tains such extreme sizes as 18 by 2 cm. and 18 by 1.3
cm. Consequently, if a set of standard tubes is kept
for comparison or callipers are used each new supply of
so-called 18 by 1.5 cm. tubes may be easily sorted out
into these three sizes, and so simplify ordering.

5. 5X0.7 cm., for use in the inverted position inside
the tubes containing carbohydrate media, as gas-
collecting tubes.

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

FIG. i. — Bohemian flask. FIG. 2.— Pear-shaped FIG. 3. — Erlenmeyer flask

flask. (narrow neck).

, 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 for the smaller sizes —
i. e., 250 and 500 c.c.

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

KOLLE'S CULTURE FLASKS 5

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,

FIG. 4. — Kolle's cul-
ture flask.

FIG. 5. — Roux's
culture bottle.

FIG. 6. — Guy's culture
bottle.

and for many purposes thin, flat culture bottles known
as Roux's bottles (Fig. 5) are to be preferred.

An even more convenient pattern is that used in the
author's laboratory (Fig. 6), as owing to the greater

FIG. 7.— Filter flask.

depth of medium which it is possible to obtain in these
flasks an exceedingly luxuriant growth is possible; the
narrow neck reduces the chance of accidental contami-
nation to a minimum and the general shape permits
the flasks to be stacked one upon the other.

6 GLASS APPARATUS IN COMMON USE

Filter Flasks or Kitasato's Serum Flasks (Fig. 7).—
Various sizes, from 250 to. 2000 c.c. capacity. These
must be of stout glass, to resist the pressure to which
they are subjected, but at the same time must be
thoroughly well annealed, in order to withstand the
temperature necessary for sterilisation.

All flasks should be either of Jena glass or the almost
equally well-known Resistance or R glass, the extra
initial expense being justified by the comparative
immunity of the glass from breakage.

Petri's Dishes or "Plates" (Fig. 8, 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
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. A batch of eighteen such plates is
sterilised and stored in a cylindrical copper box (30
cm. high by 12 cm. diameter) provided with a " pull-off"
lid. Inside each 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. 9) for removal.

Capsules (Fig. 8, b and c). — These are Petri's dishes
of smaller diameter but greater depth than those termed
plates. Two sizes will be found especially 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 construction 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.:

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

GRADUATED PIPETTES

7

2. Pipettes of i c.c. capacity graduated in o.oi c.c.
(Fig. 10, a).

FIG. 8.— Petri dish (a), and
capsules (b, c).

FIG. 9.— Plate box
with stirrup.

3. Pipettes of 10 c.c. capacity graduated in o.i c.c.
(Fig. 10, b).

These should be about 30 cm. in a b

length (i and 2 of fairly narrow bore) ,
graduated to the extreme point, and
having at least a 10 cm. length of clear
space between the first graduation and
the upper end ; the open mouth should be
plugged with cotton- wool. Each variety
should be sterilised and 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 contained pipettes.

The laboratory should also be pro-
vided with a complete set of " Standard "
graduated pipettes, each pipette in the
set being stamped and authenticated by
a certificate from one of the recognised Physical
Measurement Laboratories, such as Charlottenburg.

FIG . i o. —

Measuring pi-
pettes, a and b.

8

GLASS APPARATUS IN COMMON USE

These instruments are expensive and should be reserved
solely for standardising the pipettes in ordinary use, and
for calibrating small pipettes manufactured in the
laboratory. Such a set should comprise, at least,
pipettes delivering 10 c.c., 5 c.c., 2.5 c.c., 2 c.c., i c.c.,
0.5 c.c., 0.25 c.c., 0.2 c.c., o.i c.c., 0.05 c. c., and o.oi c.c.,
respectively.

In the immediately following sections are described
small' pieces of glass apparatus which should be prepared
in the laboratory from glass tubing of various sizes.
In their preparation three articles are essential; first
a three-square hard-steel file or preferably a glass- work-
er's knife of hard Thuringian steel for cutting glass tubes
etc. ; next a blowpipe flame, for although much can be
done with the ordinary Bunsen burner, a blowpipe
flame makes for rapid work; and lastly a bat's- wing
burner.

i . The glass-cutting knife. This article is sold in two
forms, a bench knife (Fig. u) and a pocket knife. The
former is provided with a blade some 8 cm. in length

FIG. u.— Glass-cutting knife, a. handle, b. double edged blade.
c. shaft, d. locking nut. e. spanner for nut.

and having two cutting edges. The cutting edge when
examined in a strong light is seen to be composed of small
closely set teeth, similar to those in a saw. The knife
should be kept sharp by frequent strappings on a
sandstone hone. The pocket form, about 6-cm. long

SEDIMENTATION TUBES 9

over all, consists of a small spring blade with one
cutting edge mounted in scales like an ordinary pocket
knife.

2 . For real convenience of work the blowpipe should
be mounted on a special table connected up with
cylindrical bellows operated by a pedal. That figured
(Fig. 12) is made by mounting a teak top 60 cm. square
upon the uprights of an enclosed double-action concer-
tina bellows (Enfer's) and provided with a Fletcher's
Universal gas blowpipe.

3. An ordinary bat's- wing gas-burner mounted at the
far corner of the table top is invaluable in the prepara-
tion of tubular apparatus with sharp curves, and for
coating newly-made glass apparatus with a layer of
soot to prevent too rapid cooling, and its usually asso-
ciated result — cracking.

6. Sedimentation tubes 5x0.5 cm., for sedimentation
reactions, etc., and for containing small quantities of
fluid to be centrifugalisde in the haematocrit. These
are made by taking i4-cm. lengths of stout glass tubing

FIG. 12. — Glass blower's table with Enfer's foot bellows.

of the requisite diameter and heating the centre in the
Bunsen or blow-pipe flame. When the central portion
is quite soft draw the ends quickly apart and then
round off the pointed ends of the two test-tubes thus

10

GLASS APPARATUS IN COMMON USE

formed. With the glass-cutting knife cut off whatever
may be necessary from the open ends to make the
tubes the required length.

A rectangular block of "plasticine" (modelling clay)
into which the conical ends can be thrust makes a
very convenient stand for these small tubes.

Capillary Pipettes or Pasteur's Pipettes (Fig. 13 a). —
These little instruments are invaluable, and a goodly
a. b. c. supply should be kept on hand. They
are prepared from soft-glass tubing of
various-sized calibre (the most generally
useful size being 8 mm. diameter) in the
following manner: Hold a 10 cm. length
of glass tube by each end, and whilst
rotating it heat the central portion in
the Bunsen flame or the blow-pipe
blast-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
roomy capillary tube; break the capil-
lary portion 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 completes the capil-
lary pipette. After a number have
been prepared, they are sterilised and
FIG i — Ca ii s^ore<^ in batches, either in metal cases
lary pipettes. a, similar to those used for the graduated
pipettes or in large-sized test-tubes —
sealed ends downward and plugged ends toward the
mouth of the case.

The filling and emptying of the capillary pipette
is most satisfactorily accomplished by slipping a small
rubber teat (similar to that on a baby's feeding bottle
but not perforated) on the upper end, after cutting or

BLOOD PIPETTES II

snapping off the sealed point of the capillary portion.
If pressure is now exerted upon the elastic bulb by a
finger and thumb whilst the capillary end is below the
surface of the fluid to be taken up, some of the
contained air will be driven out, and subsequent relax-
ation of that pressure (resulting in the formation of a
partial vacuum) will cause the fluid to ascend the
capillary tube. Subsequent compression of the bulb
will naturally result in the complete expulsion of the
fluid from the pipette (Fig. 1 4) .

FIG. 14. — Filling the capillary teat-pipette.

A modification of this pipette, in which a constric-
tion or short length of capillary tube is introduced
just below the plugged mouth (Fig. 13, 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. 13, c), will also be
found useful.

"Blood" Pipettes (Fig 15). — Special pipettes for
the collection of fairly large quantities of blood (as
suggested by Pakes) should also be prepared. These
are made from soft glass tubing of i cm. bore, in a
similar manner to the Pasteur pipettes, except that

12

GLASS APPARATUS IN COMMON USE

the point of the blowpipe flame must be used in order
to obtain the sharp shoulder at either end of the cen-
tral bulb. The terminal tubes must retain a diameter
of at least i mm., in order to avoid capillary action
during the collection 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. 15. — Blood pipettes
and hair-lip pin in a test-
tube.

FIG. 16. — Blood-pipette in metal
thermometer case.

it is usual to place a lance-headed hare-lip pin or a
No. 9 flat Hagedorn needle inside the tube so that the
entire outfit may be sterilised at one time.

For the collection of small quantities of blood for
agglutination reactions and the like, many prefer a
short straight piece of narrow glass tubing drawn out
at either extremity to almost capillary dimensions.
Such pipettes, about 8 cm. in length over all, are most

AUTOMATIC PIPETTES 13

conveniently sterilized in ordinary metal thermometer
cases (Fig. 16).

Graduated Capillary Pipettes (Fig. 17). — These should
also be made in the laboratory — from manometer
tubing- — of simple, convenient shape, and graduated
by the aid of "standard" pipettes (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.

Automatic ("Throttle") Pipettes.—
These ingenious pipettes, introduced
by Wright, can easily be calibrated
in the laboratory and are exceedingly
useful for graduating small pipettes, pIG< 17.— capii-
f or measuring small quantities of fluids, ! a r y graduated

. & A ,.H , ' pipettes.

in prepanng dilutions of serum for
agglutination reactions, etc. They are usually made
from the Capillary Pasteur pipettes (Fig. 13, a). The
following description of the manufacture of a 5 c.mm.
pipette will serve to show how the small automatic
pipettes are calibrated.

1. Select a pipette the capillary portion of which is
fairly roomy in bore and possesses regular even walls,
and remove the cotton- wool plug from the open end.

2. Heat the capillary portion near the free extremity
in the by-pass flame of the bunsen burner and draw it
out into a very fine hair-like tube and break this
across. This hair-like extremity will permit the pas-
sage of air but is too fine for metallic mercury to pass.

3. From a standard graduated pipette deliver
5 c.mm. clean mercury into the upper wide portion of
the pipette.

14 GLASS APPARATUS IN COMMON USE

4. Adjust a rubber teat to the pipette and by pres-
sure on the bulb gradually drive the mercury in an
unbroken column down the capillary tube until it is
stopped by the filiform extremity.

5. Cut off the capillary tube exactly at the upper
level of the column of mercury, invert it and allow
the mercury to run out.

6. Snap off the remainder of the capillary tube from
the broad upper portion of the pipette which is now

destined to form the covering tube or
air chamber, or what we may term the
" barrel." This barrel now has the lower
end in the form of a truncated cone, the
upper end being cut square. Remove
the teat.

7. Introduce the capillary tube into
this barrel with the filiform extremity
uppermost, and the square cut end pro-
jecting about 0.5 cm. beyond the taper-
ing end of the barrel.

8. Drop a small pellet of sealing wax
into the barrel by the side of the capil-
lary tube and then warm the tube at

FIG. 1 8.— the gas flame until the wax becomes
Throttle pipette softened and makes an air-tight joint

— small capacity. e J

between the capillary tube and the end
of the barrel.

9. Fit a rubber teat to the open end of the barrel,
and so complete a pipette which can be depended upon
to always aspirate and deliver exactly 5 cm. of fluid.

Slight modification of this procedure is necessary in
making tubes to measure larger volumes than say
75 c.mm. Thus to make a throttle pipette to measure
100 c.mm.:

i. Take a short length of quill tubing and draw out
one end into a roomy capillary stem, and again draw
out the extremity into a fine hair point, thus forming

AUTOMATIC PIPETTES 15

a small Pasteur pipette with a hair-like capillary
extremity.

2. With a standard pipette fill 100 c.mm. into the
neck of this pipette, and make a scratch with a writing
diamond at the upper level (a) of the mercury meniscus
(Fig. 1 9, A).

FIG. 19. — Making throttle pipettes — large capacity

Now force the mercury down into the capillary stem
•as far as it will go, so as to leave the upper part of the
tube in the region of the diamond scratch empty
(Fig. 19, B).

3 . Heat the tube in the region of the diamond scratch
in the blowpipe flame, and removing the tube from
the flame draw it out so that the diamond scratch now
occupies a position somewhere near the centre of this
new capillary portion (Fig. 19, C).

1 6 GLASS APPARATUS IN COMMON USE

4. Heat the tube in this position in the peep flame
of the Bunsen burner, and draw it out into a hair-like
extremity. Snap off the glass tube, leaving about
5 mm. of hair-like extremity attached to the upper
capillary portion (Fig. 19, D). Allow the glass to cool.

5. Lift up the bulb by the long capillary stem and
allow the mercury to return to its original position —
an operation which will be facilitated by snapping
off the hair-like extremity from the long piece of
capillary tubing.

6. Mark on the capillary stem with a grease pencil
the position of the end of the column of mercury
(Fig. 19, E.)

7. Warm the capillary tubing at this spot in the
peep flame of the Bunsen burner, and draw it out very
slightly so that when cut at this position a pointed
extremity will be obtained.

8. With a glass-cutting knife cut the capillary tube
through at the point "6," and allow the mercury to
run out.

9. Now apply a thick layer of sealing wax to the
neck of the bulb.

10. Take a piece of 5 mm. bore glass tubing and
draw it out as if making an ordinary Pasteur pipette.

11. Break the capillary portion off so as to leave a
covering tube similar to that already used for the
smaller graduated pipettes. Into this covering tube
drop the graduated bulb and draw the capillary stem
down through the conical .extremity until further
progress is stopped by the layer of sealing wax.

12. Warm the pipette in the gas flame so as to melt
the sealing wax and make an air-tight joint.

13. Fit an india-rubber teat over the open end of the
covering tube, and the automatic pipette is ready for
use (Fig. 19, F).

Sedimentation Pipettes (Fig. 20). — These are prepared
from 10 cm. lengths of narrow glass tubing by sealing

FERMENTATION TUBES

one extremity, blowing a small bulb at the centre, and
plugging the open end with cotton-wool ; after sterilisa-
tion the open end is provided with a short piece of
rubber tubing and a glass mouthpiece. When it is
necessary to observe sedimentation reactions in very
small quantities of fluid, these tubes will be found

FIG. 20. — Sedimentation pipette.

much more convenient than the 5 by 0.5 cm. test-tubes
previously mentioned.

Pasteur pipettes fitted with india-rubber teats will
also be found useful for sedimentation tests when
dealing with minute quantities of serum, etc.

Fermentation Tubes (Fig. 21). — These are used for
the collection and analysis of the gases liberated from

a b c

FIG. 21. — Fermentation tubes.

the media during the growth of some varieties of bac-
teria and may be either plain (a) or graduated (b).
A simple form (Fig. 21, 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

l8 GLASS APPARATUS IN COMMON USE

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 — or boil in the autoclave for a
similar period.

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

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.

CLEANING INFECTED TEST-TUBES 19

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

6. Place the pipettes in the hot-air oven (vide page
31), 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 37)-

(If an autoclave is not available, the tubes must be
placed in a digester, or even a large pan or pail with a
tightly fitting cover, and boiled vigorously 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.

20 GLASS APPARATUS IN COMMON USE

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 oe 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.

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
minutes.

3. Rinse thoroughly in cold water.

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

CLEANING PIPETTES

21

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

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

When dealing with graduated capillary pipettes em-
ployed for blood or serum work (whether new or in-
fected), much time is consumed in the various steps
from 5 onward, and the cleansing process can be mate-
rially hastened if the following device is adopted.

Fit up a large-sized Kitasato's filter flask to a Spren-
gel's suction pump or a Geryk air pump (see page
43). To the side tubulure of the filter flask attach
a 20 cm. length of rubber pressure tubing having
a calibre sufficiently large to admit the ends of the
pipettes.

Next fill a small beaker with distilled water. Attach
the first pipette to the free end of the rubber tubing,

FIG. 22. — Cleaning blood pipettes.

place the pipette point downward in the beaker of
water and start the pump (Fig. 22).

When all the water has been aspirated through the
pipette into the filter flask, fill the beaker with recti-
fied spirit and when this is exhausted refill with ether.
Detach the pipette and dry in the hot-air oven.

Slides and cover =slips (Fig. 23), when first purchased,

22

GLASS APPARATUS IN COMMON USE

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.

i

22«ji

38,
Vi

X
X

8-^

.yO •

\ 4 QifiTin

T/6"^*

1

t

— ' %

i

FIG. 23. — Slides and cover-slips, actual size.

If only a few slides are required for immediate use
a good plan is to rub the surface with jeweler's emery
paper (Hubert's oo). A piece of hard wood ;6X26X
26 mm. with a piece of this emery paper gummed
tightly around it is an exceedingly useful article on
the microscope bench.

Cover=slips. — The most useful sizes are the 19 mm.
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 man-
ner:

i. 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.

USED SLIDES AND COVER-SLIPS 23

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
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.

24 GLASS APPARATUS IN COMMON USE

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 to i per cent,
with hydrofluoric acid, 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-
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.

4. Now, whilst supporting the wool between the
thumb and fingers of the right hand, rotate the test-

PLUGGING TEST-TUBES. 25

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 pro-
jecting.

The plug must be firm and fit the tube or flask fairly
tightly, sufficiently tightly in fact to bear the weight

FIG. 24. — Plugging test-tubes: a, cylinder of wool being rolled; &, cylinder
of wool being doubled; c, cylinder of wool being inserted in tube.

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 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.

III. METHODS OF STERILISATION.

STERILISING AGENTS.

STERILISATION — i. e., the removal or the destruction
of germ life — may be effected by the use of various
agents. As applied to the practical requirements of
the bacteriological laboratory, many of these agents,
such as electricity, sunlight, etc., are of little value,
others are limited in their applications; others again
are so well suited to particular purposes that their use
is almost entirely restricted to such.

The sterilising agents in common use are :

Chemical Reagents. — Disinfectants (for the disin-
fection of glass and metal apparatus and* of morbid
tissues) .

Physical Agents. 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 metal apparatus) .

(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 the destruc-
tion of old cultivations of bacteria) .

26

CHEMICAL REAGENTS 27

FILTRATION. —

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 (in order of general utility)
belong :

Lysol, 2 per cent, solution;

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

Carbolic acid, 5 per cent, solution ;

Absolute alcohol;

Ether;

Chloroform ;

Camphor;

Thymol;

Toluol;

Volatile oils, such as oil of mustard, oil of garlic.
Formaldehyde is a powerful germicide, but its pene-
trating vapor restricts its use. These disinfectants are
but little used in the final sterilisation of apparatus,
chiefly on account of the difficulty of effecting their
complete removal, for the presence of even traces of
these chemicals is sufficient to so inhibit or alter the
growth of bacteria as to vitiate subsequent experi-
ments conducted by the aid of apparatus sterilised in
this manner.

28

METHODS OF STERILISATION

NOTE. — Tubes, flasks, filter flasks, pipettes, glass tubing, etc.,
may be rapidly sterilised, in case of emergency, by washing, in
turn, with distilled water, perchloride of mercury solution, alco-
hol, and ether, draining, and finally gently heating over a gas
flame to completely drive off the ether vapor. Chloroform or
other volatile disinfectants 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 invariably used for sterilising the platinum needles
(which are heated to redness) and may be employed
for sterilising the points of forceps, or other small

instruments, cover-glasses, pi-
pettes, etc., a very short ex-
posure to this heat being
sufficient.

Ether Flame. — In an emer-
gency small instruments,
needles, etc., may be sterilised
by dipping them in ether and
after removal lighting the
adherent fluid and allowing
it to burn off the surface of
the instruments. Repeat the

FIG. 25. — Muffle furnace.

process twice. It may then be safely assumed that the
apparatus so treated is sterile.

Muffle Furnace (Fig. 25). — Although this form of
heat is chiefly used for the destruction of the dead
bodies of small infected animals, morbid tissues, etc.,
it is also employed for the sterilisation of porcelain
filter candles (vide p. 42).

Filter candles are disinfected immediately after use
by boiling in a beaker of water for some fifteen or
twenty minutes. This treatment, however, leaves the
dead bodies of the bacteria upon the surface and block-
ing the interstices of the filter.

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

DRY HEAT 29

1. Roll each bougie up in a piece of asbestos cloth,
secure the ends of the cloth with a few turns of cop-
per wire, and place inside the muffle (a small muffle
76X88X163 mm: will hold perhaps four small filter
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 has
become quite cold remove the filter candles, and store
them (without removing the asbestos wrappings) in
sterile metal boxes.

NOTE. — The too rapid cooling of the candles, such as takes place
if they are removed from the muffle before it has cooled down to
the room temperature, may give rise to microscopic cracks and
flaws which will effectually destroy their efficiency.

Hot Air. — Hot air at 150° C. destroys all bacteria,
spores, etc., in about thirty minutes; a momentary
exposure to a temperature of 175° to 180° C. will
effect the same result and offers the more convenient
method of sterilisation. This method is only appli-
cable to glass and metallic substances, and the small
bulk of cotton-wool comprised in the test-tube plugs,
etc. Large masses of fabric are not effectually steril-
ised by dry heat — short of charring — as its power of
penetration is not great.

Sterilisation by hot air is effected in the hot-air oven
(Fig. 1 8). This is a rectangular, double- walled metal
box, mounted on a stand and heated from below by a
large Bunsen burner. The interior 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. 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

30 METHODS OF STERILISATION

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. An ordinary mer-
curial thermo-regulator may be used but it is prefer-
able to employ a regulating capsule of the Hearson
type (see p. 219) with a spring arm adjusted to the lever
so that when the boiling-point of the capsule (e.g.,

FIG. 26. — Hot-air oven.

175° C.) is reached the gas supply is absolutely cut off
and the jet cannot again be lighted until the spring
arm has been readjusted by hand. The thermo-
regulator is by no means a necessity, and may be
replaced by a large bore thermometer with a sliding
platinum point, connected with an electric bell, which
can be easily adjusted to ring at any given temperature.
Even if the steriliser is provided with the capsule
regulator above described the contact thermometer
should also be fitted.

DRY HEAT 3!

To USE THE HOT-AIR OVEN. —

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

To prepare a wire crate 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 the crate,
working it into the meshes and smoothing the 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 temperature of the interior up to about
1 60° C. After an interval of 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 considerably
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, in order to protect
the points of these articles from injury.

2. Close the oven door, and open the ventilating
slide, in order that any moisture left in the tubes, etc.,
may escape; light the gas below; set the electric alarm
to ring at 1 00° C.

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 1 7 5° C.

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.

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

£2 METHODS OF STERILISATION

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— Wafer 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 coagu-
late 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).

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 such spores as may be present will have germinated
and assumed the vegetative form.

MOIST HEAT

33

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

FIG. 27 . — Water sterilizer.

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" (Fig 27).

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

34

METHODS OF STERILISATION

dimension than the steriliser itself, and provided with
handles. The top of the steriliser is hinged to form a lid.

METHOD.—

1. Place the instruments, etc., to be sterilised in-
side the copper basket, and replace the basket in the
steriliser.

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

FIG. 28. — Koch's steriliser.

FIG. 29. — Arnold's steriliser.

3. After the water has boiled and steam has been
issuing from beneath the lid for at least ten minutes,
extinguish the gas, open the lid, and lift out the wire
basket by its handles and rest it diagonally on the
walls of the steriliser; 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
to the steriliser; completely disinfect by a further boil-
ing for fifteen minutes.

5. After disinfection, and whilst still hot, take out

MOIST HEAT 35

the instruments, dry carefully and 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
the cultivation of bacteria, and is carried out in a
steam kettle of special construction, known as Koch's
steam steriliser (Fig. 28) or in one of its many modifi-
cations, the most efficient of which is Arnold's (Fig. 29).

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 part for the reception of
wire baskets containing the articles to be sterilised.
The vessel is closed by a loose conical lid, provided
with handles, and perforated at its apex by a tubu-
lure ; 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.

36 METHODS OF STERILISATION

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 pre-
vent moisture condensing upon 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 organisms 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 inter-
mittent sterilisation.

Exposure to steam at 100° C. for a period of one or
two hours, or continuous sterilisation, cannot always be
depended upon and is therefore not to be recommended.

Superheated steam — i. e., steam under pressure (see
Pressure-temperature table, Appendix, page 500) 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 at-
tained in ten minutes. This method was formerly em-
ployed for the sterilisation of media (and indeed is so
used in some laboratories still), but most workers
now realise that media subjected to this high tem-
perature undergo hydrolytic changes which render
them unsuitable for the cultivation of the more deli-
cate micro-organisms. The use of superheated steam
should be restricted almost entirely to the disinfection
of such contaminated articles, old cultivations, etc.,

MOIST HEAT 37

as cannot be dealt with by dry heat or the actual
furnace. Sterilisation by means of superheated steam
is carried out in a special boiler — Chamberland's
autoclave (Fig. 30). The autoclave consists of a stout
copper ylinder, 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 interposition of a
rubber washer. The cover is perforated for a branched
tube carrying a vent cock, a manometer, and a safety
valve. The copper boiler is mounted in the upper half
of a cylindrical sheet-iron case — two concentric cir-
cular rows of Bunsen burners, each circle having an
independent gas-supply, occupying the lower half.
In the interior of the boiler is a large movable wire
basket, mounted on legs, for the reception of the
articles to be sterilised.

To USE THE AUTOCLAVE. —

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

2. Run water into the boiler to the level of the bot-
tom 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 auto-
clave 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 i2op 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. In the more expensive patterns of
autoclave this regulation of the safety valve is carried

38 METHODS OF STERILISATION

out automatically, the manometer being fitted with an
adjustable pointer which can be set to any required
pressure-temperature and so arranged that when the
index of the manometer coincides with the adjustable
hand the safety valve is opened.

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

FIG. 30. — Chamberland's Autoclave.

8. Now open the vent cock slowly, and allow the
internal pressure to adjust itself to that of the
atmosphere.

9. Remove the cover and take out the sterilised
contents.

Sterilisation Periods. — An exceedingly useful device
for the timing of sterilisation periods (and indeed for
many other operations in the laboratory) is the

ELECTRIC SIGNAL TIMING CLOCK.

This is a clock of American type in which the face
is surrounded by a metal plate having a series of 60

TIMING STERILISATION PERIODS 39

holes at equal distances apart, corresponding to the
minutes on the dial. This plate is connected with
one of the poles of a dry battery, the other pole of
which is connected to the metal case of the clock for
the purpose of actuating an ordinary magnet alarm
bell. In the centre of each of the holes in the plate
a metal rod is fixed, which then passes through an

FIG. 31. — Electric signal timing clock.

insulating ring and projects inside the clock face,
where it makes contact with the hour hand. The
clock is mounted on a heavy base, with a key-board
containing 20 numbered plugs. If one of the plugs is
inserted in a hole in the plate it makes contact with
the rod, and when the hour hand of the clock touches
the other end the circuit is completed and the bell
starts ringing. The period of this friction contact is
approximately 20 seconds. The clock can therefore
be used for electrically noting the periods of time
from one minute by multiples of one minute up to one
hour.

40 METHODS OF STERILISATION

Filtration. — (a) Cotton-wool Filter. — Practically the
only method in use in the laboratory for the sterilisa-
tion 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

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 atmos-
phere, or from liquids coming into contact with it,
micro-organisms (especially the mould fungi) com-
mence to multiply, and the long thread forms rapidly
penetrate the substance of the plug, and gain access to
and contaminate the interior of the tube.

METHOD. —

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
diphtheria bacillus, this can be readily effected as
follows :

FILTRATION 41

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 (Fig. 32), wrap a
layer of cotton-wool around each end of the tube, and
secure in position with a turn of thin copper wire or
string; then sterilise the piece of apparatus in the hot-
air oven.

2. Prepare the cultivation in a Ruffer or Woodhead
flask (Fig. 33) the inlet tube of which has its free
extremity enveloped in a layer of cotton- wool, secured

FIG. 33.— Ruffer's flask.

by thread or wire, 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. When all is ready 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.

42 METHODS OF STERILISATION

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 Filter. — The sterilisation of liquids by
filtration is effected by passing them through a cylin-
drical 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 (Berkefeld system), and
termed a "bougie" or "candle" (Fig. 34).

NOTE. — In selecting candles for use in the laboratory avoid those
with metal fittings, since during sterilisation cracks develop at the
junction of the metal and the siliceous material owing to the un-
equal expansion.

In this method the bacteria are retained 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 extremely minute, and therefore the rate
of filtration will usually be slow. Chamberland filter
candles possess finer channels than Berkefeld candles
and consequently filter much more slowly. To over-
come this disadvantage, either aspiration or pressure,
or a combination of these two forces, may be employed
to hasten the process.

Doultons white porcelain filters it may be noted are
as efficient as the Chamberland candles and filter
rather more rapidly.

Apparatus Required. —

1. Separatory funnel containing the unfiltered fluid.

2. Sterile filter candle (Fig. 34), the open end fitted with a
rubber stopper (Fig. 34, a) perforated to receive the delivery tube
of the separatory funnel, and its neck passed through a large rubber
washer"(Fig. 34, b) 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.

FILTRATION

43

4. Water injector Sprengel (see Fig. 38, c) pump, or Geryk's
pump (an air pump on the hydraulic principle, sealed by means
of low vapor-tension oil, Fig. 35).

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

5. Air filter (vide page 40) sterilised.

6. Pressure tubing.

7. Screw clamps (Fig. 36).

METHOD.—

i. Couple the exhaust pipe of the suc-
tion pump with the lateral tube of the FIG. 34.— Force-
filter flask (first removing the cotton- lainfil
wool plug from this latter), by means of pressure
tubing, interposing, if necessary, the wash-bottle of
sulphuric acid.

FIG. 35. — Geryk air pump.

44

METHODS OF STERILISATION

2. Remove the cotton-wool plug from the neck of
the filter flask and adjust the porcelain candle in its
place.

FIG. 36. — Screw clamps.

3. Attach the nozzle of the separatory funnel to the
filter candle by means of the perforated rubber stopper
(Fig. 37)-

FIG. 37. — Apparatus arranged for filtering — aspiration.

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. When the filtration is completed close the tap of

FILTRATION 45

the funnel ; 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
filtered air to enter the flask, to abolish 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 the neck with sterile
cotton- wool.

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 tyres is one of the most generally use-
ful, for this purpose) or with a cylinder of compressed
air or other gas.

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 (Fig. 38, a), to hold the filter candle itself as
well as the fluid to be filtered, should be employed.
(A vacuum must 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

46

METHODS OF STERILISATION

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. 38, a). In use the reservoir
is generally mounted on a tripod stand.

To Sterilise.—

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

FIG. 38. — Fakes' filtering reservoir — pressure and aspiration.

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.

FILTRATION

47

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.

FIG. 39. — Closed candle arranged for filtering.

4. Fill the fluid to be filtered into the cylinder and
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. 38, b), by means of pressure tubing.

6. Connect the lateral arm of the filter flask with
the exhaust pump (Fig. 38, c) and start the latter
working.

48 METHODS OF STERILISATION

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,
usually one or one and a half atmospheres, until filtra-
tion is completed, by regulating the tap on the entry
tube.

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 39.

IV. 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. 40. — Microscope stand.

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 vari9us joints and movements must work smoothly
and precisely, equally free from the defects of "loss
of time" and "slipping." All screws, etc., should con-
4 49

50 THE MICROSCOPE

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. 40, a). — Two elementary
forms — the tripod (Fig. 41, a) and the vertical column

FIG. 41. — Foot, three types.

set into a plate known as the "horse-shoe" (Fig
41, b) — serve as the patterns for countless modifica-
tions in shape and size of this portion of the stand.
The chief desiderata — stability and ease of manipula-
tion— 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 preferred. Its chief rival,
the Jackson foot (Fig. 41, c), is based upon the same
principle, and on the score of appearance has much to
recommend it.

2. The body tube (Fig. 40, b) may be either that
known as the "long" or "English" (length 250 mm.),
or the "short" or "Continental" (length 160 mm.).
Neither length appears to possess any material advan-
tage over the other, but it is absolutely necessary to
secure objectives which have been manufactured for
the particular tube length chosen. In the high-class
microscope of the present day the body tube is usually

THE FINE ADJUSTMENT 51

shorter than the Continental, but is provided with a
draw tube which, when fully extended, gives a tube
length greater than the English, thus permitting the
use of either form of objective.

For practical purposes the tube length = distance from the
end of the nosepiece to the eyeglass of the ocular. This is the
measurement referred to in speaking of "long" or "short" tube.

FIG. 42. — Coarse adjustment.

FIG. 43. — Fine adjustment.

3. The coarse adjustment (Fig. 40, c) should be a
rack-and-pinion movement, steadiness and smoothness
of action being secured by means of accurately fitting
dovetailed bearings and perfect correspondence between
the teeth of the rack and the leaves of the pinion (Fig.
42). Also provision should be made for taking up the
"slack" (as by the screws A A, Fig. 42).

4. The fine adjustment (Fig. 40, d) should on no
account depend upon the direct action of springs, but

52 THE MICROSCOPE

should be of the lever pattern, preferably the Nelson
(Fig. 43). 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 actuating the movement.

A spindle milled head (Fig. 44) will be found a very
useful device to have fitted in place of the ordinary
milled head controlling the fine adjustment. In this
contrivance the axis of the milled head is prolonged
upward in a short column, the diameter of which is
one-sixth of that of the head. The spindle
can be rapidly rotated between the fingers
for medium power adjustments while the
larger milled head can be slowly moved
when focussing high powers.

5. The stage (Fig. 40, e) should be square
in shape and large in area — at least 1 2 cm.
—flat and rigid, in order to afford a safe
support for the Petri dish used for plate
Spindle head to cultivations; and should be supplied, with
fine adjust- spring ciipS (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
concerned, as when working with high powers, and
especially when examining hanging-drop specimens,
it is almost impossible to execute sufficiently delicate
movements with the fingers. In selecting a mechan-
ical stage, preference should be given to one which
forms an integral part of the instrument (Fig. 45)
rather than one which needs to be clamped on to an
ordinary plain stage every time it is required, and its
traversing movements should be controlled by sta-
tionary milled heads (Fig. 45, A A'). The shape of the
aperture is a not unimportant point; it should be
square to allow of free movement over the substage

DIAPHRAGM.

53

condenser. The mechanical stage should be tapped
for three (removable) screw studs to be used in place
of the sliding bar, so that if desired the Vernier finder

FIG. 45. — Mechanical stage.

(Fig. 45, BE'), such as is usually fitted to this class of
stage, or a Maltwood finder, may be employed.

6. Diaphragm. — Separate single diaphragms must

FlG. 46. — Iris diaphragm.

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"

54 THE MICROSCOPE

diaphragm (Fig. 46) which enters into the construc-
tion of the substage condenser.

7. The substage condenser is a necessary part of the
optical outfit. Its purpose is to collect the beam of
parallel rays of light reflected by the plane mirror, by
virtue of a short focus system of lenses, into a cone of
large aperture (reducible 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 thick-
ness of the slides.

The form in most general use is that known as the
Abbe (Fig. 47) and consists of a plano-convex lens
mounted above a biconvex lens. This combination
is carried in a screw-centering holder known as the
substage below the stage of the microscope (Fig. 40, /) ,
and must be accurately adjusted
so that its optical axis coincides
with that of the objective. Ver-
tical movement of the entire
substage apparatus effected by
means of a rack and pinion is a

'IG-47-l?uStPof°fAbMdecided advantage, and some
means should be provided for

temporarily removing the condenser from the optical
axis of the microscope.

With the oil immersion objective, however, an
achromatic condenser, giving an illuminating cone of
about 0.9, should be used if the full value of the lens
is to be obtained. It is generally assumed that a good
objective requires an illuminating cone equivalent to
two-thirds of its numerical aperture. The best Abbe
condenser transmits a cone of about .45 whilst the
aperture of the TV inch immersion lenses of different
makers varies from i.o to 1.4, hence, the efficiency of
these lenses is much curtailed if the condenser is merely

OCULARS AND OBJECTIVES 55

the Abbe. These improved condensers must be
absolutely centered to the objective and capable of
very accurate focussing otherwise much of their value
is lost.

8. Mirrors. — Below the substage condenser is at-
tached a gymbal carrying a reversible circular frame
with a plane mirror on one side and a concave mirror
on the other (Fig. 40, 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. 48) will be sufficient for all
ordinary work without resorting to the more expen-
sive "compensation" oculars. Two or three, mag-
nifying the "real" image (formed by the objective)
four, six, or eight times respectively, form a useful
equipment.

As an accessory Ehrlich's Eyepiece is a very useful
piece of apparatus when the enumeration of cells or
bacteria has to be carried out. This is an ordinary
eyepiece fitted with an adjustable square diaphragm
operated by a lever projecting from the side of the
mount. Three notches are made in one of the sides of
the square and by moving the lever the square aperture
can be reduced to three-quarters, one-half or one-quar-
ter of the original size.

10. Objectives. — Three objectives are necessary: one
for low-power work — e.g., i inch, § inch, or J inch;
one for high-power work — e. g., -^ inch oil immer-
sion lens; and an intermediate "medium-power" lens —
e. g., J 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
aberration gives rise to an ill-defined image, due to the

THE MICROSCOPE

central and peripheral rays focussing at different
points.

(b) Correction of Chromatic Aberration. — Chromatic
aberration gives rise to a coloured fringe around the
edges of objects due to the fact that the different-
coloured rays of the spectrum possess varying refran-
gibilities and that a simple lens acts toward them as a
prism.

(c) Flatness of Field. — The ideal visual field would be
large and, above all, flat; in other words, objects at the

periphery of the field would be as
distinctly " in focus " as those in the
centre. Unfortunately, however,
this is an optical impossibility and
the field is always spherical in shape.
Some makers succeed in giving a
larger central area that .is in focus
at one time than others, and al-
though this may theoretically cause
an infinitesimal sacrifice of other
qualities, it should always be
sought for. Successive zones and
the entire peripheral ring should
come into focus with the alteration
of the fine adjustment. This simultaneous sharpness
of the entire circle is an indication of the perfect cen-
tering of the whole of the lenses in the objective.

(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

FIG. 48. — Huyghenian
eyepiece.

ACCESSORIES 57

skill and mathematical ability, and is completely
beyond the powers of the average microscopist.1

Although with the increase in power it is correspond-
ingly difficult to combine all these corrections in one
objective, they are brought to a high pitch of excel-
lence in the present-day "achromatic" objectives, and
so 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 r T" 2" 1/r
"blood film, "stained with Jenner's

or Romanowsky's stain.

2. A thin cover -slip prepara-
tion of a young cultivation of
B. diphtheria (showing segmenta-

¥

for £", -J-"
Ty oil

for

i" dry

tion) stained with methylene-
blue.

Accessories. — Eye Shade (Fig. 49). — This piece of
apparatus consists of a pear-shaped piece of blackened
metal or ebonite, hinged 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 unoccupied eye, and when carrying
out prolonged observations will be found of real service.

Nosepiece. — Perhaps the most useful accessory is a
nosepiece to carry two of the objectives (Fig. 50), or,
better still, all three (Fig. 51). This nosepiece, pref-
erably constructed of aluminium, must be of the
covered-in type, consisting of a curved plate attached
to the lower end of the body tube — a circular aperture
being cut to correspond to the lumen of that tube. To

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

58 THE MICROSCOPE

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 successively in to the
optical axis of the microscope.

FIG. 49. — Eye shade.

For critical work and particularly for photo-microg
raphy, however, the interchangeable nosepiece is by
no means perfect as it is next to impossible to secure
accurate centreing of each lens in the optical axis.
For special purposes, therefore, it is necessary to employ

FIG. 50. — Double nosepiece.

FIG. 51 — Triple nosepiece

a special nosepiece such as that made by Zeiss or Leitz
into which each objective slides on its own carrier and
upon which it is accurately centred.

Warm Stage (Fig. 52). — This is a flat metal case
containing a system of tubes through the interior of
which water of any required temperature can be cir-

WARM STAGE 59

culated. It is made to clamp on to the stage of the
microscope by the screws A Af , and is perforated with
a large hole coinciding with the optical axis of the
microscope; a short tube B, projecting from one end
of the warm stage permits water of the desired temper-
ature to be conducted from a reservoir through a length
of rubber tubing to the interior of the stage and a
similar tube at the other end B' of the stage allows
exit to the waste water. By raising the temperature
of hanging-drop preparations, etc., placed upon it,
above that of the surrounding atmosphere, the warm
stage renders possible exact observations on spore
germination, hanging-drop cultivations, etc.

FIG. 52. — Warm stage.

A better form is the electrical hot stage designed by
Lorrain Smith;1 it requires the addition of a lamp
resistance and sliding rheostat, also a delicate ammeter
reading to .01 of an ampere. It consists of a wooden
frame supporting a flat glass bulb with a long neck bent
upward at an obtuse angle (Fig. 53). The bulb is
filled with liquid paraffin, which rises in the open neck
when expanded by heat. The neck also accommo-
dates the thermometer. Two coils of manganin wire
run in the paraffin at opposite sides of the bulb (out-
side the field of vision), coupled to brass terminals on
the wooden frame by platinum wire fused into the
glass. The resistance of the two coils in series is

1 Made by Mr. Otto Baumbach, 10, Lime Grove, Manchester.

60 THE MICROSCOPE

about 10 ohms. A current of 2\ amperes is needed,
and is conducted to the coils in the stage through the
rheostat. With the help of the ammeter any desired
temperature can be obtained and maintained, up to
about 200° C. If immersion oil contact is made
between the top lens of the condenser and the lower
surface of the bulb, this stage works very well indeed
with the J^-inch oil immersion lens.

FIG. 53. — Lorrain Smith's warm stage.

Dark Ground or Paraboloid Condenser. — This is an
immersion substage condenser of high aperture by
means of which unstained objects such as bacteria
can be shown as bright white particles upon a
dense black background. The central rays of light
are blocked out by means of an opaque stop while the
peripheral rays are reflected from the paraboloidal sides
of the condenser and refracted by the object viewed.
To obtain the best results with this type of condenser
a powerful illuminant — such as a small arc lamp or an
incandescent gas lamp — is needed, together with
picked slides of a certain thickness (specified for the

MICROMETRY 6 1

particular make of condenser but generally i mm. and
specially thin cover-glasses (not more than 0.17 mm.
The objective must not have a higher NA than i.o,
consequently immersion lenses must be fitted with an
internal stop to cut down the aperture.

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.

Object Marker (Fig. 54). — This is an exceedingly
useful piece of apparatus. Made in the form of an
objective, the lenses are replaced by a diamond point,
set slightly out of the centre, which
can be rotated by means of a milled
plate. Screwed on to the nosepiece
in place of the objective, rotation of
the diamond point will rule a small
circle on the object slide to perma-
nently record the position of an in-
teresting portion of the specimen.
The diamond is mounted on a spring
which regulates the pressure, and

the size of the circle can be adjusted FlG- 54-— Diamond Ob-
ject marker,
by means of a lateral screw.

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 (some-
times, and erroneously, referred to as a micro-milli-
metre), and indicated in writing by the Greek letter
IJL. Of the many methods in use for the measurement
of bacteria, three only will be here described, viz. :

(a) By means of the Camera Lucida.

(6) By means of the ocular or Eyepiece Micrometer.

(c) By means of the Filar Micrometer (Ramsden's
micrometer eyepiece) .

62 THE MICROSCOPE

Fo^ each of these methods a stage micrometer is
necessary. This is a 3 by i inch glass slip having en-
graved 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; and
from these engraved lines the measurement in every
case is evaluated. A cover-glass is cemented over
the scale to protect it from injury.

FIG. 55. — Camera lucida, Abbe pattern.

(a) By means of the Camera Lucida.

1. Attach a camera lucida (of the Wollaston, Beale,
or Abbe pattern) (Fig. 55) 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 fi. Mark on the paper the optical combination
(ocular objective and tube length) employed to pro-
duce this particular magnification.

THE EYEPIECE MICROMETER 63

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.

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 micro,.

In place of preserving a scale for each optical com-
bination, the object to be measured and the microm-
eter scale may be projected and sketched, in turn, on
the same piece of paper, taking particular care that
the centre of the eyepiece is 25 cm. from the paper on
which the divisions are drawn.

FIG. 56. — Eyepiece micro-
meter, ordinary.

FIG. 57. — Eyepiece micrometer, net.

(b) 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. 56), or the entire surface
ruled in o.i mm. squares (the net micrometer) (Fig. 57).
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 eye lens.

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 for normal

64 THE MICROSCOPE

vision. Special eyepieces are also obtainable having
a sledging adjustment to the eye lens for focussing
the micrometer.

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

THE EYEPIECE MICROMETER 65

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.

Wright's Eikonometer is really a modification of
the eyepiece micrometer for rapidly measuring micro-
scopical objects by direct inspection, having previously
determined the magnifying power of the particular
optical combination employed. It is a small piece of
apparatus resembling an eyepiece, with a sliding eye
lens, which can be accurately focussed on a micrometer
scale fixed within the instrument. When placed over
the microscope ocular the divisions of this scale measure
the actual size of the virtual image in millimetres.

In order to use this instrument for direct measure-
ment, it is first necessary to determine the magnifying
power of each combination of ocular, tube length and
objective.

Place a stage micrometer divided into hundredths
of a millimetre on the microscope stage and focus
accurately.

Rest the eikonometer on the eyepiece. Observa-
tion through the eikonometer shows its micrometer
scale superposed on the image of the stage micrometer.

Rotate the eikonometer until the lines on the .two
scales are parallel, and make the various adjustments
to ensure that two lines on the eikonometer scale
coincide with two lines on the stage micrometer.

For the sake of illustration it may be assumed that
five of the divisions on the stage micrometer accu-
rately fill one of the divisions of the eikonometer scale ;
this indicates a magnifying power of 500 as the con-
stant for that particular optical combination, and a
record should be made of the fact.

The magnification constants of the various other
5

66

THE MICROSCOPE

optical combinations should be similarly made and
recorded.

To measure any object subsequently it should be
first focussed carefully in the ordinary way.

The eikonometer should then be applied to the eye-
piece and the size of the object read off on the eiko-
nometer scale as millimetres, and the actual size calcu-
lated by dividing the observed size by the magnification
constant for the particular optical combination em-
ployed in the observation.

(c) By means of the filar micrometer.

The Filar or cobweb Micrometer (Ramsden's microm-

FiG. 58. — Ramsden's Filar micrometer.

FIG. 59. — Ramsden's
micrometer field, a, fixed
wire; .b, reference wire
(fixed) ; c, travelling wire.

eter eyepiece (Fig. 58) consists of an ocular having a
fine "fixed" wire stretching horizontally across the
field (Fig. 59), 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
drum head is divided into one hundred parts, which
successively pass a fixed index as the head is turned.
In the lower part of the field is a comb with the inter-
vals between its teeth corresponding to one complete
revolution of this screw-head.

ILLUMINANTS 67

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 drum head. (If when the
drum head is at zero the two wires do not exactly coin-
cide they must be adjusted by loosening the drum screw
and resetting the drum.)

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 transversed 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 micro,
by reference to the recorded values for that particular
optical combination.

Microscope Illuminant. — In tropical and subtropical
regions diffuse daylight is the best illuminant. In
temperate climes however daylight of the desirable

68

THE MICROSCOPE

quantity is not always available, and recourse must be
had to oil lamps, gas lamps — preferably those with
incandescent mantles — and electricity; and of these
the last is undoubtedly the best. A handy lamp
holder which can be manufactured in the laboratory
is shown in Fig. 60. It consists of a base board
weighted with lead to which is attached the ordinary
domestic lamp holder, and behind this is fastened a

FIG. 60. — Electric microscope lamp.

curved sheet-iron reflector. An obscured metal fila-
ment lamp of about 16 candle power gives the most
suitable light, and if monochromatic light is needed,
the blue grease pencil is streaked over the side of the
lamp nearest the microscope; the current is switched
on and when the glass bulb is warm, rubbing with a wad
of cotton-wool will readily distribute the blue greasy
material in an even film over the ground glass.

V. MICROSCOPICAL EXAMINATION OF BAC-
TERIA AND OTHER MICRO-FUNGI.

APPARATUS AND REAGENTS USED IN ORDINARY
MICROSCOPICAL EXAMINATION.

The following comprises the essential apparatus
and reagents for routine work with which each student
should be provided.

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

FJG. 61. — Disinfectant Jar.

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 infected
pipettes, etc.

70 MICROSCOPICAL EXAMINATION OF BACTERIA

4. A square glazed earthenware box with a loose
lining containing 2 per cent, lysol solution for the
reception of infected material and used slides. The
bottom of the lining is perforated so that when full the
lining and its contents can be lifted bodily out of the
box, when the disinfectant solution drains away and the
slides, etc., can easily be emptied out. The empty lin-
ing is then returned to the box with its disinfectant
solution (Fig. 61).

5. Bunsen burner provided with "peep-flame"
by-pass.

6. Porcelain trough holding five or six hanging-drop
slides (Fig. 62).

FIG. 62. — Hanging-drop slides: a, Double cell seen from above; b single
cell seen from the side.

The best form of hanging-drop slide is a modification
of Boettcher's glass ring slide, and 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 (Fig. 62, a).

Another form of hanging-drop slide is made in which a circular
or oval concavity or "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.

MICROSCOPICAL EXAMINATION OF BACTERIA 71

7. Three aluminium rods (Fig. 63), 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
mm. in its short diameter, and is termed a loop or an
oese; 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. 63. — Ends of platinum rods, a, loop; b, spatula; c, needle.

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
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. [The use of a fine dental drill and small
circular saw, worked by a dental motor facilitates the
manufacture of these aluminium handled instruments.]

Now pass one end of the platinum wire through the
hole, turn up about 2 mm. at right angles and press

72 MICROSCOPICAL EXAMINATION OF BACTERIA

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
the centre of the cut end of the aluminium wire (Fig.
63). A few sharp taps with a watch maker's hammer
will now close in the sides of the two channels over the
wire and hold it securely.

FIG. 64. — Platinum rod in aluminium handle — method of mounting.

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.

8. 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.

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

10. A glass capsule with tightly fitting (ground on)
glass lid, containing clean cover-slips in absolute
alcohol.

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

12. A wooden rack (Fig. 65) with twelve drop-bottles
(Fig. 66) each 60 c.c. capacity, containing

Aniline water.

Gentian violet, saturated alcoholic solution.

Lugol's (Gram's) iodine.

Absolute alcohol.

Methylene-blue, }

Fuchsin, basic, ) saturated alcoholic solution.

MICROSCOPICAL EXAMINATION OF BACTERIA

73

Neutral red, i per cent, aqueous solution.
Leishman's modified Romano wsky stain.
Carbolic acid, 5 per cent, aqueous solution.

FIG. 65. — Staining rack, rubber change mat and lysol pot.

Acetic acid, i per cent, solution.
Sulphuric acid, 25 per cent, solution.
Xylol.

FIG. 66.— Drop bottle.

FIG. 67. — Canada balsam pot.

And two pots with air-tight glass caps (Fig. 67), each
provided with a piece of glass rod and filled respec-

74

MICROSCOPICAL EXAMINATION OF BACTERIA

lively with Canada balsam dissolved in xylol, and sterile
vaseline.

METHODS OF EXAMINATION.

Bacteria, etc., are examined microscopically.

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

2. In the "fixed" condition (i. e., 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 .

* Q

K *

U <
W

FIG. 68. — Holding tubes for removing bacterial growth,
as seen from the front.

2. Remove the tube cultivation from its
rack or jar with 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
upward, between the thumb and fingers. (This
operation is termed "flaming the plug," and is

LIVING, UNSTAINED 75

( intended to destroy any micro-organisms that
may have become entangled in the loose fibres
of the cotton-wool, and which, if not thus de-
stroyed, might fall into the tube 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 ringers 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
(Fig. 68).

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
the platinum portion by holding it in the flame
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 " sere wing" motion of
the right hand.

6. Introduce the platinum loop into the tube
and hold it in this position until satisfied that

i it is quite cool. (The cooling may be hastened

76

MICROSCOPICAL EXAMINATION OF BACTERIA

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
bottom; at the same time care must be taken
in the case of cultures on 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 the loop 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. Replace the tube cultivation in its rack or jar.

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

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

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. Lower 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 a cover-slip by
the simple method of placing a drop of the reagent in
contact with one edge of the cover-glass and apply-

BLACK AND WHITE FILMS 77

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.

The non-toxic basic dyes most generally employed
for the intra-vitam staining of bacteria are

Neutral red,

Quinoleine blue

Jt ^ , in 0.5 per cent, aqueous solutions.

Methylene green

Vesuvin,

Negative Stain (Burri). — By this method of demon-
stration the appearances presented by dark ground
illumination (by means of a paraboloid condenser)
are closely simulated, since minute particles, bacteria,
blood or pus cells etc. stand out as brilliantly white or
colourless bodies on a dark grey-brown background.

Reagent required:

Any one of the liquid waterproof black drawing inks
(Chin-chin, Pelican, etc.) . This is prepared for use as
follows:
Measure out and mix :

Liquid black ink 25 c.c.

Tincture of iodine i c.c.

Allow the mixture to stand 24 hours, centrifugalise
thoroughly, pipette off the supernatant liquid to a clean
bottle and then add a crystal of thymol or one drop of
formalin as a preservative.

METHOD. —

1. With the sterilised loop deposit one drop of the
liquid ink close to one end of a 3 by i slide.

2. With the sterilised loop deposit a drop of the
fluid culture (or of an emulsion from a solid culture)
by the side of the drop of ink (Fig. 69, a) ; mix the two
drops thoroughly by the aid of the loop.

3. Sterilise the loop.

78 MICROSCOPICAL EXAMINATION OF BACTERIA

4. Hold the slide firmly on the bench with the thumb
and forefinger of the left hand applied to the end
nearest the drop of fluid.

5. Take another clean 3 by i slide in the right hand
and lower its short end obliquely (at an angle of about
60°) transversely on to the mixed ink and culture en
the first slide, and allow the fluid to spread across the
slide and fill the angle of incidence.

6. Maintaining the original angle, draw the second
slide firmly and evenly along the first toward the end
farthest from the left hand (Fig. 69, Z>).

7. Throw the second slide into a pot of disinfectant;
allow the first slide to dry in the air.

FIG. 69. — Spreading negative film.

8. Place a drop of immersion oil on the centre of the
film, lower the i/i 2-inch objective into the oil and
examine microscopically without the intervention of
a cover-slip.

(The film of ink may be covered with a long cover-
glass and xylol balsam as a permanent preparation.)

(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.

HANGING DROP 79

4. Obtain a small quantity of the material it is
desired to examine, in the manner detailed above (pages
74-76, 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 hang-
ing-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.

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 ; when present,
spores show their typical refrangibility exceedingly
well by this method.

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

8o MICROSCOPICAL EXAMINATION OF BACTERIA

tides 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, or to the fact that the
cover-slip is not perfectly level, and although the
relative positions of the bacteria may vary, still the
flowing movement of large numbers of organisms in
some one direction will usually be sufficient to demon-
strate 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
downward, whilst still wet, on to the surface of some
Gulland 's solution or corrosive sublimate solution (vide
page 82) in a watch-glass. If the drop is large, place
it face upward 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, page 82) .

KILLED, STAINED 8 1

3. Dip the cover-glass into a beaker containing hot
water in order to remove some of the vaseline adhering
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 pages 83-85).

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

"Preparing" and "fixing" the film.

Staining.

Mounting.

Preparing the Film. —

1. Flame a cover- slip and place is on a piece of filter
paper.

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

3. Obtain a small quantity of the material to be
examined upon a sterilised platinum loop (see pages
74-76, steps 2 to n) and mix it with the drops of water
on the cover-slip.

4. 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.

5. Allow it to dry completely in the air.

Fixing. — Fix by passing the cover-slip, held in the
fingers, three or four times through the flame of a Bun-
sen burner.

In some instances (e. g., when the films after staining
are intended for micrometric observations) it is almost
essential 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.

Fixation may also be effected by immersing in some
fixative fluid, such as one of the following :
6

82 MICROSCOPICAL EXAMINATION OF BACTERIA

i. Absolute alcohol, for five to fifteen minutes.

equal parts, for five to thirty

2. Absolute alcohol, , minutes (g fof bk)od

Ether' I milk).

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

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

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

Sodium chloride 0.7 5 gramme

Hydrarg. perchloride 12.00 grammes

Acetic acid 5 . oo grammes

In distilled water loo.ooc.c.

Filter.

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

Absolute alcohol 25.00.0.

Ether 25.00.0.

Corrosive sublimate, 20 per cent, al-
coholic solution 0.4 c.c.

7 . Formalin i o per cent . aqueous solution ( = 4 per cent .
aqueous solution of formaldehyde since formalin is a 40
per cent, solution of the gas in water).

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

Clearing. — 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.)

KILLED, STAINED 83

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.)

Staining. —

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

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; (6) chemical
means — e. g., by the additon of carbolic acid, 5 per
cent, aqueous solution; caustic alkalies, 2 per cent,
aqueous solutions; 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.

(c) Fuchsin.

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

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 fuchsin
(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 fuchsin respectively to it. For aniline gentian
violet (A.G.V.) the stain is added to a saturated solu-
tion of aniline oil in water.

84 MICROSCOPICAL EXAMINATION OF BACTERIA

(d) Thionin blue.

(e) Bismarck brown.

(f) Neutral red.
(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.

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

Decolour isation. — 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., Je<lualParts-

Hydrochloric, i per cent, aqueous

solution.
Hydrochloric, i per cent, alco-

s TV/,-. 1 . -, holic (oo per cent.) solution.

6. Mineral acids : _ . ,y

Sulphunc, 25 per cent, aqueous

solution.

Nitric, 33 per cent, aqueous solu-
tion.

Counter staining. — Use colours which will contrast
with the first stain; e. g.,
Vesuvin,

for films stained by methylene-blue or
Gram's method.

Neutral red,

Eosin,

Fuchsin,

Methylene-blue, I ,

^ , . . t } for films stained by fuchsm.

Gentian violet, J

IMPRESSION FILMS 85

8. 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
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 (Klatschpraeparat) 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 230) 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. Lower 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

86 MICROSCOPICAL EXAMINATION OF BACTERIA

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 82).

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.

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 that position
by means of the spring clips.

3. Use the |-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 rinding
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

DARK GROUND ILLUMINATION 87

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 contrary,
withdraw his eye, rack the tube up, and commence
again from step 2.

Dark Ground Illumination.—

1 . Set up the microscope stand in the vertical position
and insert the highest eyepiece available.

2. Remove the nosepiece from the microscope tube
and fit the § inch objective in place.

3 . Remove the substage condenser and replace it by
the dark ground condenser.

4. Fit up the source of illumination some 30-50 cm.
distant from the microscope. (This should be the Lili-
put Arc Lamp (Leitz) , Nernst Lamp or incandescent
gas lamp ; if either of the two latter are employed, a bull's
eye condenser to produce parallel rays must be inter-
posed between light and microscope) ; and adjust illumi-
nant and microscope so that the substage plane mirror is
completely filled with light.

5. Focus the two concentric rings engraved upon the
upper surface of the condenser and centre them accu-
rately by means of the centring screws.

6. Prepare a "fresh" specimen (see pages 74-76) of the
material it is desired to observe, using selected, new,
3 by i glass slips of less than i mm. thickness, and No.
i cover-glasses (0.17 mm. thick), which should be
cleaned with a piece of soft washleather and not with the
emery paper, as scratches on the glass produce haziness
in the preparation).

7. Deposit a large drop of immersion oil (or pure
water) on the upper surface of the condenser and rack it
down a few millimetres.

8. Adjust the fresh preparation on the microscope
stage and fasten it in position with the stage clips.

9. Rack up the condenser until the immersion

88 MICROSCOPICAL EXAMINATION OF BACTERIA

fluid makes contact with the under surface of the slide ;
avoid the formation of air bubbles.

10. Adjust the substage mirror so that the light is
reflected upward. A bright spot will be seen on the
fresh preparation near the centre of the field.

11. Replace the §-inch objective by the TV-mch
oil immersion lens which has been fitted with the special
stop to reduce its N. A. ; place a drop of immersion oil
upon the centre of the cover-glasses of the fresh prepa-
ration and lower the microscope tube until the front
lens of the objective has entered the oil drop.

12. Focus the bright spot referred to
in step 10. If it no longer occupies the
centre of the field, alter the angle of the
sub-stage mirror until it does.

13. Now focus the lens accurately on
the film, cautiously vary the height of
the dark ground condenser until the best
position is found. The intensely illumi-
nated bacteria will stand out in vivid
contrast to the dark background.

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.

The immersion oil is pure cedarwood oil, and is kept in a small
bottle of stout glass (Fig. 70), the cavity of which is shaped like an
inverted cone, and is provided with a safety funnel (so that the oil
does not escape if the bottle is accidently overturned) and a dust
cap of boxwood fitted with a wooden rod with which the drop of
oil is applied to the coverglass or lens.

3 . Use the TV-inch oil immersion lens of the micro-
scope. Rack down the body tube till the front lens

STAINED FILMS 89

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.

VI. STAINING METHODS.

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 Gruebler, 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 — those
relegated to small type comprise such as are not so
generally useful, but give excellent results in the hands
of the experienced worker.

BACTERIA STAINS.

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

90

SIMPLE STAINS 9!

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.

3. Carbolic Meikylene-blue (Kuehne).
Weigh out

Methylene-blue ........ i.$ grammes

Carbolic acid 5.0 grammes

and dissolve in

Distilled water 100.0 c.c.

and add

Absolute alcohol 10.0 c.c.

Filter.

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

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

Filter.

Gentian Violet. —

5. Saturated Aqueous Solution.
Weigh out

Gentian violet 2.25 grammes

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

6. Saturated Alcoholic Solution.
Weigh out

Gentian violet 5.0 grammes

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

92

STAINING METHODS

7. 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 c.c.

Filter.

8. Anilin Water Solution (Koch-Ehrlich) .
Measure out

Distilled water 100 c.c.

Add anilin oil drop by drop (shaking well after the
addition of each drop) until the solution is opaque.

Filter until clear,
and add

Absolute alcohol 10 c.c.

Saturated alcoholic solution gentian violet . n c.c.

Filter.

NOTE. — This solution will not keep longer than 14 days.

Thionine Blue (or Lauth's Violet) . —

9. Carbolic Thionine Bine (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.

Fuchsin (Basic). —

10. Saturated Aqueous Solution.
Weigh out

Basic fuchsin 1.5 grammes

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

CONTRAST STAINS 93

11. Saturated Alcoholic Solution.
Weigh out

Basic fuchsin .3.5 grammes

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

1 2 . 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 c.c.

Filter.

CONTRAST STAINS.

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

Eosin, aqueous yellowish.

Eosin, aqueous bluish.

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

Weigh out

Eosin, aqueous i.o gramme

dissolve in

Distilled water 100.0 c.c.

and add

Absolute alcohol 5.0 c.c.

Filter.

94 STAINING METHODS

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

Eosin, alcoholic 0.5 gramme

and dissolve in

Alcohol (70 per cent.) 100.0 c.c.

Filter.

Safranine. —

15. A queous Solution.
Weigh out.

Safranine 0.5 gramme

and dissolve in

Distilled water ......... 100.0 c.c.

Filter.

Neutral Red.—

1 6. Aqueous Solution.
Weigh out

Neutral red i.o gramme

and dissolve in

Distilled water . . 100.0 c.c.

Filter.

Vesuvin (or Bismarck Brown). —

17. Saturated Aqueous Solution.
Weigh out

Vesuvin 0.5 gramme

and dissolve in

Distilled water 100.0 c.c.

Filter.

H^EMATIN

95

TISSUE STAINS.

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

Gentian violet . i . o gramme

and dissolve in

Absolute alcohol I5-° c-c.

Distilled water 80.0 c.c.

then add

Aniline oil 3.0 c.c.

Shake well and filter before use.

Haematoxylin (Ehrlich). —

1. Weigh out

Haematoxylin ..2.0 grammes

and dissolve in

Absolute alcohol 100.0 c.c.

2. Weigh out

Ammonium alum 2.0 grammes

and dissolve in

Distilled water loo.oc.c.

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

4. Add

Glycerine 85.0 c.c.

Acetic acid, glacial ic.oc.c.

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

Hsematin (Mayer's). —
A. Weigh out

Haematin i . o gramme

and dissolve in

Alcohol 90 per cent, (warmed to 37° C.) . 50 c.c.

96 STAINING METHODS

B. Weigh out

Potash alum 50 grammes

and dissolve in

Distilled water 100 c.c.

Prepare these two solutions in separate flasks.
Take a clean flask of 250 c.c. capacity and insert a large
funnel in its neck. Pour the solutions A and B simul-
taneously and slowly into the funnel to mix thoroughly.
Store for future use.

NOTE. — If acid haematin is required, introduce glacial acetic
acid (3 c.c.) into the mixing flask before adding the solutions A
and B.

Alum Carmine (Mayer) .—
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.

Lithium Carmine (Orth).—
Weigh out

Carmine 2.5 grammes

and dissolve in

Lithium carbonate, cold saturated solution . 100.0 c.c.

Filter.
Picrocarmine. —

Weigh out

Picrocarmine 2.0 grammes

BLOOD STAIN 97

and dissolve in

Distilled water 100.0 c.c.

BLOOD STAINS

When watery solutions of medicinal methylene blue
and water soluble eosins are mixed a precipitate is
formed which is soluble only in alcohol, and solutions of
this precipitate impart a peculiar reddish-purple
colour to chromatin. This compound was first used
by Romano wsky to demonstrate malarial parasites, but
various modifications are now employed for staining
blood films generally, and also for bacteria and pro-
tozoa. The best modifications of the original Roman-
owsky are those of Jenner and Leishman — Jenner
being most suitable for the histological study of the
blood, and Leishman for the demonstration of protozoa.

Jenner 's Stain.—

A. Weigh out:

Eosin aqueous yellow ... .6.0 grammes

Dissolve in

Distilled water (non-alkaline) . . 250 c.c.

This will make a thick solution.

B. Weigh out :

Methylene blue (medicinally pure) Hoechst . 5.0 grammes

Dissolve in

Distilled water (non-alkaline) ... 250 c.c.

1. Add B to A very slowly, stirring all the time. A
viscous precipitate forms which frequently loses its
viscosity when heat is applied. (This explains the
necessity of mixing slowly) .

2. Evaporate slowly in a porcelain basin, stirring
occasionally, on a water bath at 55° C. When a paste

7

98 STAINING METHODS

begins to form scrape and break up occasionally.
(On no account must the paste be allowed to fuse.)

3. Grind the resulting mass into an amorphous
powder.

4. Weigh out:

Amorphous powder 0.5 grammes

Dissolve in

Methylic alcohol (Merck's puriss. for analysis) . 100 c.c.

Allow time for true solution. (About three days is
sufficient.)

METHOD.—

1 . Prepare film, dry, but do not fix.

2. Flood the unfixed film with the stain, allow it to
act for 3 minutes (the methylic alcohol of the stain
fixes the film) .

3 . Pour off the stain and wash in distilled water until
the film presents a pink colour.

4. Dry and mount.

Irishman's Stain. —

A. Weigh out:

Methylene blue (medicinal) . . . i gramme
Dissolve in

Sodium carbonate, o. 5 per cent, aqueous solution . . 100 c.c.

Keep at 65° C. for 12 hours in either a hot incubator
or a water-bath; then stand in dark place at room
temperature (20° C.) for ten days.

B. Weigh out :

Eosin, extra B. A o.i gramme

Dissolve in

Distilled water 100 c.c.

i. Mix the two solutions A and B in equal volumes,

CAPSULE STAINS 99

and allow the mixture to stand for 12 hours with
occasional stirring.

2. Filter, and collect precipitate on filter paper.

3. Wash precipitate thoroughly with distilled water,
and dry.

4. Weigh out 0.15 gramme of the dried precipitate;
rub up in a mortar with 5 c.c. of methylic alcohol
(Merck's puriss, for analysis) .

Allow undissolved powder to settle, then decant the
supernatant fluid to a clean 100 c.c. measuring cylinder.

5. Add further 5 c.c. alcohol to sediment in mortar
and repeat the process, and so on until all the sediment
has been dissolved.

6. Now make up the fluid in the measuring cylinder
to 100 c.c. by the addition of more methylic alcohol.

. METHOD.—

1. Prepare film, dry, but do not fix.

2. Flood the unfixed film with stain, allow it to act
30 seconds.

3. Add double the volume of distilled water to the
stain on the film, and mix with glass rod or platinum
loop.

4. Allow this diluted stain to act five minutes.

5. Wash off with distilled water.

6. Leave some water on film for thirty seconds to in-
tensify the colour contrasts.

7. Dry and mount.

METHODS OF DEMONSTRATING STRUCTURE OF
BACTERIA, ETC.

To Demonstrate Capsules.
1. MacConkey. —

Stain. —
Weigh out

Dahlia - 0.5 gramme

Methyl green (oo crystals) . 2.5 grammes

100 STAINING METHODS

rub up in a mortar with

Distilled water 100.0 c.c.

Add

Fuchsin, saturated alcoholic solution ... 10.0 c.c.

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. Muir's Method.—

1. Prepare, dry and fix film in the ordinary manner.

2. Flood the film with carbolic fuchsin, warm until steam
begins to rise. Allow the stain to act for thirty seconds.

3. Wash quickly with methylated spirit.

4. Wash thoroughly with water.

5. Subject the film to the action of the following mordant for
five seconds:

Corrosive sublimate, saturated aqueous solution . 2 c.c.
Tannic acid, 20 per cent, aqueous solution ... 2 c.c.
Potash alum saturated aqueous solution . . . . 5 c.c.

6. Wash thoroughly in water.

7. Treat with methylated spirit for about sixty seconds. (The
preparation should now be pale red.)

8. Wash thoroughly in water.

9. Counterstain in methylene blue, aqueous solution thirty
seconds.

10. Wash in water.

11. Dehydrate in alcohol.

1 2 . Clear in xylol and mount in xylol balsam.

FLAGELLA STAINS IOI

3. 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 stain.

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

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

5. Wash in water.

6. Dry and mount.

4. Ribbert's Method.—

Stain. —

Measure out and mix:

Acetic acid, glacial 12.5 c.c.

Alcohol, absolute 50.0 c.c.

Distilled water 100.0 c.c.

Warm to 36° C. (e. g.t in the "hot" incubator) and saturate
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 c.c.

Alum, saturated aqueous solution 5c.c.

Carbolic fuchsin (Ziehl) 5 c.c.

Mix thoroughly.

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

Decant off the clear fluid into tubes and centrifugalise
thoroughly.

102 STAINING METHODS

This solution is at its best some four or five days
after manufacture ; it 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 c.c.

Filter.

This stain must be freshly prepared.

METHOD. — The cultivations employed should be
smear agar cultures, twelve to eighteen hours old if
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. Some workers
prefer to use tap water, others employ normal saline
solution, but distilled water gives the best emulsion.

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.

FLAGELLA STAINS 103

9. Wash well in water.

10. Dry and mount.

2. "Pitfield" Original Method.—

(a) Mordant. —

Tannic acid i gramme

Water 10 c.c.

(b) Stain. —

Saturated aqueous solution of alum 10 c.c.

Saturated alcoholic solution of gentian violet. . i c.c.
Distilled water 5 c.c.

Mix equal parts of a and b before using.

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

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 . . 10 c.c.
Potash alum, saturated aqueous solution . 10 c.c.
Tannin, 10 per cent, aqueous solution . . 10 c.c.

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. Loeffler's Method.—

(a) Mordant. —

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

Ferrous sulphate, saturated aqueous solution . 5 c.c.

Haematoxylin solution 3C.c.

Carbolic acid, i per cent, aqueous solution . . 4 c.c.

This solution must be freshly prepared.

Hcematoxylin solution is prepared by boiling i gramme logwood

104 STAINING METHODS

with 8 c.c. distilled water, filtering and replacing the loss from
evaporation.

Alternative Mordant (B tinge's Mordant). —

Tannic acid, 20 per cent, aqueous solution . . 10 c.c.
Ferrous sulphate, saturated aqueous solution . . 5 c.c.
Fuchsin, saturated alcoholic solution i c.c.

(6) Stain —

Weigh out

Methylene-blue |

Or methylene-violet .... [• 4 grammes

Or fuchsin J

and dissolve in

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

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 discharged;
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 demonstrated
better by means of an alkaline stain or an acid stain — a point to
be determined for each. Speaking generally, those bacilli which
give rise to an acid reaction in the culture medium require an
alkali; those which form alkali in cultivation require an acid.
According to requirements, therefore, LoefHer recommends the
addition of sodium hydrate, i per cent, aqueous solution, i c.c.;
or an equal quantity of an exactly comparable solution of sul-
phuric acid.

5. Van Ermengem's Method. — This method, being merely a
precipitation of a silver salt on the micro-organisms and not a
true stain, creates a false impression as to the relative proportions
of bacteria and flagella.

(a) Fixing Fluid. —

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

FLAGELLA STAINS 105

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

(b) Sensitising Solution. —

Silver nitrate, 0.5 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 grammes

Potassium acetate, fused 10 grammes

and dissolve in

Distilled water 350 c.c.

Filter.

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 as above in the preced-
ing 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 contain-
ing 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 sensitising
solution, this time until the film commences to turn black.

9. Wash in distilled water.

10. Dry and mount.

To Stain Nuclei of Yeast Cells.

1. Prepare and fix film in the usual manner.

2. Soak in ferric ammonia sulphate 3 per cent,
aqueous solution for two hours.

106 STAINING METHODS

3. Wash thoroughly in water.

4. Stain in haematoxylin solution (see page 95) for
thirty minutes.

5. Wash in water.

6. Differentiate in ferric ammonia sulphate solu-
tion for iJ-2 minutes, examining wet under micro-
scope during the process.

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.

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 J-inch objective. (Spores should

SPORE STAINS

I07

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. Moeller'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 solution,
for five seconds.

7. Wash in water.

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

9. Wash in water.

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

4. Abbott's Method.—

1. Prepare and fix films in the usual manner.

2. Pour Loeffler'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 (alcohol
80 per cent.) solution.

108 STAINING METHODS

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 combina-
tion which results in the formation of a new blue-black
pigment, only very sparingly soluble in absolute
alcohol. Such organisms are said to " stain by Gram,'*
or to be "Gram positive."

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

2 . Stain in aniline gentian violet three to five minutes.
Filter as much aniline water on to the cover-slip as

it will hold ; then add the smallest quantity <3f alcoholic
solution of gentian violet which suffices to saturate the
aniline water and form a "bronze scum" upon its
surface — if too much of the alcoholic gentian violet is
added the alcohol present redissolves this scum.

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

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 proc-
ess until the film ceases to darken with the fresh appli-
cation of iodine solution.

Lugol's solution is prepared by dissolving

Iodine i gramme

Iodide of potassium 3 grammes

In distilled water 300 c.c.

DIFFERENTIAL STAINS IOQ

5. Wash in water.

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

The following mixture may be substituted for abso-
lute alcohol as a decolouriser

Acetone 10 c.c.

Absolute alcohol ^100 c.c.

7. Wash in water.

8. Counterstain very lightly with aqueous solution of
Neutral Red. Other counterstains may be used such
as 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.

Gram=Claudius Method.—

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

2. Stain in methyl violet, i per cent, aqueous solution
for three to five minutes.

3. Treat with two lots picric acid, saturated aqueous
solution.

4. Wash in water and dry.

5. Decolourise with clove oil.

6. Wash off clove oil witk xylol.

7. Mount in xylol balsam.

Qram=Weigert Method. —

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.

HO STAINING METHODS

Modified Gram=Weigert Method. — (To demonstrate
trichophytain hair.)

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

2. Stain thirty minutes in a tar-like solution of
aniline gentian violet (prepared by adding 1 5 drops of
the alcoholic solution of gentian violet to 3 drops of
aniline water) .

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 to remove excess of stain.
(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 obtain the best differentiation the preparation
should be repeatedly examined microscopically (with
a J-inch objective) between steps 5 and 9, as the actual
time involved varies with different specimens.

Ziehl=Neelsen's Method. — (To demonstrate tubercle
and other acid- fast bacilli.)

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.) Avoid over-heating.

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
and the film turns yellow.)

DIFFERENTIAL STAINS III

5. Wash in water. (A pale red colour returns to
the film).

6. Wash in alcohol till no more colour is discharged.
(This often, 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.
Pappenheim's Method. —

This method is supposed to differentiate between
B. tuberculosis and other acid-fast micro-organisms.

1. Prepare and fix film in the usual way.

2. Stain in carbol-fuchsin without heat for three
minutes.

3. Without previously washing in water treat the
film with three or four successive applications of coralin
(Rosolic acid) solution.

Corallin i gramme

Methylene-blue

(saturated alcoholic solution) . 100 c.c.

Glycerine 20 c.c.

4. Wash in water.

5. Dry and mount.

Neisser's Method — Modified. — (To demonstrate diph-
theroid bacilli.)
Stain I. —
Measure out and mix

Methylene-blue, saturated alcoholic

solution 4-0 C-C.

Acetic acid, 5 per cent, aqueous solu-
tion 96.0 c.c.

Filter.
Stain II. —
Weigh out

Neutral red 2.5 grammes

112 STAINING METHODS

and dissolve in

Distilled water 1000 c.c.

Filter.
METHOD. —

1. Prepare and fix films in the usual way.

2. Pour stain I on the film and allow it to act for
two minutes.

3. Wash thoroughly in water.

4. Treat with Lugol's iodine for ten seconds.

5. Wash thoroughly in water.

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

7. Wash thoroughly in water.

8. Dry and mount.

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

The bacilli are stained a light red by the neutral red,
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.

Wheal and Chown (Oxford) Method. — (To demonstrate actino-
myces.)

1. Stain briefly with Ehrlich's hsematoxylin (until nuclei are
faint blue after washing with tap water) .

2. Wash in tap water.

3. Stain in hot carbol-fuchsin (as for tubercle bacilli) for five
to ten minutes.

4. Wash in tap water.

5. Decolourise with Spengler's picric acid alcohol. This is
prepared by mixing:

Alcohol, absolute 20 c.c.

Picric acid, saturated aqueous solution 10 c.c.
Distilled water 10 c.c.

During the progress of steps 1-5 the preparation must be re-
peatedly examined microscopically with the £-inch objective.

DIFFERENTIAL STAINS 113

When properly differentiated the clubs appear brilliant red on
greenish ground.

6. Dehydrate in alcohol.

7. Clear in xylol.

8. Mount in xylol balsam.

This method serves equally well for films and for sections.

VII. METHODS OF DEMONSTRATING BACTERIA
IN TISSUES.

FOR bacteriological purposes, sections of tissue 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, — 2 to 5 mm.
cubes when possible, but in any case 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 82) ;
formaldehyde, 4 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,
absolute alcohol.

In both these processes a large excess of fluid should
always be used.

114

HARDENING 1 15

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

FIG. 71. — Washing tissues.

the bottle and replace it with fresh absolute alcohol.
Allow the tissues to remain therein for twenty-four
hours.

NOTE. — If not needed for cutting immediately, the hardened
tissues can be stored in 75 per cent, alcohol.

3 . Remove the alcohol 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

Il6 DEMONSTRATING BACTERIA IN TISSUES

and place under a tap of running water. The water of
course, overflows, but the tissues remain in the bottle
(Fig. 71).

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 arabic 5 grammes

Saccharose i gramme

Boric acid i gramme

Water 100 c.c.

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 with ether, or
for preference, carbon dioxide, 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 90 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).

9. Mount in xylol balsam.

Alternative Rapid Method. —

1. Cut very small blocks of the tissue.

2. Fix in formalin 10 per cent, aqueous solution (fixation fluid
No. 7, page 82) for 24 hours.

3. Transfer block to plate of freezing microtome and freeze with
carbon dioxide vapour.

4. Float the sections off the knife into a glass dish of tepid
water.

5. Stain the sections in glass capsules containing selected stains.

6. Place the stained section in a dish of clean water and
introduce a glass slide obliquely beneath the section; with a
mounted needle draw the section on to the slide and hold it there;

CLEARING

117

gently remove the slide from the water, taking care that any
folds in the section are floated out before the slide is finally
removed from the water.

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

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

9. Drop on xylol to clear the section.

10. Place a large drop of xylol balsam on the section and care-
fully lower a cover-glass on to the balsam.

PARAFFIN METHOD.

1. 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.

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.

FIG. 72. — L-shaped brass moulds.

FIG. 73. — Paraffin kettle.

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. Dehydration is effected by transferring the tissues
to fresh absolute alcohol.

5. Clearing. Half fill a wide-mouthed bottle with

Il8 DEMONSTRATING BACTERIA IN TISSUES

chloroform. On the surf ace, of the chloroform float
a layer of absolute alcohol about five to ten millimetres
in depth. Place the pieces of tissue in the layer of
alcohol and when they have sunk through this layer,
transfer them to pure chloroform for from six to
twenty-four hours according to the size of the pieces.
When "cleared," the tissue becomes more or less
transparent.

6. Infiltration. Place the cleared tissues in fresh
chloroform with several pieces of paraffin wax and
stand in a warm place, such as on the top of the warm
incubator. The warmth gradually melts the paraffin
and the tissues should remain in the mixture about
twenty-four hours.

7. Transfer the tissues to a vessel containing pure
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
four to six 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.

8. 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. 72).

(6) Pour fresh melted paraffin into the mould from a
special vessel (Fig. 73).

(c) Lift the piece of tissue from the paraffin bath
and arrange it in the mould.

(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

MOUNTING PARAFFIN SECTIONS 1 19

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 stout copper, 10 cm., 5 cm., and
2.5 cm. square respectively, and 0.75 cm. deep (Fig. 74)
will be found extremely useful.
These placed upon plates of glass
replace the pair of L's in the above
process. When the paraffin has set
firmly the screw a should be loosened
to allow the two halves of the flange b
to separate slightly — this facilitates FIG. 74.— Paraffin
removal of the paraffin block.

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 section
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, touching
the under surface of the glass from time to time on the
back of the other hand. As soon as the slide 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 warming over the flame as here
described.)

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

120

DEMONSTRATING BACTERIA IN TISSUES

4. Place the slide in a wire rack (Fig. 75), section
downward, 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.

NOTE. — If large, thick sections have to be manipulated, or if
time is of importance or acids are used during the staining
process, 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. The albuminous fluid is prepared
as follows:

FIG. 75. — Section rack.

Mayer's Albumin.—

Weigh out

Salicylate of soda i gramme

and dissolve in

Glycerine 50 c.c.

Add

White of egg 50 c.c.

Mix thoroughly by means of an egg whisk.

Filter into a clean bottle.

As an alternative method paint a thin layer of Schallibaum's
solution on the slide with a camel's hair pencil; lay the section
carefully on this film and heat gently to fix the section.

DOUBLE STAINING 121

Schallibaum's solution:

Clove oil 30 c.c.

Collodion 10 c.c.

Keep in a dark blue bottle in a cool place.
Staining Paraffin Sections. —

1. Warm paraffin section over the Bunsen flame to
soften (but not to melt) the paraffin, then dissolve out
the wax with xylol poured on from a drop bottle.

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 Lugol's iodine solution for two minutes and sub-
sequently immersed in 90 per cent, alcohol to remove
all traces of yellow staining.

4. Wash in water.

5. Stain deeply, if using a single stain, as the subse-
quent processes decolourise.

6. Wash in water, decolourise if necessary.

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

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

9. Mount in xylol balsam.

SPECIAL STAINING METHODS FOR SECTIONS.
Double=staining Carmine and Gram=Weigert. —

1. Prepare the section 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. 76).

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 c.c.

Alcohol (90 per cent.) 160 c.c.

122

DEMONSTRATING BACTERIA IN TISSUES

4. Wash in water.

5. Wash in alcohol.

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.

FIG. 76. — Staining pot.

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

12. Clear with xylol.

13. Mount in xylol balsam.

Alternative Gram=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.

TO DEMONSTRATE CAPSULES 123

9. Wash thoroughly with xylol.

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

11. Clear with xylol.

12. 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.)

Unna=Pappenheim Method. —

Stain. —

Weigh out and mix

Methylene green 0.15 gramme

Pyronin 0.25 gramme

and dissolve in

Carbolic acid 0.5 per cent, aqueous solution . . 78 c.c.

Measure out

Alcohol 2.5 c.c. 1

„, > and add to the stain.

Glycerine 20.0 c.c. J

Method.-

1. Place tissue in the above stain for ten minutes.

2. Differentiate and dehydrate with absolute alcohol.

3. Clear in xylol.

4. Mount in xylol balsam.

To Demonstrate Capsules.—

1. MacConkey's Method. — Stain precisely as for
cover-slip films (vide page 100).

2. Friedldnder's Method. —

Stain. —

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

Acetic acid, glacial 10 c.c.

Distilled water . . 100 c.c.

124 DEMONSTRATING 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.

To Demonstrate Spirochsetes in Tissues.
Piridin Method (Levaditi).—

1. Cut slices of tissue i mm. thick.

2. Fix in 10 per cent, formalin solution for twenty-
four hours.

3. Wash in water for one hour.

4. Place in 96 per cent, alcohol for twenty-four hours.

5. Measure into a dark green or amber bottle 100 c.c.
silver nitrate solution i per cent., and 10 grammes pyri-
din puriss. Transfer slices of tissue to this. Stopper
and keep at room temperature three hours, then in
thermostat at 50° C. for four to six hours.

6. Wash quickly in 10 per cent, pyridin solution.

7. Reduce silver by transferring slices of tissue to
following solution for forty-eight hours.

TO STAIN PROTOZOA IN SECTIONS 125

Pyrogallic acid 4 grammes

Acetone 10 c.c.

Pyridin puriss 15 grammes

Distilled water 100 c.c.

8. Wash well in water.

Take through alcohols of increasing strength up to
absolute, keeping in each strength for twenty-four
hours.

9. Clear, embed, cut very thin sections, mount,
remove paraffin, again clear and mount in xylol balsam.

The spirochaetes if present are black and show up
against the pale yellow color of the background.

Weak carbol fuchsin, neutral red or toluidin blue
can also be used to stain the background if desired,
after the removal of the paraffin in step 9.

To Demonstrate Protozoa in Sections (Leishman) .—

Reagents required :

Leishman' s Polychrome stain.
Acetic acid i in 1500 aqueous solution.
Caustic soda i in 7000 aqueous solution.
Distilled water.

1. Mount section, remove paraffin and take into
distilled water as usual (vide page 121).

2. Drain off the excess of water.

3. Cover the section with diluted Leishman (i part
stain, 2 parts distilled water) and allow to act for five
to ten minutes (until tissue appears a deep blue) .

4. Decolourise with acetic acid solution until only
the nuclei appear blue (examine the section wet, with
low power objective) .

5. If the eosin colour is too well marked treat with
the caustic soda solution until the desired tint is ob-
tained (as seen with the J-inch objective).

6. Wash with distilled water.

7. Rapidly dehydrate with alcohol.

8. Clear with xylol.

9. Mount in xylol balsam.

VIII. CLASSIFICATION OF FUNGI.

For practical purposes FUNGI may be divided into:

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

2. Hyphomycetes (moulds).

3. Blastomycetes (yeasts and torulae) .

4. Schizomycetes (bacteria).

NOTE. — Formerly myxomycetes were included in the fungi;
they are now recognized 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.
Hyphae are —

(a) Nutritive or submerged.

(b) 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 .

Mucorinae. — Mucor (Fig. 77). — Note the branching
filaments — "mycelium" (a), " hyphae" (b).
Note the asexual reproduction.

126

PERISPORACE^E

127

1. A filament grows upward. 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. 77. — Mucor mucedo.

FIG. 78. — Aspergillus

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

Finally the membrane ruptures and spores escape.

Perisporaceae. — Aspergillus (Fig. 78). — Note the
branching filaments — "mycelium" (a).

FIG. 79. — Penicillium.

Note the asexual reproduction.

i. A filament (6) grows upward, its termination be-
comes clubbed ; on the clubbed extremity flask-shaped
cells appear — " sterigmata " (c) .

128 CLASSIFICATION OF FUNGI

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. Two or more gonidia
may be supported upon each sterigma.

Penicillium (Fig. 79). — Note the branching filaments
—"mycelium" (a) (frequently containing globules).

Note the asexual reproduction.

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

2. At the apex of each basidium 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.

Ascomycetae. — Oidium (Fig. 80). — (This family is

FIG. So.— Oidium.

perhaps as nearly related to the blastomycetes as it is
to the hyphomycetes.)

Note the branching filaments — " pseudomycelium "
(a). Here and there filaments are broken up at their
ends into oval or rod-shaped segments, "oidia," and
behave as spores.

Note the asexual reproduction. From the pseudo-
mycelium arise true hyphae (6), each of which in turn
ends in a chain of spores (c).

SACCHAROMYCES I2Q

MORPHOLOGY OF THE BLASTOMYCETES.

The tlastomycetes are composed of spherical or ova
cells (8 to 9.5 fi in diameter), which, when rapidly
multiplying by budding, may form a spurious mycelium.
A thin cell-wall encloses the granular protoplasm, in
which vacuoles and sometimes a nucleus may be
noted. This latter is best seen when stained with
hsematoxylin (see page, 105).

During their growth and multiplication the blasto-
mycetes split up solutions containing sugar into alcohol
and CO2.

Saccharomyces (Fig. 81). — Note the round or oval
cells of granular protoplasm (a) containing solid par-
ticles and vacuoles (c), and surrounded by a definite
envelope.

Reproduction. — Budding ; ascospores — asexual.

Note the asexual reproduction.

i. "Gemmation" — that is, the budding out of
daughter cells (6) from various parts of the gradually
enlarging mother cell. These are eventually cast off

FIG. Si. — Saccharomyces with ascospores. FIG. 82. — 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 cerevisise, thirty
hours at 25° to 37° C., or ten days at 12° C.

130 CLASSIFICATION OF FUNGI

Torulae (Fig. 82). — Torulae whilst resembling yeasts
in almost every other respect, never form endo-spores.
Note the elongated, sausage-shaped cells (a) the larger
oval cells (b) and the globular cells (c) the former two
often interlacing and growing as a film.

Note the absence of ascospore formation.

IX. 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 food requirements into—

Prototrophic, requiring no organic food (e. g.,

nitrifying bacteria) ;
Metatrophic, requiring organic food (e. g.,

saprophytes and facultative parasites) ;
Paratrophic, requiring living food (obligate

parasites) ;

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 classification,
as under —

1. Cocci. (Fig. 83). — Rounded or oval cells, sub-
divided according to the arrangement of the individuals
after fission, into —

I32

SCHIZOMYCETES

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.

Sarcince, where division takes place in three planes

123456

FIG. 83. — Types of bacteria — cocci: i, Diagram of sphere indicating
planes of fission; 2, diplococci; 3, streptococci; 4, tetrads; 5, sarcinae; 6,
staphylococci.

successively, and the individuals remain attached in
cubical packets of eight and its multiples.

Micrococci or Staphylococci, where division takes place
in three planes, but with no definite sequence; conse-

I 23456

FIG. 84. — Types of bacteria — bacilli, etc.: i, Bacilli; 2, diplobacilli; 3
streptobacilli; 4, spirilla; 5, vibrios; 6, spirochaetse.

quently the individuals remain attached in pairs, short
chains, plates of four, cubical packets of eight, and
irregular masses containing numerous cocci.

2. Bacilli (Fig. 84, i to 3) .—Rod-shaped cells. A
bacillus, however short, can usually be distinguished

SPIRILLA 133

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.

(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 leptothricise (vide infra) .

3. Spirilla (Fig. 84, 4 to 6). — Curved and twisted
filaments. Classified, according to shape, into —

Spirillum.
Vibrio (comma).
Spirochasta.

Many Spirochaetes appear to belong to the animal
kingdom and are grouped under protozoa ; other organ-
isms to which this name has been given are undoubt-
edly bacteria.

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

(a) Differentiation between base and apex;

(b) Growth apparently apical ;

(c) Exaggerated pleomorphism ;

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

1. Beggiotoa. 1 Free swimming forms, which

2. Thiothrix. J contain sulphur granules.

3. Crenothrix.

These forms do not contain

4. Cladothnx. ,

T , . sulphur granules.

5. Leptothnx.

6. Streptothrix. A group which exhibits true but
not dichotomous branching, and contains some patho-
genic species.

The morphology of the same bacterium may vary
greatly under different conditions.

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

134 SCHIZOMYCETES

culture of the same bacillus, but grown under different
conditions, may consist almost entirely of long fila-
ments or threads. This variation in morphology is
known as "pleomorphism."

Some of the factors influencing pleomorphism are :

1. The composition, reaction, etc., of the nutrient
medium in which the organism is growing.

2. The atmosphere in which it is cultivated.

3. The temperature at which it is incubated.

4. Exposure to or protection from light.

The various points in the anatomy morphology and
physiology of bacteria upon which stress is laid in the
following pages should be studied as closely as is possible
in preparations of the micro-organisms named in con-
nection with each.

ANATOMY.

1. Capsule (Fig. 85, b). — A gelatinous envelope
(probably akin to mucin in composition) surrounding
each individual organism, and preventing absolute con-
tact between any two. In some species the capsule
(e. g., B. pneumonias) 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 (e. g., Streptococcus mesen-
teroides) . In some species colouring matter or ferric
oxide is stored in the capsule.

2. Cell Wall (Fig. 85, c). — A protective differen-
tiation of the outer layer of the cell protoplasm;
difficult to demonstrate, but treatment with iodine or
salt solution sometimes causes shrinkage of the cell
contents — " plasmolysis " — and so renders the cell wall
apparent (e. g., B. megatherium) in the manner
shown in figure 85. Stained bacilli, when examined
with the polarising microscope, often show a doubly

STRUCTURE OF BACTERIA

135

refractile cell wall (e. g., B. tuberculosis and B.
anthracis) .

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

FIG. 85. — Diagrammatic sketch of
composite bacterium to illustrate
details of anatomical structure.

FIG. 86. — Plasmolysis.

from proteid in that it is not precipitated by C2H6O.
It is usually homogeneous in appearance — sometimes
granular — and may contain oil globules or sap vacuoles
(Fig. 85, 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-

136 SCHIZOMYCETES

sively proved to exist, but in some bacteria chromatin
particles have been observed near the centre of the
bacterial cell and denser masses of protoplasm situated
at the poles which exhibit a more marked affinity than
the rest of the cell protoplasm for aniline dyes. These
latter are termed polar granules or Polkoerner (Fig. 85,
e). Occasionally these aggregations of protoplasm
alter the colour of the dye they take up. They are
then known as metachromatic bodies or Ernstschen
Koerner (e. g., B. diphtherias).

5. Flagella (Organs of Locomotion, Fig. 85, a). —
These are gelatinous elongations of the cell protoplasm

(or more probably of the cap-
sule), occurring either at one
pole, at both poles, or scat-
tered around the entire periph-
ery. Flagella are not pseu-
dopodia. The possession of
flagella was at one time sug-
,-, „, , ... . gested as a basis for a system of

FIG. 87. — Types of ciliation. *

classification, when the follow-
ing types of ciliation were differentiated (Fig. 87) :

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).

(c) Lophotrichous (a tuft or bunch of flagella situated
at each pole; e. g., B. cyanogenus).

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

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

REPRODUCTION 137

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.

000

CD CD

OO

FIG. 88. — Fission of cocci. FIG. 89. — Fission of bacteria.

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

Cultures on artificial media,
after gro wing in the same me-
dium for some time — i. e., when
the pabulum is exhausted — show
"involution forms" (Fig. 90),
well exemplified in cultures of B.
pestis on agar two days old, B.
diphtheria? on potato four to
six days old.

They are of two classes, viz. :

(a) Involution forms charac-
terised by alterations of shape
(Fig. go). (Not necessarily .

v & y ' v ^ FIG. 90. — Involution forms.

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, so that the species might
not become extinct, when

(a) The supply of nutrient was exhausted.

138 SCHIZOMYCETES

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

(c) The environment became unfavourable; e. g.,
change of temperature.

This is not altogether correct; e. g., the temperature
at which spores are best formed is constant for each
bacterium, but varies with different 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 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,". n<?r 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
one end (some 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. 91, a).

a b c d e

IWT

SPORE FORMATION 139

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

1. Clostridium (Fig. 91, b): Rod swollen at the centre
and attenuated at the poles; spindle shape; e. g., B.
butyricus.

2. Cuneate (Fig. 91, c) : Rods swollen slightly at one
pole and more or less pointed at the other; wedge-
shaped.

3. Clavate (Fig. 91, d) : Rods
swollen at one pole and cylin-
drical (unaltered) at the other;
keyhole-shaped; e. g., B.
chauvei.

4. Capitate (Fig. 91, e) : Rods FlG-
with a spherical enlargement

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

(a) Low water contents of plasma of the spore.

(b) Low heat-conducting power 1 of the spore

(c) Low permeability J membrane.
This resistance varies somewhat with the particular

species — e. g., some spores may resist boiling for a few

140 SCHIZOMYCETES

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
which may be followed in hanging-drop cultures on a
warm stage:

1. Swell up slowly and enlarge, through the absorp-
tion 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.
leptosporus.

(b) It loses its spore membrane by solution; e. g., B.
anthracis.

(c) It loses its spore membrane by rupture.

In this process the rupture may be either polar (at
one pole only e. g., B. butyricus), or bipolar (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.

(b) The outer layers of the cell protoplasm, which
become differentiated for that purpose; e. g., B. mega-
therium.

The new bacillus now increases in size, elongates,
and takes on a vegetative growth — i. e., undergoes
fission — the bacilli resulting from which may in their
turn give rise to spores.

GERMINATION

141

O 0

FIG. 92. Simple.

OQE

FIG. 93. Solution.

~ii ~

O

FIG. 94. Polar.

0

FIG. 95. Bipolar.

0

FIG. 96. Equatorial.

142 SCHIZOMYCETES

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.

3. 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.

1. 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.

2. Temperature. — Practically no bacterial growth
occurs below 5° C., and very little above 40° C. 30° C.

THE METABOLISM OF BACTERIA 143

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. is°C. 30° C.

2. Mesophilic bacteria (includes patho-

genic bacteria) 15° C. 37° 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 (see
pages 298-301).

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 extent
the biochemical characters of the organisms — e. g.,
chromogenicity or power of liquefaction. Pathogenic
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
excrementitious substance. Occasionally, however, it

144 SCHIZOMYCETES

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 ' ' photogenic. ' '

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 Inseparate) Toxins : those which are
so closely bound up with the cell protoplasm of the
bacteria elaborating them that up to the present time

THE METABOLISM OF BACTERIA 145

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 276 et seq.).

Jo

X. 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 organisms, thus isolated, 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
pabulum of the organism.

The general requirements of bacteria as to their
food-supply have already been indicated (page 142)
and many combinations of proteid and of carbohy-
drate have been devised, from time to time, on those
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.t 10 per
cent, gelatine, 2 per cent. agar. Gelatine is employed

146

NUTRIENT MEDIA 147

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.

Media, on the other hand which have been solidified
by the addition of agar, only become liquid when ex-
posed to 90° C. for about ten minutes, and again solidify
when the temperature falls to 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 proportion 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, in ordinary use (see also
Chapter XI), those which are only occasionally required
for more highly specialised work are grouped together
in Chapter XII. It must be premised that scrupulous
cleanliness is to be observed with regard to all ap-
paratus, vessels, funnels, etc., employed in the prepara-
tion of media; although in the preliminary stages of

148 NUTRIENT MEDIA

the preparation of most media absolute sterility of the
apparatus used is not essential.

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 it
has been determined empirically that its preparation
shall be carried out by extracting half a kilo of moist
meat with one litre of water. For many purposes,
however, it is more convenient to have a more concen-
trated extract; one kilo of meat should therefore be
extracted with one litre of water, to form " Double
Strength" meat extract.

It was customary at one time, and is even now in
some laboratories to use either " shin of beef" or "beef-
steak"— both contain muscle sugar which often needs
to be removed before the nutrient medium can be
completed. Heart muscle (bullock's heart or sheep's
heart) is much to be preferred and from the point of
economy, ease and cleanliness of manipulation, and
extractive value, the imported frozen bullock's hearts
provide the best extract.

Meat extract (Fleischwasser) 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
1000 grammes (roughly, z\ pounds) of fresh lean
meat — e. g., 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

REACTION OF MEAT EXTRACT 149

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, 5 c.c. of Brand's meat juice
or 3 grammes of Wyeth's beef juice, or 10 grammes Liebig's
extract of meat (Lemco) may be dissolved in 1000 c.c. distilled
water, and heated and filtered as above to form ordinary or
single strength meat extract.

Media, prepared from such meat extracts are, however,
eminently unsatisfactory when used for the cultivation of the
more highly parasitic bacteria; although when working in tropical
and subtropical regions their use is well-nigh compulsory.

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.

150 NUTRIENT MEDIA

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 phenglphthalein.
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 100° C. for forty-five minutes.

2. The estimation be performed at the boiling-point.

3 . Phenolpthalein be used as the indicator.

The estimation is carried out by means of titration
experiments against standard solutions of caustic soda,
in the following manner :

Method of Estimating the Reaction. —

Apparatus Required: Solutions Required:

1. 25 c.c. burette graduated i. icN NaOH, accurately
in tenths of a centimetre. standardised.

2. i c.c. pipette graduated in 2. 2 NaOH, accurately stand-
hundredths, and provided ardised

with rubber tube, pinch-
cock, and delivery nozzle. n ^T

, ,. j 3. — NaOH, accurately stand-

3. 2 5 c.c. measure (cylinder or 3* 10

pipette, calibrated for 98° ardised.

C. not 15° C.). 4> Q 5 per cent soiution of

4. Several 60 c.c. conical phenolphthalein in 50 per
beakers or Erlenmeyer cent> alcohoL

flasks.

5. White porcelain evaporat-
ing basin, filled with boil-
ing water and arranged

TITRATING MEAT EXTRACT 151

Apparatus Required: — (Continued.}

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 boil-
ing on a tripod stand.

METHOD. — Arrange the apparatus as indicated in
figure 97.

(A) i. Fill the burette with £ NaOH.
2. Fill the pipette with -f NaOH.

FIG. 97. — Arrangement of apparatus for titrating media.

3. Measure 25'c.c. of the meat extract (previously
heated in the steamer at 100° C. for forty-five minutes)
into one of the beakers by means of the measure;
rinse out the measure with a very small quantity of boil-
ing 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
reached, as indicated by the development of a pinkish

152 NUTRIENT MEDIA

tinige, shown in figure 98 (6). Note the amount of
decinormal 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. 98, c), which is the so-
called "end-point" of the American Committee of Bacteriologists.

a & c

FIG. 98. — 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
become 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.

(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'-f NaOH from the pipette, just short of
the equivalent of the amount of deci-normal soda
solution required to neutralise the 2 5 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
7- NaOH.) Immerse the beaker in the water-bath.

3. Complete the titration by the aid of the £ NaOH.

REACTION OF MEAT EXTRACT

153

4. Note the amount of ~0 NaOH solution required
to complete the titration, and add it to the equivalent
of the -f 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

FIG. 99. — Stock bottle for deka-normal soda solution.

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 solution
exactly neutral to phenolphthalein.

The sign + (plus) is prefixed to this number if the
original solution reacts acid, and the sign — (minus) if
it reacts alkaline.

154 NUTRIENT MEDIA

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 alkaline 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.
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 +
(i.e., + 20), to signify that it is acid, indicates the re-
action of the meat extract.

NOTE. — The standard soda solutions should be prepared by
accurate measuring operations, controlled by titrations, from a
stock solution of zoN NaOH, which should be very carefully
standardised. If a large supply is made or the consumption is
small this stock solution must be kept in an aspirator bottle 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 arrangement as
is shown in figure 99, which also shows a constant burette arrange-
ment for the delivery 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. For most bacteria, however, the
"optimum" usually approximates fairly closely to
H- 10 ; and as experiment has shown that this reaction is
the most generally useful for routine laboratory work,
it is the one which may be adopted as the standard for
all nutrient media derived from meat extract.

STANDARDISING BOUILLON 155

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 +10.

Standardising Nutrient Bouillon. — For example, 1000
c.c. bouillon are prepared; at the first titration it is
found

1. 25 c.c. require the addition of 5.50 c.c. £ NaOH
to neutralise.

Two controls give the following results:

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.

Averaging these two controls, 25 c.c. require the
addition of 5.65 c.c. •— NaOH to neutralise, and there-
fore 1000 c.c. require the addition of 226 c.c. ~ NaOH,
or 22.60 c.c. Y NaOH, or 2.26 c.c. 10 or NaOH.

Initial litre of the bouillon = +22.6, and as such
requires the addition of (22.6 c.c. — 10 c.c.) = 12. 6 c.c.
of ~ 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 bouillon to (1000
— 75 c.c.) = 925 c.c. The amount of -f- NaOH required
to render the reaction of this quantity of medium +10
may be deduced thus :

1000 c.c. : 925 c.c. :: 12.6 c.c. :x.

Then* =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
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-

156 NUTRIENT MEDIA

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 or
even decinormal 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 either
0.05 c.c. of normal soda solution or 0.5 c.c. of deci-
normal soda solution.

Standardising Nutrient Agar and Gelatine. — The
method of standardising agar and gelatine is precisely
similar to that described under bouillon.

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 ex-
actly through its centre to form a semicircle (Fig. ioo,a).

2. Fold the semicircle exactly in half to form a quad-
rant ; make the crease 2, 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 3 and 3 a, thus formed and again open out
to a semicircle.

1'OLDING FILTERS

157

4. The semicircle now appears as in figure 100, a, the
dark lines indicating the creases already formed.

5. Fold the point i over to the point 3, and la to 3a,
to form the creases 4 and 40, indicated in the diagram
by the light lines. Fold point i over to 3 a, and ia to 3,
to form the creases 5 and 5 a.

6. Thus far the creases have all been made on the
same side of the paper. Now subdivide each of the

FIG. 100. — Filter folding: a, Filter folded in half, showing creases; &,
appearance of filter on completion of folding; c, filter opened out ready for
use.

eight sectors by a crease through its centre on the op-
posite side of the paper, indicated by the faint broken
lines 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 100, b.

158 NUTRIENT MEDIA

When opened out the filter assumes the shape repre-
sented in figure 100, c.

The folded filter is next placed inside a glass funnel
supported on a retort stand, and moistened with hot
distilled water before the filtration of the medium is
commenced.

Liquef iable solid media are filtered through a specially
made filter paper — "papier Chardin" — which is sold
in boxes of twenty-five ready-folded filters.

Gelatine, when properly made, filters through this

FIG. 101. — Hot- water filter funnel and ring burner.

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
"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

STORING MEDIA

159

walls being filled with water at about 90° C. — and
supporting the latter on a ring gas burner fixed to a
retort stand (Fig. 101). The gas is lighted and the
water jacket maintained at a high temperature until
filtration is completed. If the steam steriliser of the
laboratory is sufficiently large, it is sometimes more
convenient to place the flask and filtering funnel bodily
inside, close the steriliser and allow filtration to proceed
in an atmosphere of live steam, than to use the gas ring
and hot- water funnel.

STORING MEDIA IN BULK.

After filtration fill the medium into sterile litre flasks
with cotton-wool plugs and sterilise in the steamer for
twenty minutes on each of three
consecutive days. After the
third sterilisation, and when the
flasks and contents are cool, cut
off the top of the cotton-wool
plug square with the mouth of
the flask; push the plug a short
distance down into the neck of a b

the flask and fill in with melted d(S^ sto«T^le ."", bS
paraffin wax to the level of the fore> and b> after sterilizing.
mouth. When the wax has set the flasks are stored
in a cool dark cupboard for future use.

This plan is not' absolutely satisfactory, although
very generally employed on occasion, and it is prefer-
able to fill the medium into long-necked flint glass
bottles (the quart size, holding nearly 1000 c.c., such as
those in which Pasteurised milk is retailed) and to
close the neck of the bottle by a special rubber cap.1
This cap is made of soft rubber, the lower part,
dome-shaped with thin walls, being slipped over the
neck of the bottle (Fig. 102, a) . The upper part is solid,

1 This rubber cap has been made for me by the Holborn Surgical Instnr
ment Co., Thavies Inn, London, W. C.

l6o NUTRIENT MEDIA

but with a sharp clean-cut (made with a cataract or
tenotomy knife) running completely through its axis
from the centre of the disc to the top of the dome.
During sterilisation the air in the neck of the bottle,
expanded by the heat, is driven out through the valvu-
lar aperture in the solid portion of the stopper. On
removing the bottle from the steam chamber, the
liquid contracts as it cools, and the pressure of the
external air drives the solid piece of rubber down into
the neck of the bottle, and forces together the lips of the
slit (Fig. 102,6). Thus sealed, the bottle will preserve its
contents sterile for an indefinite period without loss
from evaporation.

TUBING NUTRIENT MEDIA.

After the final filtration, the nutrient medium is
usually " tubed" — i. e., filled into sterile tubes in defi-
nite measured quantities, usually 10 c.c. This process
is sometimes carried out by means of a large separator
funnel fitted with a "three-way" tap which communi-
cates 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 asTreskow's
funnel, renders it particularly liable to damage. It is
better, therefore, to arrange a less expensive piece of
apparatus which will serve the purpose equally well
(Fig. 103).

A Geissler's three-way stop-cock has the tube on one
side of the tap ground obliquely at its extremity, and
the tube on the opposite side cut off within 3 cm. of the
tap. The short tube is connected by means of a per-
forated rubber cork with a 10 cm. length of stout glass
tubing (1.5 cm. 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-

STORING "TUBED" MEDIA

161

ated in cubic centimetres up to 20, by pouring into it
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. 21); or into ordinary
media tubes which already have smaller tubes, inverted,
inside them (Fig. 104) , 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

FIG. 103. — Separatory funnel and three-way tap arranged for tubing media.
FIG. 104. — Gas tube (Durham).

sterilisation nearly all the air is replaced by the medium,
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. Each
box (Fig. 105) has a movable partition formed by the

162

MEDIA BOXES

vertical face of a weighted triangular block of wood,
sliding free on the bottom (Fig. 105, A) ; or by a flat
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. 105, B). The front of the box is pro-

FIG. 105 . — Medium box, showing alternative partitions A and B.

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.

XI. CULTURE MEDIA.

ORDINARY OR STOCK MEDIA.

Nutrient Bouillon.—

1 . Measure out double strength meat extract, 500 c.c.,
into a litre flask and add 300 c.c. distilled water.

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 distilled water pre-
viously heated to 60° C. (Be careful to leave no un-
broken globular masses of peptone.)

. 3. Add the peptone emulsion to the meat extract in
the flask and heat in 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
+ io (vide page 155).

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 35).

NOTE. — As an alternative method when neither fresh nor frozen
meat is available nutrient bouillon may be prepared from a com-
mercial meat extract, as follows:

Lemco Broth. —

1. Measure out 250 c.c. distilled water into a litre flask.

2. Weigh out 10 grammes Liebig's Lemco Meat Extract on a

163

164

CULTURE MEDIA

piece of clean filter paper and add to the water in the flask.
Shake the flask well to make an even emulsion of the meat
extract.

3. Weigh out Witte's peptone (10 grammes), salt (5 grammes).
Mix into smooth paste with 100 c.c. distilled water previously
heated to 60° C.

5. Add the peptone salt emulsion to the meat extract emulsion
in the flask and add 650 c.c. distilled water. Heat in the steamer
for forty-five minutes.

6. Standardise the medium and complete as for nutrient
bouillon.

Nutrient Gelatine.—

i. Weigh a 2 -litre flask on a trip balance (Fig. 106)
and note the weight, or counterpoise carefully.

FIG. 1 06. — Trip balance.

An extremely useful counterpoise is a small sheet-
brass cylinder about 38 mm. high and 38 mm. in di-

u

^— ' ^

FIG. 107. — Counterpoise; weight when empty, 35 grammes; when full of dust
shot, 200 grammes.

ameter, with a funnel-shaped top and provided with a
side tube by which its contents, fine "dust" shot, may
be emptied out (Fig. 107).

DISSOLVING GELATINE 165

2 . Measure out double strength meat extract, 500 c.c.,
into the " tared" flask.

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; place the flask in the water-
bath and raise to the boil.

FIG. 108. — Arrangement of steam can and water-bath for the preparation

of media.

4. Arrange a 5 -lit re tin can (with copper bottom,
such as is used in the preparation of distilled water)
by the side of the water bath, 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 delivery tube, bent twice at right angles;
adjust the tube to reach to the bottom of the interior
of the flask containing the gelatine, etc. (Fig. 108).

1 66 CULTURE MEDIA

5. Keep the water in the steam can vigourously boil-
ing, and so steam at 100° C., bubbling through the
medium mass, for ten minutes, by which time complete
solution of the gelatine is effected. A certain amount
of steam will condense as water in the medium flask
during this process — hence the necessity for the use
of double strength me.at extract — but if the water
bath is kept boiling this condensation will not exceed
100 c.c.

6. Weigh the flask and its contents; then (in 5*
grammes + weight of the flask) minus (weight of the
flask and its contents) equals the weight of water re-
quired to make up the bulk to i litre. The addition of
the requisite quantity of water is carried out as follows :

In one pan of the trip balance place the counterpoise
of the tared flask (or its equivalent in weights) to-
gether with the weights making up the calculated
medium weight. In the opposite pan place the flask
containing the medium mass. Now add boiling
distilled water from a wash bottle until the two pans
are exactly balanced.

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., calculate 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, to
precipitate the phosphates, etc.

9. 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

1 This figure is obtained by adding together i litre water, 1000 grammes;
10 per cent, gelatine, 100 grammes; i per cent, peptone, 10 grammes; 0.5
per cent, salt, 5 grammes; total, 1115 grammes. Modifications of the above
process, as to quantities and percentages, will require corresponding altera-
tions of the figures. The average weight of a measured litre of 10 per cent,
nutrient gelatine when prepared in this way after filter at ion is 1080 grammes.

NUTRIENT AGAR-AGAR 167

and formed large, firm masses floating on and in clear
gelatine) ,

10. Filter through papier Chardin into a sterile flask.

11. Tube in quantities of 10 c.c.

12. Sterilise in the steamer at 100° C. for twenty
minutes on each of three consecutive days — i. e., by
the discontinuous method.

Nutrient Agar=agar. —

1 . Weigh a 2 -litre flask and note the weight — or coun-
terpoise exactly.

2. Measure out double strength meat extract, 500 c.c.,
into the " tared" flask.

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; place
the flask in a water-bath.

4. Arrange the steam can and water-bath as already
directed (for the preparation of gelatine) and figured.

5. Bubble live steam (at 100° C.) through the
medium mass, for twenty-five minutes, by which time
complete solution of the agar is effected.

6. Now weigh the flask and its contents; then (I0351
grammes + weight of flask) minus (weight of flask and
its contents) equals the weight of water required to
make up the bulk of the medium to i litre. Add the
requisite amount (see preparation of gelatine, page
1 66, step 6).

7. Titrate, and estimate the reaction of the medium
mass; control the result. Calculate the amount of

1 This figure is obtained by adding together i litre of water (meat ex-
tract), looo grammes; 2 per cent, agar, 20 grammes; i per cent, peptone,
10 grammes; 0.5 per cent, salt, 5 grammes — total 1035 grammes. Modi-
fications of the process as to quantities or percentages will nessitate corres-
ponding alterations in the calculated medium figure. The average weight
of a measured litre of 2 per cent, agar when prepared in this way, after
filtration, is 1010.5 grammes.

1 68 CULTURE MEDIA

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
replace in the steamer for twenty minutes (to complete
the precipitation of the phosphates, etc.) .

9. 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 one hour (until the egg-albumen has coagulated
and formed large, firm masses floating on and in clear
agar.)

10. Filter through papier Char din, by the aid of a
hot-water funnel, if necessary (Fig. 101), into a sterile
flask.

11. Tube in quantities of 10 c.c. or 15 c.c.

12. Sterilise in the steamer at 100° C. for thirty
minutes on each of three consecutive days — i. e., by the
discontinuous method.

Blood=serum (Inspissated). —

1. Sterilise cylindrical glass jar (Fig. 109) and its
cover by dry heat, or by washing first with ether and
then with alcohol and drying.

2 . Collect blood at the slaughter house from ox or
sheep in the sterile cylinder.

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.

5. Remove the serum with sterile pipettes, or syphon
it off, and fill into sterile tubes (5 c.c. in each) or
flasks.

BLOOD-SERUM

169

6. Heat tubes containing serum to 56° C. in a water-
bath for half an hour on each of two successive days.

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.)

FIG. 109. — Blood-serum jar with wicker basket for transport.

The serum inspissator (Fig. no) 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
serum may be solidified at any desired " slant," and
is heated from below by a Bunsen burner controlled by
a thermoregulator. The more elaborate forms resemble
the hot-air oven (Fig. 26) in shape and are provided
with adjustable shelves so that any desired obliquity
of the serum slope can be obtained.

8. Place the tubes in the incubator at 37° C. for

CULTURE MEDIA

forty-eight hours in order to eliminate those that have
been contaminated. Store the remainder in a cool
place for future use.

Alternative Method.

Steps 1-5 as above.

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.

7. Coagulate in the inspissator when needed.

:::•::• '

FIG. no. — Serum inspissator.

Serum Water. —

This forms the basis of many useful media, and is prepared as
follows :

1. Collect blood in the slaughterhouse (see page 168) and when
firmly clotted collect all the expressed serum and measure in a
graduated cylinder.

2. For every 100 c.c. of serum add 300 c.c. distilled water and
mix in a flask.

3. Heat the mixture in the steamer at 100° C. for thirty min-
utes. (This destroys any diastatic ferment present in the serum
and partially sterilises the fluid.)

4. Filter if turbid.

5. If not needed at once complete the sterilisation of the serum
water by two subsequent steamings at 100° C. for twenty minutes
at twenty-four hour intervals.

CITRATED BLOOD AGAR 171

Citrated Blood Agar. Guy's. —

1. Kill a small rabbit with chloroform vapour, and
nail it out on a board (as for a necropsy) ; moisten the
hair thoroughly with 2 per cent, solution of lysol.

2. Sterilise several pairs of forceps, scissors, etc. by
boiling.

3. Reflect the skin over the thorax with sterile
instruments.

4. Open the thoracic cavity by the aid of a fresh
set of sterile instruments.

5. Open the pericardium with another set of sterile
instruments.

6. Sear the surface of the left ventricle with a red-
hot iron.

7. Take a steiile capillary pipette (Fig. 13,^); break
off the sealed extremity with a pair of sterile forceps.

8. Steady the heart in a pair of forceps and thrust
the point of the pipette through the wall of the ven-
tricle and through the seared area, apply suction to
the plugged end of the pipette and fill it with blood.

9. Transfer the entire quantity of blood collected
from the rabbit's heart to a small Erlenmeyer flask
containing a number of sterile glass beads and 5 c.c.
concentrated sod. citrate solution. (See page 378.)

10. Agitate thoroughly and set aside for a couple of
hours.

1 1 . Melt up several tubes of nutrient agar (see page
167) and cool to 42° C.

12. With a sterile 10 c.c. graduated pipette transfer
i c.c. citrated blood from the Erlenmeyer flask to each
tube of liquefied agar. Rotate the tube between the
hands in order to diffuse the citrated blood evenly
throughout the agar.

13 . Place the tubes in a sloping position and allow the
medium to set.

14. Place tubes of blood agar for forty-eight hours in

172 CULTURE MEDIA

the incubator at 37° C. and at the end of that time
eliminate any contaminated tubes.

15. Store such tubes as remain sterile for future use.

Milk.- .:

1. Pour i litre of fresh cow's or goat's milk into a
large separating funnel, and heat in the steamer at
100° C. for one hour.

2. Remove from the steamer and estimate the re-
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
supply of milk from a different source.

Reject 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 subnatant 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.

Nutrose Agar (Eyre). —

(This is a modification of the well known Drigalski-
Conradi medium originally introduced for the isolation
of B. typhosus).

i. Collect 250 c.c. perfectly fresh ox serum (vide

NUTROSE AGAR 173

Blood Serum, page 168, steps i to 5) and add to it
450 c.c. sterile distilled water.

2. Weigh out agar powder, 20 grammes, and emulsify
it with 2 50 c.c. of the cold serum water.

3. Weigh out

Witte's peptone 10 grammes

Sodium chloride . 5 grammes

Nutrose 10 grammes

and dissolve in 200 c.c. of serum water heated to 80° C.

4. Mix the agar emulsion and the peptone-nutrose
solution in a " tared " flask of 2 -litre capacity and add a
further 100 c.c. serum water.

5. Complete the solution of the various ingredients
by bubbling live steam through the flask as in making
nutrient agar.

6. Add further 250 c.c. serum water.

7. Weigh the flask and its contents: then (1045
grammes + weight of flask) minus (weight of flask and
its present contents) = weight of fluid required to make
up the bulk of the medium to i litre. Add the requisite
amount of sterile distilled water.

8. Titrate and estimate the reaction of the medium
mass. Then standardise to reaction of +2.5.

9. Clarify with egg, and filter as for nutrient agar.
(In clarifying, after the addition of the egg white
the mixture should be in the steamer for full two
hours.)

10. After filtration is complete measure the filtrate,
and to every 150 c.c. of the medium add:

Litmus solution (Kahlbaum) 20 c.c.

Krystal violet aqueous solu-
tion (1:1000) (B. Hoechst) 1.5 c.c.

Lactose 1.5 grammes

11. Tube in quantities of 15 c.c.

12. Sterilise in the steamer at 100° C. for thirty min-
utes on each of three successive days — i.e., by the dis-
continuous method for three days.

174 CULTURE MEDIA

Egg Medium (Dorset).—

1. Prepare 1000 c.c. of a 0.85 per cent, solution of
sodium chloride in a stout 2 -litre flask.

2. Sterilise in the autoclave at 120° C for twenty
minutes. Cool to 20° C.

3. Take 12 fresh eggs; wash the shells first with
water then with undiluted formalin: allow the shells
to dry.

4. Break the eggs into a sterile graduated cylinder
and measure the total volume of the mixed whites
and yolks. Add one part sterile saline solution to three
parts mixed eggs.

5. Transfer this mixture to a large wide-mouthed
stoppered bottle previously sterilised. Add sterile glass
beads and shake thoroughly in a mechanical shaker for
about thirty minutes, or whip with an egg- whisk.

6. Filter through coarse butter muslin into a sterile
flask.

NOTE. — A few drops of alcoholic solution of basic fuchsin (suffi-
cient to give a definite pink colour) , or a few drops of waterproof
Chinese ink added to the medium at this stage facilitates the sub-
sequent "fishing" of colonies.

7. Tube in quantities of 10 c.c.

8. Solidify in the sloping position in the inspissator
at 75° C. for one hour.

9. Place the tubes for forty-eight hours in the incu-
bator at 37° C., and eliminate any contaminated tubes.

To prevent drying, 0.5 c.c. glycerine bouillon (see
page 209) may be added to each tube between steps 8
and 9.

10. Cap those tubes of media which remain sterile
with india-rubber caps and store for future use.

Potato.—

i. Choose fairly large potatoes, wash them well, and
scrub the peel with a stiff nail-brush.

BEER WORT

175

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).

The reaction of the fresh potato is strongly acid to
phenolphthalein. If, therefore, the potatoes are re-
quired to approximate +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. in).

6. Sterilise in the steamer at 100° C. for
twenty minutes on each of five consecutive
days.

NOTE. — The cork borer reserved for cutting the
potato cylinders should be silver electro-plated both
inside and out, and the knife used for dividing the
cylinders should be of silver or silver plated. When
these precautions are adopted the potato wedges
will retain their white color and will not show the discoloration
so often observed when steel instruments are employed.

Beer Wort. — Wort is chiefly used as a medium for
the cultivation of yeasts, moulds, 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.

FIG. in. —
Potato tube.

1 76 CULTURE MEDIA

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.

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"1
beer wort direct from the brewery. In this case it is diluted
with an equal quantity of distilled water, steamed for an hour,
filtered, filled into sterile flasks or tubes, and sterilised by the
discontinuous method.

Wort Gelatine.—

1. Measure out wort (prepared as above), 900 c.c.,
into a sterile flask.

2. Weigh out gelatine, 100 grammes ( = 10 per cent.),
and add it to the wort in the flask.

3. Bubble live steam through the mixture for ten
minutes, to dissolve the gelatine.

4. Cool to 60° C. ; clarify with egg as for nutrient
gelatine (vide page 164).

5. Filter through papier Chardin.

6. Tube, and sterilise as for nutrient gelatine.

Wort Agar. —

1. Measure out wort (as above), 700 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 . Bubble live steam through the mixture for twenty
minutes, to dissolve the agar.

1 "Hopped" wort exerts a toxic effect upon many bacteria, including the
lactic acid bacteria.

177

4. Cool to 60° C. ; clarify with egg as for nutrient
agar (vide page 167).

5. Filter through papier Chardin, using the hot-
water funnel.

6. Tube, and sterilise as for nutrient agar.

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.

"Sugar" or "Carbohydrate" Media.-

Formerly the ability of bacteria to induce hydrolytic
changes in carbohydrate substances was observed only
in connection with a few well-defined sugars, but of
recent years it has been shown that when using
litmus as an indicator these so-called "fermentation
reactions" facilitate the differentiation of closely
allied species, and the list of substances employed
in this connection has been considerably extended.
The media prepared with them are now no longer
regarded as special, but are comprised in the " stock
media" of the laboratory. The chief of these sub-
stances are the following, arranged in accordance with
their chemical constitution :

Monosaccharides Dextrose (glucose), laevulose, galactose, „.

mannose, arabinose, xylose.

Disaccharides Maltose, lactose, saccharose.

Trisaccharides Raffinose (mellitose) .

Poly sac char ides Dextrin, inulin, starch, glycogen, arnidon.

Glucosides Arnygdalin, coniferin, salicin, helicin,

phlorrhizin.
12

178 CULTURE MEDIA

Polyatomic alcohols Trihydric, Glycerin.

Tetrahydric, Erythrite.

Pentahydric, Adonite

Hexahydric, Dulcite, (dulcitol or mel-
ampitite) , isodulcite (rhamnose) , rnan-
nite (mannitol), sorbite (sorbitol),
inosite.

These substances should be obtained from Kahlbaum
(of Berlin); in the pure form, and when possible
as large crystals, and the method of preparing a
medium containing either of them may be exem-
plified by describing Dextrose Solution.

Dextrose Solution. —

1. Weigh out

Peptone 20 grammes

Glucose 10 grammes

and grind together in a mortar ; then emulsify in 100 c.c.
of distilled water heated to 60° C.

2. Place in a flask and add

Distilled water 850 c.c.

3. Steam in the steamer at 100° C. for twenty
minutes to dissolve the peptone and glucose.

4. Add

Kubel-Tiemann litmus solution (Kahlbaum) . . . 50 c.c.

(The substances enumerated above react acid to
phenolphthalein, but variously toward the neutral
litmus solution. To such as react acid, add very
cautiously ° sodium hydrate solution to the medium
in bulk until the neutral tint has returned) .

5. Fill into tubes in which have previously been
placed the inverted Durham's gas tubes.

6. Sterilise in the steamer at 100° C. for twenty
minutes on each of three successive days.

NOTE. — On no account should these media be sterilised in the
autoclave, as temperatures above 100° C. themselves induce
hydrolytic changes in the substances in question. It is equally

NEUTRAL LITMUS SOLUTION 179

important that the twenty minutes should not be exceeded in
sterilisation, as neglect of this precaution may discolour the
litmus or lead to the production of yellowish tints when the
tubes are subsequently inoculated with acid-forming bacteria.

Neutral Litmus Solution.

The most satisfactory is the Kubel-Tiemann, pre-
pared by Kahlbaum. It can however be made in the
laboratory as follows :

1. Weigh out

Commercial litmus 50 grammes,

and place in a well stoppered 500 c.c. bottle; measure
out and add 300 c.c. alcohol 95 per cent.

2. Shake well at least once a day for seven days —
the alcohol acquires a green colour.

3. Decant off the green alcohol and fill a further 300
c.c. 95 per cent, alcohol into the bottle and repeat the
shaking.

4. Repeat this process until on adding fresh alcohol
the fluid only becomes tinged with violet.

5. Pour off the alcohol, leaving the litmus as dry as
possible. Connect up the bottle to an air pump and
evaporate off the last traces of alcohol.

6. Transfer the dry litmus to a litre flask, measure in
600 c.c. distilled water and allow to remain in contact
24 hours with frequent shakings.

7. Filter the solution into a clean flask and add one or
two drops of pure concentrated sulphuric acid until the
litmus solution is distinctly wine-red in colour.

8. Add excess of pure solid baryta and allow to stand
until the reaction is again alkaline.

9. Filter.

10. Bubble CO2 through the solution until reaction
is definitely acid.

n. Sterilise in the steamer at 100° C. for thirty
minutes on each of three consecutive days. This
sterilises the solution and also drives off the carbon
dioxide, leaving the solution neutral.

l8o CULTURE MEDIA

Media for anaerobic cultures. In addition to the
foregoing media, all of which can be, and are employed
in the cultivation of anaerobic bacteria, certain special
media containing readily oxidised substances are com-
monly used for this purpose. The principal of these
are as follows :

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 per cent.),
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, usually 13 per cent, required.

6. Fill, in quantities of 10 c.c., into tubes containing small
gas tubes (vide Fig. 104, page 161). Sterilise in the steamer at
100° C. for twenty minutes on each of three consecutive days.

Glucose Formate Bouillon (Kitasato). —

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163,
sections i to 6).

2. Weigh out glucose, 20 grammes ( = 2 per cent.), sodium
formate, 4 grammes ( = 0.4 per cent.), and dissolve in the fluid.

3. Tube, and sterilise as for bouillon.

Glucose Formate Gelatine (Kitasato). —

1. Prepare nutrient gelatine (vide page 164, sections i to 7) and
measure out IOQO c.c.

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.

Glucose Formate Agar (Kitasato). —

i. Prepare nutrient agar (vide page 167, 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 l8l

Sulphindigotate Bouillon (Weyl).—

1. Measure out nutrient bouillon (vide page 163, sections i
to 6 1000 c.c.).

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.

Sulphindigotate Gelatine (Weyl).—

1. Prepare nutrient gelatine (vide page 164, sections i to 7).
Measure out 1000 c.c.

2. Weigh out glucose, 20 grammes ( = 2 per cent.), and sodium
sulphindigotate, i gramme ( = o . i per cent.) , and dissolve in
the hot gelatine.

3. Filter through papier Chardin.

4. Tube, and sterilise as for nutrient gelatine.

Sulphindigotate Agar.—

1. Prepare nutrient agar (vide page 167, sections i to 8).
Measure out 1000 c.c.

2. Weigh out glucose, 20 grammes ( = 2 per cent.), sodium
sulphindigotate, i gramme ( = o . i per cent.) , and dissolve in
the hot agar.

3. Tube, and sterilise as for nutrient agar.

NOTE. — The Sulphindigotate media are of a blue colour, which
during the growth of anaerobic bacteria is oxidised and decolour-
ised to a light yellow.

XII. SPECIAL MEDIA.

In this chapter are collected a number of media
which have been elaborated by various workers for
special purposes, grouped together under headings
which indicate their chief utility. In many instances
the name of the originator of the medium is given, but
without reference to his original instructions, since
these are in many cases inadequate to the require-
ments of the isolated worker, who would probably fail
to reproduce the medium in a form giving the results
attributed to it by its author. Such modifications
have therefore been introduced as make for uniformity
between the different batches of media.

A considerable number of coloured media, chiefly in-
tended for work with intestinal bacteria, have been
included ; but beyond the fact that the author's modi-
fication of the Drigalski-Conradi medium has been in-
cluded amongst the routine media of the laboratory,
no comment has been made upon their relative values,
since only by observation and practice can the skill
necessary to utilise their full value be acquired.

The instructions as to sterilisation are raiely given
in full; the routine method of exposure in the steam
steriliser at 100° C. (without pressure) for twenty min-
utes on each of three successive days for all fluid
media, and thirty minutes on each of three successive
days for all liquefiable or solid media must be carried
out ; and only when these general rules are to be de-
parted from are further details given.

182

INOSITE-FREE MEDIA. 183

Media for the Study of the Chemical Composition of Bacteria.
Asparagin Medium (Uschinsky). —

1. Weigh out and mix

Asparagin 3-4 grammes

Ammonium lactate 10.0 grammes

Sodium chloride 5.0 grammes

Magnesium sulphate 0.2 gramme

Calcium chloride o . i gramme

Acid potassium phosphate (KH2PO4) i . o gramme

2. Dissolve the mixture in distilled water 1000 c.c.

3. Add glycerine, 40 c.c.

4. Tube, and sterilise as for nutrient bouillon.

Asparagin Medium (Frankel and Voges). —

1. Weigh out and mix

Asparagin 4 grammes

Sodium phosphate, (Na2HPOj 1 2OH. . 2 grammes

Ammonium lactate 6 grammes

Sodium chloride 5 grammes

and dissolve in

Distilled water 1000 c.c.

2. Tube, and sterilise as for nutrient bouillon.

NOTE. — Either of the above asparagin media, after the addition
of 10 per cent, gelatine or 1.5 per cent, agar, may be advan-
tageously employed in the solid condition.

Proteid Free Broth (Uschinsky).—

1. Weigh out and mix

Calcium chloride . . . . o . i gramme

Magnesium sulphate . 0.2 gramme

Acid potassium phosphate (KH2PO4) . 2.0 grammes

Potassium aspartate 3.0 grammes

Sodium chloride 5.0 grammes

Ammonium lactate .6.0 grammes

2. Dissolve the mixture in distilled water 1000 c.c.

3. Add glycerine 30 c.c.

4. Tube and sterilise as for nutrient broth.

Media for the Study of Bio-chemical Reaction.

Inosite-free Media — Bouillon (Durham). —

1. Prepare meat extract, 1000 c.c. (vide page 148), from
bullock's heart which has been "hung" for a couple of days.

2. Prepare nutrient boullion (+10), 1000 c.c. (vide, page 161),
from the meat extract, and store in i -litre flask.

1 84 SPECIAL MEDIA

3. Inoculate the bouillon from a pure cultivation of the B.
lactis aerogenes, and incubate at 37° C. for forty-eight hours.

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 + 10.

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 con-
dition; but if acid or gas is present, again make the bouillon in
the flask + 10, reinoculate with one or other of the above-men-
tioned bacteria, and incubate; then test again. Repeat this till
neither acid nor gas appears in the medium when used for the
cultivation of either of the bacilli referred to above.

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 prepared nutrient
sugar-free gelatine or agar, by dissolving in it the required per-
centage of gelatine or agar respectively and completing the medium
according to directions given on pages 166 and 167. The most im-
portant application of inosite-free bouillon is its use in the prep-
aration of sugar bouillons, whether glucose, maltose, lactose, or
saccharose, of exact percentage composition.

Sugar (Dextrose) Bouillon. —

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163,
sections i to 6) or sugar-free bouillon (vide svprd).

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
it causes the medium to gradually deepen in colour.

NOTE. — In certain cases a corresponding percentage of lactose,
maltose, or saccharose is substituted for glucose.

Sugar Gelatine. —

1. Prepare nutrient gelatine (vide page 164, sections i to 7).
Measure out 1000 c.c.

2. Weigh out glucose, 20 grammes (=2 per cent.), and dissolve
in the hot gelatine.

IRON PEPTONE SOLUTION 185

3. Filter through papier Chardin.

4. Tube, and sterilise as for nutrient gelatine.

Sugar Agar. —

1. Prepare nutrient agar (vide page 167, 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. — Other "sugar" media are prepared by substituting a
corresponding percentage of lactose, maltose (or any other of the
substances referred to under "Sugar Media," page 177) for the
glucose.

Iron Bouillon. —

1. Measure out nutrient bouillon, 1000 c.c. (vide page 141,
sections i to 6).

2. Weigh out ferric tartrate, i gramme ( = 0.1 per cent.), and
dissolve it in the bouillon.

3. Tube, and sterilise as for bouillon.

NOTE. — The lactate of iron may be substituted for the tartrate.

Lead Bouillon. —

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163,
sections i to 6).

2. Weigh out lead acetate, i gramme ( = 0.1 per cent.), and
dissolve it in the bouillon.

3. Tube, and sterilise as for bouillon.

Nitrate Bouillon.—

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163,
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 sub-
stituted 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 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 800 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,

1 86 SPECIAL MEDIA

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 solution
of ferric tartrate. (A yellowish-white precipitate forms.)

7. Sterilise as for nutrient bouillon.

Lead Peptone Solution. —

Prepare as for iron peptone solution but in step 6 substitute
o.i c.c. of a i per cent, neutral aqueous solution of lead acetate.

Nitrate Peptone Solution (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 1000 c.c.,
with ammonia-free distilled water.

3. Heat in the steamer at 100° C. for twenty minutes.

4. Weigh out sodium nitrate, i gramme, and dissolve in the
contents of the flask.

5. Filter through Swedish filter paper.

6. Tube, and sterilise as for nutrient bouillon.

Litmus Bouillon. —

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163,
sections i to 6).

2. Add sufficient sterile litmus solution to tint the medium a
dark lavender colour. (Media rendered + 10 will usually react
very faintly alkaline or occasionally neutral to litmus.)

3. Tube, and sterilise as for bouillon.

Rosolic Acid Peptone Solution.—

1 . Weigh out rosolic acid (corallin) ,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 nutrient bouillon.

Capaldi=Proskauer Medium, No. I. —

i. Weigh out and mix

Sodium chloride 2.0 grammes

Magnesium sulphate o.i gramme

Calcium chloride 0.2 gramme

Monopotassium phosphate ....2.0 grammes

a. Dissolve in water 1000 c.c. in a 2 -litre flask

URINE GELATINE 187

3. Weigh out and mix

Asparagin 2 grammes

Mannite 2 grammes

and add to contents of flask.

4. Measure out 25 c.c. of the solution and titrate it against
decinormal sodic hydrate, using litmus as the indicator. Control
the result and estimate the amount of sodic hydrate necessary
to be added to render the remainder of the solution neutral to
litmus. Add this quantity of sodic hydrate.

5. Filter.

6. Add litmus solution 47.5 c.c. ( = 5 per cent.).

7. Tube, and sterilise as for nutrient bouillon.

Capaldi=Proskauer Medium No. II. —

1. Weigh out and mix

Peptone 20 grammes

Mannite i gramme

2. Dissolve in water 1000 c.c. in a 2-litre flask.

3. Neutralise to litmus as in No. i (vide supra, Step 4).

4. Filter.

5. Add litmus solution 47.5 c.c. ( = 5 per cent.).

6. Tube, and sterilise as for nutrient bouillon.

Urine Media. Bouillon.—

1. Collect freshly passed urine in sterile flask.

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 as for nutrient bouillon.
(Leave the reaction unaltered.)

Urine Gelatine.—

1. Collect freshly passed urine in sterile flask.

2. Take the specific gravity, and, if above lojo, dilute with
sterile water until that gravity 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 166).

8. Filter through papier Chardin.

9. Tube, and sterilise as for nutrient gelatine.

1 88 SPECIAL MEDIA

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 166).

8. Filter through papier Chardin.

9. Tube, and sterilise as for nutrient gelatine.

Urine Agar. —

1. Collect freshly passed urine in sterile flask.

2. Take the specific gravity and if above 1010, dilute with sterile
water till this gravity is reached.

3 . Weigh out i . 5 per cent, or 2 per cent, powdered agar, and add
it to the urine.

4. Heat in the steamer at 100° C. ior ninety minutes to dissolve
the agar.

5. Cool to 60° C. and clarify with egg as for nutrient agar (vide
page 1 6 8).

6. Filter through papier Chardin, using the hot-water funnel.

7. Tube, and sterilise as for nutrient agar.
(Leave the reaction unaltered.)

Serum Sugar Media (Hiss).—

In these media the fermentation of carbohydrate substance by
bacterial action is indicated by the coagulation of the serum
proteids in addition to the production of an acid reaction.

Serum Dextrose Water (Hiss).—

1. Measure out into a litre flask

Serum water (See page 1 70) 1000 c.c.

2. Weigh out

Dextrose 10 grammes

and dissolve in the serum water.

3. Filter through Swedish filter paper.

4. Measure out and add to the medium

Litmus solution (Kahlbaum) .... 50 c.c.

MILK RICE 189

5. Tube in quantities of 10 c.c. and sterilise in the steamer at
100° C. for twenty minutes on each of three successive days.

Lasvulose, galactose, maltose, lactose, etc., can be substituted
in similar amounts for dextrose and the medium completed as
above.

Omeliansky's Nutrient Fluid (For Cellulose Fermenters) . —
i. Weigh out and mix

Potassium phosphate . . .

. . . 4.0 grammes

Magnesium sulphate

2 o grammes

Ammonium sulphate . . .

. . . 4.0 grammes

Sodium chloride
ssolve in distilled water

. . .0.25 gramme
4000 c.c.

2.

3. Flask in quantities of 250 c.c.

4. Weigh out and add 5 grammes precipitated chalk to each
flask.

5. Sterilise in the steamer at 100° C. for twenty minutes on
each of three successive days.

Media for the Study of Chromogenic Bacteria.

Milk Rice (Eisenberg).—

1. Measure out nutrient bouillon, 70 c.c., and milk, 210 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 about 0.5 cm. thick, 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 1 00° C. for thirty minutes on each of three consecutive days.

(A solid medium of the colour of cafe au lait is thus produced.)

Milk Rice (Soyka).—

1. Measure out nutrient bouillon, 50 c.c., and milk, 150 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, 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 thirty minutes on
each of three consecutive days.

(A pure white, opaque medium is thus formed.)

IQO SPECIAL MEDIA

Media for the Study of Phosphorescent and Photogenic Bacteria.

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 500 c.c. distilled water. Add the solution to the
fish in the beaker.

3 . Place the beaker in a water-bath and proceed as in 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, if it is to be used as fluid
medium, make up to 1000 c.c. by the addition of distilled water.
If, however, it is to be used as the basis for agar or gelatine media
store it in the "Double Strength" condition.

9. Tube and sterilise as for nutrient bouillon.

As an alternative method "Marvis" fish food (16 grammes)
may be substituted for the 500 grammes of fresh fish.

Fish Gelatine.—

1. Measure out double strength fish bouillon, 500 c.c., into a
"tared" 2-litre flask.

2. Add sheet gelatine, 100 grammes, cut into small pieces.

3 . Bubble live steam through the mixture for fifteen minutes to
dissolve the gelatine.

4. Weigh the flask and its contents; adjust the weight to the
calculated figure for one litre of medium (1135.5 grammes) by the
addition of distilled water at 100° C. (vide page 166).

5. Cool to below 60° C., and clarify with egg.

6. Filter through papier Chardin.

7. 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. double strength fish bouillon.

2. Wash the emulsion into a "tared" 2-litre flask with 300 c.C.
fish bouillon.

NAEGELI'S SOLUTION IQI

3. Weigh out sheet gelatine, 70 grammes, cut it into small
pieces and add it to the contents of the flask.

4. Bubble live steam through the mixture to dissolve the gela-
tine and agar.

5. Weigh the flask and contents. Adjust the weight to the
calculated figure for one litre of medium (1110.5 grammes) by the
addition of distilled water at 100° C. (vide page 166).

6. Cool to below 60° C. and clarify with egg.

7. Filter through papier Chardin.

8. Tube, and sterilise as for nutrient gelatine.

Shake well after the final sterilisation, to aerate the medium.

Media for the Study of Yeasts and Moulds.
Pasteur's Solution. —

(Reaction alkaline).

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 as for nutrient bouillon.

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 nutrient bouillon.

Cohn's Solution. —

1. Weigh out and mix

Acid potassium phosphate (KH2PO4) .5.0 grammes

Calcium phosphate 0.5 gramme

Magnesium sulphate 5.0 grammes

Ammonium tartrate 10.0 grammes

and dissolve in

Distilled water 1000 c.c.

2. Tube, or flask and sterilise as for nutrient bouillon.
Naegeli's Solution.—

1. Weigh out and mix

Dibasic potassium phosphate (K2HPO4) i.o gramme

Magnesium sulphate o . 2 gramme

Calcium chloride o . i gramme

Ammonium tartrate 10.0 grammes

and dissolve in

Distilled water . . . . - 1000 c.c.

2. Tube or flask; sterilise as for nutrient bouillon.

192

SPECIAL MEDIA

Plaster =of=Paris Discs. —

i. Take large corks, 2 .5 cm. diameter, and roll a piece of stiff
note-paper round each, so that about a centimetre projects as a
ridge above the upper surface of the cork, and secure in position
with a pin (Fig. 112).

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 raise the plaster
discs.

4. Place the plaster discs on a piece of
asbestos board and sterilise by exposing in the
hot-air oven to 150° C. for half an hour.

Fig. 112. — Cork 5. Remove the sterile discs from the oven
and paper mould for by means of sterile forceps, place each inside
plaster-of-Paris disc, a sterile capsule, and moisten with a little
sterile water.

6. Sterilise in the steamer at 100° C. for thirty minutes on
each of three consecutive days.

Gypsum Blocks (Engel and Hansen). —

These are in the form of truncated cones and for their prepara-
tion small tin moulds are required, each having a diameter of 5 . 5
cm. at the base and 4 cm. at the truncated apex. The height
(or depth) of a mould is 4 . 5 to 5 cm.

1. Mix powdered calcined gypsum into a stiff paste with dis-
tilled water.

2. Fill the paste into the moulds and allow it to set and dry by
exposure to air.

3. Remove the block from the mould and transfer it to a
double glass dish of adequate size (7 cm. diameter X 7 cm. high).

4. Sterilise block in its dish for one hour in the hot-air oven at
115° C.

5. Carefully open the dish and add sterile distilled water to
moisten the block and form a layer in the bottom of the dish i cm.
deep.

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 as for nutrient bouillon.

GELATINE AGAR 193

Wheat Bouillon (Gasperini). —

1. Weigh out and mix wheat flour, 150 grammes; magnesium
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 as for nutrient bouillon.

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 one centimetre 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 four consecutive days.

Media for the Study of Parasitic Moulds.

French Proof Agar (Sabouraud). —

1. Weigh out Chassaing's peptone, 10 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 tared 2-litre flask
with 600 c.c. distilled water.

4. Bubble live steam through the mixture for twenty minutes,
to dissolve the agar.

5. Cool to 60° C. and clarify with egg as for nutrient agar
(vide page 168).

6. Filter through Papier Chardin, using the hot-water funnel.

7. Weigh out French maltose, 40 grammes, and dissolve in the
agar.

8. Tube, and sterilise as for nutrient agar.

English Proof Agar (Blaxall). — Substitute Witte's peptone for
that of Chassaing, and proceed as for French proof agar.

French Mannite Agar, Sabouraud.— (For cultivation of Favus.)
Proceed exactly as in preparing French Proof agar vide supra
substituting Mannite (38 grammes) for maltose.

Media for the Study of Milk Bacteria.

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

13

194 SPECIAL MEDIA

to employ an incubating temperature of 30°, C., 10 per cent,
gelatine and o . 5 per cent, agar must be dissolved in the meat
extract before the addition of the peptone 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, otherwise the action upon the medium of such organisms
as elaborate a liquefying ferment may be retarded or completely
absent.

1. Measure out 400 c.c. double strength meat extract into a
"tared" 2-litre flask, and add to it gelatine, 100 grammes.

2. Weigh out powdered agar, 5 grammes, emulsify with 100 c.c.,
cold distilled water and add to the contents of the flask.

3. Dissolve the agar and gelatine by bubbling live steam
through the flask for twenty minutes.

4. Weigh out peptone, 10 grammes; salt, 5 grammes ; emulsify
with 100 c.c. double strength meat extract previously heated to
60° C., and add to the contents of the flask.

5. Replace in the steamer for fifteen minutes. Then adjust the
weight to the calculated figure for one litre (in this instance 1120
grammes) by the addition of distilled water at 100° C.

6. Estimate the reaction; control the result. Then add
sufficient caustic soda solution to render the reaction + 10.

7. Replace in the steamer at 100° C. for twenty minutes.

8. Cool to 60° C. Clarify with egg as for nutrient agar.

9. Filter through papier Chardin, using the hot-water funnel.

10. Tube, and sterilise as for nutrient agar.

Agar Gelatine (Guarniari). —

1. Measure out double strength meat extract, 400 c.c., into a
"tared" 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 . Dissolve the agar and gelatine by bubbling live steam through
the flask for twenty minutes.

4. Weigh out Witte's peptone, 25 grammes; salt, 5 grammes,
and emulsify with 100 c.c. double strength meat extract previously
heated to 60° C., and add to the contents of the flask.

5. Replace in the steamer for fifteen minutes.

6. Weigh the flask and make up the medium mass to the cal-
culated figure for one litre (1083 grammes) by the addition of
distilled water at 100° C.

7. Neutralise carefully to litmus paper by the successive
additions of small quantities of normal soda solution.

8. Replace in the steamer at 100° C. for twenty minutes.

9. Cool to 60° C. Clarify with egg as for nutrient agar.

10. Filter through papier Chardin, using the hot-water funnel.

11. Tube, and sterilise as for nutrient agar.

LITMUS WHEY

195

Whey Gelatine.—

1. Curdle fresh milk by warming to 60° C., and adding rennet;
filter off the whey into a sterile "tared" 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. Bubble live steam through the mixture fifteen minutes to
dissolve the gelatine; and weigh.

5. Estimate the reaction of the medium mass; then add
sufficient caustic soda solution to restore the reaction of the
medium mass (i.e., total weight minus weight of flask) to the
equivalent of the original whey.

6. Cool to 60° C. and clarify with egg as for nutrient gelatine
(vide page 166).

7. Filter through papier Chardin.

8. Tube, and sterilise as for nutrient gelatine.

Whey Agar. —

1. Curdle fresh milk by warming to 60° C., and adding rennet;
filter off the whey into a sterile flask.

2. Weieh out agar, 1.5 or 2 per cent., and add it to the whey
in the flask.

3. Bubble live steam through the mixture for twenty minutes,
. to dissolve the agar.

4. Cool to 60° C. ; clarify with egg as for nutrient agar (vide
page 1 6 8).

5. Filter through papier Chardin, using the hot-water funnel.

6. Tube, and sterilise as for nutrient agar.

Litmus Whey.—

1. Curdle fresh milk by warming to 60° C. and adding rennet.

2. Filter off the whey through butter muslin into a sterile flask.

3. Neutralise to litmus by the cautious addition of citric acid
solution 4 per cent. (Do not neutralise with mineral acid.)

4. Heat in the steamer at 100° C. for one hour to coagulate all
the proteid.

(If the whey is cloudy when removed from the steamer allow
it to stand for forty-eight hours in the ice chest and then decant
off the clear fluid — or filter through a Berkfeld filter candle.)

5. Filter into a sterile flask.

6. Tint the whey with litmus solution to a deep purple red.

7. Tube, and sterilise as for milk.

Litmus Whey (Petruschky).—

i. Measure out into a flask

Fresh milk 1000 c.c.

196 SPECIAL MEDIA

2. Add

Hydrochloric acid (or glacial acetic acid) . 1.5 c.c.
and boil.

3. Filter off coagulated casein.

4. Neutralise to litmus by the addition of 3 caustic soda so-
lution and boil. Whey now cloudy and acid again.

5. Again neutralise to litmus by addition of ^L caustic soda
solution.

6. Filter.

7. Tint the whey with neutral litmus solution to a deep purple
colour.

8. Tube and sterilise as for milk.

Litmus Whey Gelatine.—

1. Measure out milk 1000 c.c. into a tared 2-litre flask.

2. Add hydrochloric acid (or glacial acetic acid) 1.5 c.c. and
boil for five minutes.

3. Filter off the casein, and make the whey faintly alkaline to
litmus.

4. Weight out

Peptone 10 grammes

and emulsify in a few cubic centimeters of the whey and return
to the flask.

5. Weight out

Gelatine 50 grammes

add it to the whey in the flask and incorporate the mixture
by bubbling through live steam.

6. Clear with egg and filter.

7. Make the weight of the medium mass to the calculated fig-
ure for one litre (1060 grammes) by the addition of distilled water.

8. Weigh out

Dextrose 15 grammes

and dissolve in the fluid whey gelatine.

9. Add sterile litmus solution to the required tint.

10. Tube and sterilise for twenty minutes in steamer at 100° C.
on each of five successive days.

This medium will remain semi-fluid at the room temperature,
and may be used for cultures in the cool or hot incubator.

Litmus Whey Agar is prepared in a similar manner to Whey
Gelatine, with the substitution of 15 grammes of agar for the
gelatine.

Malt Extract Solution (Herschell).—

1. Measure into a flask distilled water 1000 c.o.

2. Weigh out

Extractum malti (malt extract) . . 25 grammes
and add to distilled water in flask.

BEYRINCK'S SOLUTION 197

3. Boil for five minutes, allow to stand, and decant off clear
fluid from sediment.

4. Tube and sterilise as for nutrient bouillon.

Media for the Study of Earth Bacteria, Nitrogen Fixers.

Earthy Salts Agar (Lipman and Brown). — (For the enumeration
of soil organisms.)

1. Measure out

Agar 20 grammes.

Emulsify in 200 c.c. distilled water.

2. Wash the agar emulsion into a tared 2-litre flask with 400 c.c.
distilled water.

3. Weigh out

Peptone 0.5 gramme.

Emulsify in 50 c.c. distilled water and add to the contents
of the flask.

4. Bubble live steam through the mixture for twenty minutes
to dissolve the agar.

5. Weigh out and mix

Dextrose. 10.0 grammes.

Potassium phosphate .... 0.5 gramme.

Magnesium sulphate 0.2 gramme.

Potassium nitrate 0.06 gramme.

and add to the contents of the flask.

6. Adjust the weight of the medium mass to the calculated fig-
ure for one litre (1025 grammes) by the addition of distilled water
at 100° C.

7. Titrate the medium mass and adjust the reaction to +5.

8. Cool to 60° C. Clarify with egg and filter.

9. Tube in quantities of 10 c.c. and sterilise as for nutrient agar.

Beyrinck's Solution. I. — (For the cultivation of nitrogen fixing
organisms.)

1. Weigh out and mix i gramme potassium hydrogen phos-
phate, 0.2 gramme magnesium sulphate, and 0.02 gramme sodium
chloride.

2. Dissolve in water 1000 c.c., in a 2-litre flask.

3. Add i c.c. of a one per thousand aqueous solution of ferrous
sulphate.

4. Add i c.c. of a one per thousand solution manganese
sulphate.

5. Weigh out 20 grammes dextrose and add to the contents of
the flask (dextrose up to 40 grammes may be used for the different
organisms) .

6. Steam for twenty minutes, filter.

7. Tube, and sterilise as for nutrient bouillon.

198 SPECIAL MEDIA

Beyrinck's Solution. II. — (For growth of Azobacter.}
Proceed as in preparing solution No. i, substituting mannite for
dextrose in step 5.

Winogradsky's Solution (for Nitric Organisms) . —

1. Weigh out and mix.

Potassium phosphate i . o gramme

Magnesium sulphate 0.5 gramme

Calcium chloride o.oi gramme

Sodium chloride 2.0 grammes

and dissolve in

Distilled water 1000 c.c.

2. Fill into flasks, in quantities of 20 c.c. and add to each a
small quantity of freshly washed magnesium carbonate.

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. Store the remainder for future use.

Winogradsky's Solution (for Nitrous Organisms). —

1. Weigh out and mix

Ammonium sulphate i gramme

Potassium sulphate i gramme

and dissolve in

"Distilled water 1000 c.c.

2. Add 5 to 10 grammes basic magnesium carbonate, previously
sterilised by boiling.

3. Fill into flasks and sterilise, etc., as for previous solution.

Silicate Jelly (Winogradsky).—

1. Weigh out and mix

Ammonium sulphate 0.40 gramme

Magnesium sulphate 0-05 gramme

Calcium chloride o.oi gramme

and dissolve in

Distilled water 50 c.c.

Label — Solution A.

2. Weigh out and mix

Potassium phosphate o.io gramme

Sodium carbonate 0.60 gramme

and dissolve in

Distilled water 50 c.c.

Label — Solution B.

BILE SALT BROTH 199

3. Weigh out

Silicic acid 3.4 grammes

and dissolve in

Distilled water 100 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 at 100° C. for thirty minutes on each
of three consecutive days.

Media for the Study of Water Bacteria.

Naehrstoff Agar (Hesse and Niedner) . — (For enumeration of water
organisms.)

1. Weigh out: agar, 12.5 grammes and emulsify in 250 c.c.
distilled water.

2. Wash the agar emulsion into a tared 2 -litre flask with a
further 250 c.c. distilled water.

3. Dissolve by bubbling live steam through the mixture.

4. Emulsify Naehrstoff-Heyden (albumose) 7 . 5 grammes in
200 c.c. cold distilled water and add to melted agar.

5. Adjust weight of medium mass to the calculated figure for one
litre (1020 grammes) by addition of distilled water at 100° C.

6. Clarify with white of egg and filter.

7. Tube in quantities of 10 c.c. and sterilise in the steamer at
100° C. for twenty minutes on each of three successive days.

Bile Salt Broth— Double Strength.—

1. Weigh out Witte's peptone, 40 grammes, and emulsify with
300 c.c. distilled water previously warmed to 60° C.

2. Wash the peptone emulsion into a litre flask with 600 c.c.
distilled water.

3. Weigh out sodium taurocholate, 10 grammes, and glucose,
10 grammes; dissolve in 100 c.c. distilled water and add to the
peptone emulsion in the flask.

4. Heat in the steamer at 100° C. for twenty minutes.

5. Filter through Swedish filter paper into a sterile flask.

6. Add sterile neutral litmus solution sufficient to colour the
medium to a deep purple.

7. Fill into small Erlenmeyer -flasks in quantities of 25 c.c.

8. Sterilise as for nutrient bouillon.

200 SPECIAL MEDIA

Media for the Study of Plant Bacteria.

Beetroot.— 1

Carrot. — [ are prepared tubes and sterilised in a manner pre-
Turnip. — [ cisely similar to that described for potato.
Parsnip. — J

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 as for nutrient bouillon.

Haricot Bouillon. — (For cultivation of bacteria from tubercles of
Legumes.)

1. Measure out 1000 c.c. distilled water into a 2-litre flask.

2. Weigh out 250 grammes haricot beans and add to the water
in the flask.

3. Weigh out 10 grammes sodium chloride and add to the
contents of the flask.

4. Add i c.c. of a i per cent, solution of sodium bicarbonate.

5. Place in the steamer at 100° C. for thirty minutes.

6. Filter.

7. Weigh out 20 grammes saccharose and add to the filtrate.

8. Tube, and sterilise as for nutrient bouillon.

Haricot Agar. —

1. Measure out 400 c.c. distilled water into a "tared" 2-litre
flask.

2. Weigh out 15 grammes agar and mix into a thick paste with
too c.c. cold distilled water, and add to the flask.

3. Dissolve the agar by bubbling live steam through the mixture
as in making nutrient agar.

4. Weigh out 250 grammes haricot beans, place in the flask with
the agar mixture.

5. Add i c.c. of i per cent, aqueous solution sodium bicarbonate.

6. Weigh out 10 grammes sodium chloride and add to the
contents of the flask.

7. Place in the steamer at 100° C. for thirty minutes.

8. Adjust the weight of the medium mass to 1030 grammes (the
figure per litre obtained experimentally) by the addition of distilled
water at 100° C.

OLEIC ACID AGAR 2OI

9. Cool to 60° C., clarify with egg and filter.

10. Weigh out 20 grammes saccharose and add to the contents
of the flask.

u. Tube, and sterilise as for nutrient agar.

Wood Ash Agar. —

-i. Measure 400 c.c. distilled water into a tared 2-litre flask.

2. Weigh out 10 grammes agar and make into a thick paste
with 100 c.c. cold distilled water.

3. Add this agar paste to the distilled water in the flask.

4. Dissolve the agar by passing live steam through it, as in
preparing nutrient agar.

5. Weigh out 5 grammes clean wood ash and place in a second
flask containing 200 c.c. distilled water with some sterile glass
beads: shake thoroughly in a mechanical shaker for ten minutes.

6. Heat in steamer at 100° C., for thirty minutes.

7. After removal from the steamer dry the outside of the flask
thoroughly, place it over a Bunsen flame and boil for one minute.

8. Filter directly into the flask containing the melted agar
mixture.

9. Weigh out 4 grammes maltose. Add to the contents of the
flask.

10. Adjust the weight of the medium mass to the calculated
figure for one litre (1019 grammes) by the addition of distilled
water at 100° C.

11. Replace the flask in the steamer for twenty minutes, cool
to 60° C., and clarify with egg and filter.

12. Tube, and sterilise as for nutrient agar.

Media for the Study of Special Bacilli.

B. Acnes.
Oleic Acid Agar (Fleming).—

1. Measure out into a sterile stout glass bottle which already
contains about 10 sterile glass beads

Ascitic fluid 250 c.c.

2. Weigh out

Oleic acid 25 grammes

and add it to the ascitic fluid in the bottle.

3. Emulsify evenly by shaking (either by hand or in a shaking
machine) for ten minutes.

4. Liquefy and measure out into a flask

Nutrient agar 750 c.c.

then cool to 55° C.

5. Mix the oleic acid emulsion with the agar.

202 SPECIAL MEDIA

6. Add 10 c.c. sterile neutral red, i per cent! aqueous solution.

7. Tube in quantities of 10 c.c., slant, and allow to set.

8. Incubate for forty-eight hours at 37° C. and reject any
contaminated tubes. Store the sterile tubes for future use.

Coli-typhoid Group.
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 163).

3. Incubate at 37° C. for forty-eight hours to eliminate con-
taminated tubes. Store the remainder for future use.

Carbolised Bouillon. —

1. Prepare nutrient bouillon (vide page 163, sections i to 6).
Measure out 1000 c.c.

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.

Carbolised Gelatine.—

1. Prepare nutrient gelatine (vide page 164, sections i to 7).
Measure out 1000 c.c.

2. Weigh out carbolic acid, 5 grammes ( = 0.5 per cent.), and
dissolve it in the gelatine.

3. Filter if necessary 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.

Carbolised Agar. —

1. Prepare nutrient agar (vide page 167, sections i to 8).
Measure out 1000 c.c.

2 . Weigh out i gramme pure phenol and dissolve in the medium.

3. Filter if necessary through papier chardin.

4. Tube, and sterilise as for nutrient agar.

Litmus Gelatine. —

1. Prepare nutrient gelatine (vide page 164, 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.

GLYCERINE POTATO BOUILLON 203

Lactose Litmus Bouillon (Lakmus Molke). —

1. Weight out peptone, 4 grammes, and emulsify it with
200 c.c. meat extract (vide page 148), previously heated to 60° C.

2. Weigh out salt, 2 grammes, and lactose, 20 grammes, 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 com-
pletely dissolve the peptone, etc.

5 . Neutralise carefully to litmus paper by the successive 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.

Lactose Litmus Gelatine (Wurtz). —

1. Prepare nutrient gelatine (vide page 164, 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. 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.

Lactose Litmus Agar (Wurtz). —

1. Prepare nutrient agar (vide page 167, 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.

Glycerine Potato Bouillon.—

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,

204 SPECIAL MEDIA

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 dis-
tilled 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 as for nutrient bouillon.

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
bubble live steam through the mixture for ten minutes.

5. Estimate the reaction; adjust the reaction of the medium
mass to + 25.

6. Cool the medium to below 60° C. ; clarify with egg as for
nutrient gelatine (vide page 166).

7. Add i per cent, potassium iodide (powdered) to the medium.

8. Filter through papier Chardin.

9. Tube and sterilise as for nutrient gelatine.

Aesculin Agar. — (B. coli and allied organisms give black
colonies surrounded by black halo.)

1. Measure out 400 c.c. distilled water into a tared 2-litre flask.

2. Weigh out

Agar 15 grammes

Peptone 10 grammes

Sodium taurocholate 5 grammes

and make into a thick paste with 150 c.c. distilled water.

3. Add this paste to the distilled water in the flask.

4. Dissolve the ingredients by bubbling live steam through the
mixture.

5. Weigh out

Aesculin i . o gramme

Ferric citrate 0.5 gramme

and dissolve in a second flask containing 100 c.c. distilled water.

6. Mix the contents of the two flasks — adjust the weight to

FUCHSIN A GAR

205

the calculated medium figure (in this case 1031.5 grammes) by
the addition of distilled water at 100° C.

7. Clarify with egg and filter.

8. Tube and sterilise as for nutrient agar.

Bile Salt Agar (MacConkey).—

1. Weigh out powdered agar, 15 grammes (= 1.5. per cent.)*
and emulsify with 200 c.c. cold tap water.

2. Weigh out peptone, 20 grammes ( = 2 per cent.) , and emulsify
with 200 c.c. tap water previously warmed to 60° C.

3. Mix the peptone and agar emulsions thoroughly.

4. Weigh out sodium taurocholate, 5 grammes ( = 0.5 per
cent.), dissolve it in 300 c.c. tap water, and use the solution
to wash the agar-peptone emulsion into a tared 2-litre flask.

5. Bubble live steam through the mixture for twenty minutes.

6. Adjust the weight of the medium mass to the calculated
figure for one litre (1040 grammes).

7. Cool to 60° C. and clarify with egg as for nutrient agar
(vide page 168).

8. Filter through papier Chardin, using the hot-water funnel.

9. Weigh out lactose, 10 grammes (= i per cent.), and dissolve
it in the agar.

If desired, add 5 c.c. of a i per cent. ( = 0.5 per cent.) aqueous
solution of neutral red.
10. Tube, and sterilise as for nutrient agar.

Litmus Nutrose Agar (Drigalski=Conradi).—

This medium should be prepared in precisely the same manner
as the Nutrose agar described .on page 172 substituting meat ex-
tract for serum water, and increasing the percentage of agar added
per litre to 3 per cent.

Fuchsin Agar (Braun). —

1. Liquefy and measure out into a sterile flask:

Nutrient agar 1000 c.c.

2. Weigh out: lactose 10 grammes and dissolve in the fluid
agar.

3. Adjust the reaction to —5 and filter.

4. Measure out and mix thoroughly with agar:

Fuchsin, alcoholic solution 5 c.c.

The fuchsin solution is prepared by mixing:

Fuchsin (basic) 3 grammes.

Absolute alcohol 60 c.c.

Allow to stand twenty-four hours, then centrifugalise
thoroughly and decant the supernatant fluid into a
well-stoppered bottle, j

2O6 SPECIAL MEDIA

5. Measure out and add to the nutrient agar, sodium sulphite,
10 per cent, aqueous solution, freshly prepared . . 25 c.c.

6. Tube and sterilise as for nutrient agar.

7. Store in a dark cupboard.

Fuchsin Sulphite Agar (Endo).—

1. Liquefy and measure out into a sterile flask:

Nutrient agar 1000 c.c.

2. Weigh out

Lactose 10 grammes.

and dissolve in the fluid agar.

3. Adjust the reaction to +3 and filter.

4. Measure out and mix thoroughly with the fluid agar.

Fuchsin, alcoholic solution (vide supra) .... 5 c.c. •

5. Measure out and add to the medium

Sodium sulphite, 10 per cent, aqueous solution . .25 c.c.

6. Tube and sterilise as for nutrient agar.

Brilliant Green Agar (Conradi) . —

1 . Liquefy and measure out into a sterile flask

Nutrient agar 1000 c.c.

2. Adjust reaction to + 30 by the addition of normal phosphoric
acid ; and filter.

3. Measure out and mix thoroughly with the fluid medium

Brilliant green (Hoechst) i per thousand aque-
ous solution 6.5 c.c.

4. Measure out and add to the medium

Picric acid (Gruebler) , i per cent, aqueous solution . 6.5 c.c.

5. Tube and sterilise as for nutrient agar.

Brilliant Green Bile Salt Agar (Fawcus).—

1. Weigh out agar 20 grammes and emulsify in 100 c.c. cold
distilled water.

2. Wash the emulsion into a "tared" 2-litre flask with 500 c.c.
distilled water.

3 . Dissolve the agar by bubbling live steam through the flask.

4. Cool, clarify with egg and filter.

5. Weigh out

Sodium taurocholate 5 grammes

Peptone 20 grammes

and add to the medium in the flask.

DOUBLE SUGAR A GAR 207

6. Weigh out

Lactose 5 grammes

and add to the medium in the flask.

7. Adjust reaction to + 15 and filter if necessary.

8. Measure out

Brilliant green, i per thousand aqueous solution. 20 c.c.
and mix thoroughly with the fluid agar.

9. Measure out and add to the medium

Picric acid, i per cent, aqueous solution .... 20 c.c.

10. Tube and sterilise as for nutrient agar.
China Green Agar (Werbitski) .—

1. Liquefy and measure out into a sterile flask

Nutrient agar 1000 c.c.

2. Adjust the reaction accurately to +13 and filter.

3. Measure out and mix thoroughly with the fluid agar
China green 0.2 per cent, aqueous solution. . . 15 c.c.

4. Tube and sterilise as for nutrient agar.

Malachite Green Agar (Loeffler). —

1. Liquefy and measure out into a sterile flask

Nutrient agar 1000 c.c.

2. Weigh out

Dextrose 10 grammes.

and dissolve in nutrient agar.

3. Adjust the reaction to +3, and filter.

4. Measure out and mix thoroughly in the fluid agar

Malachite green, o.i per cent, aqueous solution. .16 c.c.
for "weak" medium.

40. To the filtered agar add

Malachite green, 2 per cent, aqueous solution . . .25 c.c.
for " strong " medium.

5. Tube and sterilise as for nutrient agar.
Double Sugar Agar (Russell). —

1. Liquefy and measure out into a sterile flask

Nutrient agar 1000 c.c.

2. Add 100 c.c. litmus solution to the fluid agar.

3. Weigh out and dissolve in the fluid agar.

Lactose 10 grammes

Dextrose 10 grammes.

208 SPECIAL MEDIA

4. Render the reaction of the medium neutral to litmus
paper by the cautious addition of normal caustic soda.

5. Tube in quantities of 10 c.c. and sterilise in the steamer at
100° C. for twenty minutes on each of three successive days.

6. Store for use in a cool dark place.

B. Diphtheria.

Glycerine Blood=serum. —

1. Prepare blood-serum as described, page 168, sections i to 4.

2. Add 5 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. — are used for special purposes. Five per cent, is
that usually employed.

Blood =serum (Loeffler). —

1. Prepare nutrient bouillon (vide page 163), 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 168, 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 168, 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> °f 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.

Blood Serum (Councilman and Mallory). —

1. Collect blood serum in slaughterhouse, coagulate, remove
serum and tube (vide page 168).

Great care must be taken to avoid the inclusion of air bubbles
— indeed if only a few tubes are filled at one time, it is a good plan
to stand them upright in the receiver of an air pump and to ex-
haust as completely as possible before transferring to the serum
inspissator.

2. Heat the tubes in a slanting position in hot-air steriliser at
90° C. till firmly coagulated, say half an hour.

GLYCERINATED POTATO 2OQ

3. Sterilise in steam steriliser at 100° C. for 20 minutes on each
of three successive days.

Resulting medium not translucent, but opaque and firm.

B. Tuberculosis.

Egg Medium (Lubenau). —

This modification of Dorset's egg medium (quod mde page
1 74) is preferred by some for the growth of the tubercle bacillus of
the human type. It consists in the addition of one part of 6 per
cent, glycerine in normal saline solution, to the egg mixture be-
tween steps 4 and 5.

Glycerine Bouillon. —

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163,
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.

Glycerine Agar. —

1. Prepare nutrient agar (vide page 167, 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.

Glycerine BIood=serum. —

1. Prepare blood-serum as described, page 168, sections i to 4.

2. Add 5 per cent, pure glycerine.

3. Complete as described above for ordinary blood-serum, sec-
tions 5 to 7.

NOTE. — Different percentages of glycerine — from 4 per cent, to
8 per cent. — are used for special purposes. Five per cent, is that
usually employed.

Glycerinated Potato. —

1. Prepare ordinary potato wedges (vide page 174, sections
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.

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.

14

210 SPECIAL MEDIA

Animal Tissue Media (Frugoni). —

1. Take a number of sterile test-tubes 16X3 or 4 cm-> P

with cotton wool, and into each insert a 2 cm. length of stout glass
tubing (about i cm. diameter); fill in glycerine (6 per cent.)
bouillon to the upper level of the piece of glass tubing. Sterilise
in the steamer at 100° C. for twenty minutes on each of three suc-
cessive days.

2. Kill a small rabbit by means of chloroform vapour.

3. Under strictly aseptic precautions remove the lungs, liver and
other solid organs and transfer them to a sterile double glass dish.

4. With the help of sterile scissors and forceps divide the organs
into roughly rectangular blocks 3X1.5X1 cm.

5. Pour into the dish a sufficient quantity of sterile glycerine
solution (6 per cent, in normal saline), cover, and allow to stand
for one hour.

6. Introduce a block of tissue into each tube so that it rests
upon the upper end of the piece of glass tubing. (The surface of
the tissue will now be kept moist by capillary attraction and
condensation) .

7. Sterilise in the autoclave at 120° C. for thirty minutes.

8. Cap the tubes and store them in the ice chest for future use.
Tissues obtained at postmortems can also be used after pre-
liminary sterilisation by boiling or autoclaving.

Media for the Study of Special Cocci.

Diplococcus Gonorrhoea.
Ascitic Bouillon (Serum Bouillon). —

1. Collect ascitic fluid (pleuritic fluid, hydrocele fluid, etc.), by
aspiration directly into sterile flasks, under strictly aseptic pre-
cautions.

2. Mix the serum with twice its bulk of sterile nutrient bouillon
(vide page 163).

3. If considered necessary (on account of the presence of
blood, crystals, etc.), filter the serum bouillon through porcelain
filter candle.

4. Tube, and sterilise in the water bath at 56° C. for half an
hour on each of five consecutive days.

5. Incubate at 37° C. for forty-eight hours and eliminate con-
taminated tubes. Store the remainder for future use.

Serum Agar (Heiman). —

i. Prepare nutrient agar (vide page 167), to following formula:

Agar 2.0 per cent.

Peptone 1.5 per cent.

Salt 0.5 per cent.

Meat extract quantum sufficit.

SERUM AGAR 211

2. Make reaction of medium + 10.

3. Filter; tube in quantities of 6 c.c.

4. Sterilise as for nutrient agar.

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 (Wertheimer) . —

1. Prepare nutrient agar (vide page 167), to the following
formula :

Agar 2.0 per cent.

Peptone 2.0 per cent.

Salt 0.5 per cent.

Meat extract quantum sufficit.

2. Make reaction of medium + 10.

3. Filter; tube in quantities of 5 c.c.

4. Sterilise as for nutrient agar.

5. After the last sterilisation cool to 42° C., then add 5 c.c.
sterile blood-serum from human placenta (sterilised, if necessary,
by the fractional method) to each tube; slope the tubes.

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 to stand in the ice-chest for twelve hours to sediment.

2. Decant 1000 c.c. of the clear fluid into a measuring cylinder
and transfer to sterile litre flask.

3. Add 0.5 c.c. dekanormal NaOH solution for every 100 c.c.
serum (i.e., 5.0 c.c.), and mix thoroughly.

4. Heat in the steamer for twenty minutes.

5. Weigh out 15 grammes agar, emulsify in a separate vessel
with 200 c.c. of the alkaline fluid previously cooled to about
20° C., and then add to the remainder of the fluid in the flask.

6. Bubble live steam through the mixture for twenty minutes
to dissolve the agar.

7. Filter through papier Chardin, using a hot-water funnel.

8. Weigh out glucose 10 grammes ( = 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 as for nutrient agar.

212 SPECIAL MEDIA

Serum Agar (Libman). —

1. Prepare nutrient agar (vide, page 167) using, however, 1.5
per cent, peptone (that is 15 grammes per litre instead of 10
grammes) .

2. Adjust the reaction to o (i. e., neutral to phenolphthalein) .

3. Filter and transfer 1000 c.c. liquefied medium to a sterile
flask.

4. Weigh out dextrose 20 grammes and dissolve in the fluid
agar.

5. Tube in quantities of 6 c.c.; and sterilise in the steamer at
100° C. for thirty minutes on each of three consecutive days.

6. After the third sterilisation cool to 42° C. and add to each
tube 3 c.c. of sterile hydrocele fluid, ascitic fluid or pleuritic effu-
sion (previously sterilised, if necessary, by the fractional method) ;
allow the tubes to solidify in a sloping position.

7. When solid, incubate at 37° C. for forty-eight hours, and
eliminate any contaminated tubes. Store the remainder for
future use.

Egg=albumen, Inspissated. —

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).

30. 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 inspissator.

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 solu-
tion 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 sterilised
forceps to a Petri dish or large capsule.

ASCITIC FLUID AGAR 213

6. Sterilise in the steamer at 100° C. for twenty minutes on
each of three consecutive days.

Egg Albumin Broth (Lipschuetz).—

1. Weigh out

Egg albumin (extra fine powder, Merck) . . 4 grammes

and place in a 2 -litre flask with a number of sterile glass beads.

2. Measure out distilled water 200 c.c. into a half-litre flask
and warm to 37° C. in the incubator.

3. Add the water to the flask containing the albumin and beads
and dissolve by shaking.

4. Add -^ NaOH, 40 c.c. Allow the mixture to stand for
thirty minutes with frequent shaking.

5. Filter through Swedish filter paper.

6. Sterilise by boiling two or three times at intervals of two
hours.

7. Add ordinary nutrient bouillon 600 c.c.

8. Fill into small Erlenmeyer flasks in quantities of 50 c.c.

9. Incubate for forty-eight hours at 37° C. — discard any con-
taminated flasks and store the remainder for future use.

Egg Albumin Agar. —

1. Prepare egg albumin solution as above 1-6.

2. Liquefy and measure out ordinary nutrient agar 600 c.c. and
add to the egg albumin solution (in place of the nutrient broth).

3. Complete as above 8-9.

Diplococcus Meningitidis Intracellularis.

Ascitic Fluid Agar (Wassermann) Synonym N=as=gar (Mervyn
Gordon) .

1. Liquefy and measure out into a sterile flask:

Nutrient agar 600 c.c.

2. Measure out into a half litre flask

Distilled water 210 c.c.

and add to it

Ascitic fluid 90 c.c.

Nutrose 6 grammes

3. Heat over a bunsen flame, shaking constantly until the fluid
boils, and the nutrose is dissolved,

4. Add the nutrose ascitic solution to the fluid agar.

5. Heat in the steamer for thirty minutes, then filter.

6. Tube and sterilise as for nutrient agar.

NOTE. — The finished medium in this case measures 900 c.c. only
since inconvenient fractions would be introduced in making up to
one litre exactly.

214 SPECIAL MEDIA

D^plococcus Pneumonias.

Blood Agar (Washbourn).—

1. Melt up several tubes of nutrient agar (vide page 167) 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 thoroughly 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 instruments.

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. 13, 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 slanting 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.

Media for the Study of Mouth Bacteria Generally.

Potato Gelatine (Goadby).—

1. Prepare glycerine potato broth (see page 203, sections i to 5).

2. Add 10 per cent, gelatine to the potato decoction and
bubble live steam through the mixture for ten minutes.

3. Estimate the reaction; adjust the reaction of the medium
to +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.

Media for the Study of Protozoa.

Tissue Medium (Noguchi). — For spirochcetes (cultivations must be
grown anaerobically} .

1. Plug and sterilise test-tubes 20 X 2 cm.

2. Kill a small rabbit with chloroform vapour. Open the abdo-

TISSUE MEDIUM 215

men with all aseptic precautions, remove kidneys and testicles and
transfer to a sterile glass dish. Cut up the organs with sterile
scissors into small pieces — say 4 millimetre cubes. The four organs
should yield from 25 to 30 pieces of tissue.

3. Drop a small piece of sterile tissue into the bottom of each
sterilised tube.

4. Take a flask containing about 400 c.c. nutrient agar (+ 10
reaction) , liquefy the medium by heat and cool in a water bath
to 50° C.

5. Add 200 c.c. ascitic or hydrocele fluid (horse or sheep serum
may be employed, but' is not so good) to the liquid agar and mix
carefully to avoid formation of air bubbles.

6. Fill about 20 c.c. of the ascitic agar into each of the sterilised
tubes which already contains a piece of sterile rabbit's tissue, stand
all the tubes upright in racks or a j ar, and allow agar to set.

7. After solidification pour sterile paraffin oil on the surface of
the medium in each tube to the depth of 3 centimetres.

8. Incubate tubes at 37° C. for several days and discard any
which prove to be contaminated.

9. Store such tubes as are sterile for future use.

XIII. INCUBATORS.

AN incubator (Fig. 113) consists essentially of a cham-
ber 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. 113. — Incubator-

wooden casing. The water in the jacket is heated
by gas or electricity and maintained at some constant
temperature by a thermo-regulator. The cellular
incubator (Fig. 114) which was made for me1 some
years ago is of the greatest practical utility. Here the

1 Made by the firm of Chas. Hearson & Co., 235 Regent St., London, W.

2l6

THE RMO-REG ULATORS

2I7

central cavity is subdivided by five double -walled
partitions (in which water circulates in connection with
the water tanks at the top and base of the incubator)
and again by iron shelves to form twenty-four pigeon
holes. Into each of these slides an iron drawer 35 cm.
long X 12 cm. wide X 22 cm. high forming a self-
contained incubator. The drawer is fitted with a
wooden form to which is fixed a handle and a numbered
label. The thermo-regulating apparatus is the well-
known Hearson capsule.

Fio. 114. — Cellular incubator.

Two incubators at least are required in the labora-
tory, for the cultivation of bacteria the one regulated
to maintain a temperature of 37° C., and known as
the "hot" incubator; the other, 20° C. to 22° C., and
known as the "cool" or "cold" incubator.

Two other incubators, regulated to 42° C. and 60° C.
respectively, whilst not absolutely, necessary very soon
justify their purchase.

Thermo regulators. — The thermo-regulator is the

2l8 INCUBATORS

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 mercury within a glass
bulb ; in the other, by alterations in
the position of the walls of a metal-
lic capsule containing a fluid, the
boiling-point of which corresponds
to the temperature at which the in
cubator is intended to act. They
are:

(a) Reichert's (Fig. 115), consists of
a bulb containing mercury which is
FIG. 115.— Reichert's to be suspended in the medium,
thermo-regulator. whether air or water, the temperature
of which it is desired to regulate. Gas enters at A, and
passes out to the jet by B. As the temperature rises
the mercury expands and cuts off the main gas supply.
As the temperature falls the mercury contracts and re-
opens 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) Hear son's capsule regulator consists of a metal cap-
sule hermetically sealed and filled with a liquid which
boils at the required temperature, this is adjusted in the
interior of the incubator. Soldered to the upper side of
the capsule is a thick piece of metal having a central

THERMO-REGULATORS

cup to receive the lower end of a rigid rod, through
which the movements of the walls of the capsule are
transmitted to the gas valve fixed outside the incubator.

The gas valve or governor is shown in figure 1 16. 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 rigid rod
which enters the socket below the screw P.

The construction of the valve is such that, when-
ever the short arm 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, however, a very small portion of
gas passes from A to C, through an aperture inside the
valve, the size of which aperture can be adjusted by

FIG. 116. — Capsule thermo-regulator.

the screw needle S, hence the gas flame below the incu-
bator is never extinguished.

The expansion of the metal walls of the capsule,
which takes place upon the boiling of its contents, pro-
vides the motive force, transmitted through the rigid
rod to raise the long arm of the lever B D, and as this
expansion only takes place at a predetermined temper-
ature, the lever will only be acted upon when the criti-
cal temperature is reached, no sensible effect being pro-
duced at even i° C. below that at which the capsule is
destined to act.

W is a weight sliding on the lever rod D ; by increas-
ing the distance between the weight and the fulcrum

220 INCUBATORS

of the lower increased pressure is brought to bear upon
the walls of the capsule with the result that the boiling-
point of the liquid in the capsule is slightly raised, and
a range of about two degrees can thus be obtained
with any particular capsule.

XIV. 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 3 7° C. (that is, in the ' ' hot " incubator) .
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 em-
ployed for all media.

Tube cultivations are usually packed in the incubator
in small tin cylinders, such as those in which American
cigarettes are sold, or in square tin boxes. Beakers or
tumblers may be used for the same purpose, but being
fragile are not so convenient. Metal test-tube racks,
long enough to just fit into the interior of the incuba-
tor and each accommodating two rows of tubes, are
also exceedingly useful.

221

222 METHODS OF CULTIVATION

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.

(b) 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. Morbid material previously collected (vide page

373)-

3 . Fluids, tissues, etc. , or from 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 containing

FIG. 117. — Inoculating tubes, seen from the front.

the cultivation arid 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

SOLID MEDIA

223

the tubes as nearly horizontal as is possible without
allowing the fluid in the bouillon tube to reach the
cotton-wool plug (Fig. 117).

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.

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
remove a small portion of the growth; withdraw the
loop from the tube, keeping the infected side of the
loop downward.

7. Pass the loop into the bouillon. tube almost down
to the level of the fluid, reverse the loop so that the
infected side faces upward, 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 :

1. Obb'que tube or slanted tube (Fig. 118), in which
the medium has been allowed to solidify whilst the tube
was retained in an inclined position, so forming an
extensive surface of medium extending from the bot-
tom of the tube almost to its mouth.

This is employed for " streak" or "smear" cultiva-
tions (Strichcultur) .

2. Straight tube (Fig. 119), in which the medium

1 See also method of opening and closing culture tubes, pages 74-76.

224 METHODS OF CULTIVATION

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 platinum loop (or
spatula). Open the tubes and charge the loop as in
previous inoculation.

2. Pass the infected loop to the bottom of the tube
to be inoculated and draw it, as lightly as possible,
along the centre of the surface of the medium, ter-
minating 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 cul-
ture, but rub the infected loop all over the surface of
the medium, instead of restricting the inoculation to
a narrow line.

NOTE. —Gelatine and agar oblique tubes should be freshly
"slanted" before use.

Stab Culture. —

1. 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
possible in the axis of the cylinder of medium. Then
withdraw the needle.

3. Replug the tubes. Sterilise the platinum needle.

TUBE CULTURES

225

4. Label the newly planted tube and incubate.

NOTE. — When gelatine is stored for some time the upper
surface of the cylinder becomes concave owing to evaporation.
Tubes showing this appearance should be liquefied and again
allowed to set before use for stab culture, otherwise when the
needle enters the medium, the surface tension will cause the
gelatine cylinder to%split.

FIG. 118. — Sloped or slant-
ed medium for streak or
smear culture.

FIG. 119. — Straight tube.

Shake Culture. —

1. Liquefy a tube of nutrient gelatine (or agar, or
other similar medium), by heating in a water-bath
(Fig. 121).

2. Inoculate the liquefied medium and label it, etc.,
precisely as if dealing with a tube of bouillon.

15

226

METHODS OF CULTIVATION

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.

Esmarch's Roll Cultivation. —

1. Liquefy three tubes of gelatine by heat.

2. Prepare three dilutions of the inoculum (as described for
plate cultivations, page 228, steps 4 to 7).

3. Roll the tubes, held almost horizontally, in a groove made
in a block of ice, until the gelatine has set in a thin film on the
inner surface of tube (Fig. 120) ; or under the cold-water tap.

FIG. 1 20. — Esmarch's roll culture on block of ice.

In order that the medium may adhere firmly to the glass, the
agar used for roll cultivation 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 obsolete in modern laboratories and are
merely mentioned for the benefit of students, since examiners who
are interested in the academic and historical aspects of bacteriology
sometimes expect candidates to be acquainted with the method of
preparing them.

The Preparation of Plate Cultures.

If a small number of bacteria 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
surface and allowed to solidify, each individual micro-
organism becomes fixed to a certain spot and its
further development is restricted to the vicinity of this
spot. After a variable interval the growth of this

227

organism becomes visible to the naked eye as a " colony."
This is the principle upon which the method of plate cul-
tivation is based and its practice enables the bacteri-
ologist to study the particular manner of development
affected by each species of microbe when growing
(a) unrestricted upon the sur-
face of the medium, (b) in the
depths of the medium. The
method itself is as follows :

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) previously 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 introduced.

6. Tube of cultivation to be planted
from.

7. Platinum loop.

8. Bunsen burner.

9. Grease pencil.

Method of " Pouring" Plates.— water baf fo.r ™eltins *

agar and gelatine previous to

Plapp tVip alfl d rl* Vi rm tVip slanting them; or to making
• • •* "shake cultures or pouring

levelling tripod (Figs. 122, 123); plates,
if gelatine plates are to be poured fill the dish with
ice water — gelatine solidifies so slowly that it is
necessary to hasten the process ; if agar is to be used
fill with water at 50° C. — agar sets almost immediately
at the room temperature and by slightly retarding the
process lumpiness is avoided ; cover the dish 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.

FIG. 121. — Handy form of

228

METHODS OF CULTIVATION

This leveling is an important matter since the de-
velopment of a colony is to some extent proportionate
to the supply of medium available for its nutrition..
Thus in a "smear" on sloped tube culture, the colonies
at the upper part of the medium are stunted and small

FIG. 122. — Plate-levelling stand.

but increase in size and luxuriance of growth the
nearer they approach to the bottom of the tube,
where there is the greatest depth of medium.

3. Place three sterile Petri dishes in a row on the
surface of the glass plate and number them i, 2, and
3, from left to right.

FIG. 123. — Plate-levelling stand, side view.

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.

5. Add one loopful of the inoculum to tube No. i,

PLATES

229

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-
lum throughout the medium; avoid jerky movements,
as these cause bubbles of air to form in the medium.

FIG. 124. — Mixing emulsion for plates.

The knack of mixing evenly without producing air
bubbles, is not always easily acquired, by this method,
An alternative plan is to hold the inoculated tube
vertically upright between the opposed palms and to
rotate it between them by rapid backward and forward
movements of the two hands (Fig. 124).

FIG. 125. — Pouring plates.

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
liquefied medium from tube No. 2 to tube No. 3, and
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,

230 METHODS OF CULTIVATION

elevating the bottom of the tube, pour the liquefied
medium into the Petri dish, to form a thin layer.
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, also the date; the number of
the dilution having been previously indicated (step 3).

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.

Agar plates are poured in a similar manner, but the
agar must be melted in boiling water and then allowed
to cool t045°C. or42°C. in a carefully regulated water-
bath before being inoculated, and the entire process
must be carried out very rapidly, otherwise the agar
will have solidified before the operation is completed.

NOTE.— In pouring plates, since tube No. i (for the first dilu-
tion) rarely gives a plate that is of any practical value it is fre-
quently replaced by a tube of bouillon or sterile salt solution, and
in such case plate No. i is not poured.

Surface Plates.—

This method of pouring what may be termed " whole "
plates (since colonies may appear both on the surface
and in the depths of the medium) is essential to the
accurate study of the formation of colonies under

SURFACE PLATES

23I

various conditions, but when the main object of the
separation of the bacteria is to obtain subcultivations
from a number of individual bacteria, "surface" plates
must be prepared, since here colony formation is
restricted to the surface of the medium. The method
adopted varies slightly according to whether the
medium employed is gelatine or agar, or one of the de-
rivatives or variants of the latter.

(a) Gelatine Surface Plates.—

1. Liquefy three tubes of nutrient gelatine.

2. Pour each tube into a separate Petri dish and al-
low it to solidify. Then turn each plate and its cover
upside down.

3. When quite cold .
raise the bottom of plate

i , revert it and deposit a
drop of the inoculum

/ , ,1 n • 1 i, FIG. 126. — Surface plate spreader.

(whether a fluid culture

or an emulsion from solid culture) upon the surface
of the gelatine with a platinum loop — close to one side
of the plate ; replace the bottom half of the Petri dish
in its cover.

4. Take a piece of thin glass rod, stout platinum wire
or best of all a piece of aluminium wire (say 2 mm.
diameter) about 28 cm. long. Bend the terminal 4 cm.
at right angles to the remainder, making an L-shaped
rod (Fig. 126). Sterilise the short arm and adjacent
portion of the long arm, in the Bunsen flame, and allow
it to cool.

5. Now raise the bottom of the Petri dish in the left
hand, leaving the cover on the laboratory bench, and
holding it vertically, smear the drop of inoculum all
over the surface of the gelatine with the short arm of
the spreader by a rotatory motion, (Fig. 127). Replace
the dish in its cover.

6. Raise the bottom of plate 2 and rub the infected

232 METHODS OF CULTIVATION

spreader all over the surface of the gelatine — then go
on in like manner to the third plate in the series.

7. Sterilise the spreader.

8. Label and incubate the plates.

After incubation, plate No. i will probably yield an
enormous number of colonies; plate 2 will show fewer
colonies, since only those bacteria adhering to the rod

FIG. 127. — Spreading surface plate.

after rubbing over plate i would be deposited on its
surface, and by the time the rod reached plate 3 but
very few organisms should remain upon it. So that
the third plate as a rule will only show a very few
scattered colonies, eminently suitable for detailed
study.

(b) Agar Surface Plates. —

1. Liquefy three tubes of nutrient agar — nutrose
agar or the like.

2 . Pour each tube into a separate Petri dish and allow
it to solidify.

3 . When quite solid invert each dish, raise the bottom
half and rest it obliquely on its inverted cover (Fig. 128)
and place it in this position in an incubator at 60° C.
for forty- five minutes (or in an incubator at 42° C. for

HANGING-DROP CULTURES 233

two hours) . This evaporates the water of condensation
and gives the medium a firm, dry surface.

4. On removing the plates from the incubator close
each dish and place it — still upside

down — on the laboratory bench.

5. Inoculate the plates in series of
three, as described for gelatine sur-
face plates 3-8. FlG> ing sur.

face plate of agar.

Hanging=drop Cultivation.

Apparatus Required. —

Hanging drop slides.

Caver-slips.

Section rack (Fig. 75).

Blotting paper.

Bell glass to cover slides.

Original culture.

Tubes of broth, or liquefied gelatine or agar.

Forceps.

Platinum loop.

Bunsen burner.

Grease pencil.

Sterile vaseline.

Lysol.

(a) Fluid Media. —

1. Prepare first and second dilutions of the inoculum
as directed for plate cultivations (vide pages 228-229,
sections 4 to 6) , substituting tubes of nutrient broth
for the liquefied gelatine.

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

234 METHODS OF CULTIVATION

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. 53).

(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, when the circular cell on the
hanging-drop slide should be replaced by a rectangu-
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 and numbered to facilitate
the subsequent identification of the colonies which are
observed to develop from solitary germs.

HANGING-BLOCK CULTURE 235

Hanging-block Culture (Hill).—

Apparatus required: As for hanging-drop cultivation
with the addition of a scalpel.

Carry out the method as far as possible under cover
of a bell glass, to avoid aerial contamination.

1. Liquefy a tube of nutrient agar (or gelatine) and
pour into a Petri dish to the depth of about 4 mm. and
allow to set.

2. With a sharp scalpel cut out a block some 8 mm.
square, from the entire thickness of the agar layer.

3. Raise the agar block on the blade of the scalpel
and transfer it, under side down, to the centre of a
sterile slide.

4. Spread a drop of fluid cultivation (or an emulsion
of growth from a solid medium) over the upper surface
of the agar block as if making a cover-slip film.

5 . Place the slide and block covered by the bell glass
in the incubator at 37° C. for ten minutes to dry
slightly.

6. Take a clean dry sterile cover- slip in a pair of for-
ceps, and with the help of a second pair of forceps lower
it carefully on the inoculated surface of the agar (avoid-
ing air bubbles) , so as to leave a clear margin of cover-
slip overlapping the agar block.

7. Invert the preparation and with the blade of the
scalpel remove the slide from the agar block.

8. With a platinum loop run a drop or two of melted
agar around the edges of the block. This solidifies at
once and seals the block to the cover-slip.

9. Prepare a sterile hanging-drop slide, and smear
hard vaseline or melted white wax on the rim of the
metal cell.

10. Invert the cover-slip with the block attached on
to the hanging-drop slide, and seal the cover-slip firmly
in place.

11. Observe as for hanging-drop cultivations.

236 METHODS OF CULTIVATION

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., cultivation
in vacua.

(c) Absorption of oxygen from the air in contact with
the cultivation by means of pyrogallic acid rendered alka-
line with caustic soda — i. e., cultivation in an atmos-
phere 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, pages 189-190).

Further, it is advisable to seal all joints between
india-rubber stoppers and tubulures or the mouths
of the tubes with melted paraffin; glass stoppers and
taps should be lubricated with resin ointment or a
mixture of beeswax i part, olive oil 4 parts.

ANAEROBIC CULTURES

237

(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. — 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 exclud-
ing 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.

Method III (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 IV (Roux's Biological Method).—

1. Plant a deep stab, as in method I.

2. Pour a layer, i or 2 cm. deep, of broth cultivation
of a vigourous aerobe — e. g., B. aquatilis sulcatus or B.

238

METHODS OF CULTIVATION

prodigiosus — upon the surface of the medium; or an
equal depth of liquefied gelatine, which is then inocu-
lated 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 consequently remain
in an anaerobic condition.

(B) Method V.-

i . Prepare tube or flask cultivations in the usual way.

2. Replace the cotton- wool plug by
an india-rubber stopper perforated 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. 129).
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 WulfFs bottle
fitted as a wash-bottle and containing
sulphuric acid.

4. Exhaust the air from the culture
vessel.

5- Before disconnecting the appa-
ratus, seal the glass tube from the cul-
at the constriction, using the blowpipe

Fl°*

ture vessel
flame.

6. Incubate.

(C) Method VI (Buchner's Method).—

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. 130).

Pyrogallic acid in compressed tablets each containing i gram.
Dekanormal solution of caustic soda.

ANAEROBIC CULTURES

239

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 stopper from the caustic soda bottle.

4. Drop one of the pyrogallic acid tablets into the
Buchner 's tube (roughly, use i gramme pyrogallic acid
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. The pyrogallic tablet acts as a

buffer and prevents damage to either
the inoculated tube or the Buchner's
tube even should it be slipped in hur-
riedly.

7. Fit the india-rubber stopper tightly
into the mouth of the Buchner 's tube.

The pyrogallic acid tablet dissolves
slowly in the soda solution and its oxida-
tion proceeds very slowly at first so that
ample time is available when this method
is adopted.

8. Restopper the caustic soda bottle.

9. Place Buchner's tube in a wire sup-
port, and incubate.

the

FIG. 130. — Buch-
ner's tube.

Method VII (Wright 's Method) .-

1. Prepare tube cultivation in
usual way.

2 . Cut off that portion of the cotton- wool plug project-
ing above the mouth of the tube with scissors, then
push the plug into the tube for a distance of 2 or 3 cm.

1 If compressed tablets of pyrogallic acid cannot be obtained make up a
stock "acid pyro" solution

Pyrogallic acid, 10 grammes

Hydrochloric acid, 1.5 c.c.

Distilled water, 100 c.c.
and at step 4, run in 10 c.c. of the solution.

240

METHODS .OF CULTIVATION

3 . By means of a pipette drop about i c.c. of pyrogal-
lic acid 10 per cent, aqueous solution on to the plug.
It will immediately 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.

Method VIII (McLeod's Method).—

Apparatus and Solutions Required. —

McLeod's plate base (a hollow glazed earthenware disc 9 cm. in
diameter and 2 cm. deep: the upper surface is pierced by a central
hole, 2 cm. in diameter, giving access to the interior, the lower
part of which is divided into two by a low partition. A shallow
groove encircles the upper surface near to the edge) .

FIG. 131. — McLeod's anaerobic plate base with half petri dish inverted

in situ

Plasticine.

Pyrogallic acid (i gramme) compressed tablets.
Sodic hydroxide (0.4 gramme) compressed tablets.
Wash bottle of distilled water.

Surface plates of one or other agar medium (in petri dishes of
8 cm. diameter).
Surface plate spreader.

METHOD. —

i. Roll out a long cylinder of plasticine and fit it
into the groove on the upper surface of the earthenware
base.

ANAEROBIC CULTURES 241

2. Place a tablet of pyrogallic acid in one division
of the interior of the plate base, and two tablets of
sodic hydroxide in the other.

3. Prepare surface plate culture of the organism to
be cultivated.

4. Run a few cubic centimetres of distilled water
into that division of the plate base containing the
sodic hydroxide.

5. Invert the bottom half of the surface plate over
the plate base and press its edges firmly down into the
plasticine filling the groove.

6. Label and incubate.

(D) Method IX.-

Apparatus Required. —

Small Ruffer's or Woodhead's flask (Fig. 33).

Sterile india-rubber stopper.

India-rubber tubing.

Glass tubing.

Metal screw clips.

Cylinder of compressed hydrogen ; or hydrogen gas 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.

5. Connect up the india-rubber tubing on one of
the side tubes with a cylinder of compressed hydrogen

16

242

METHODS OF CULTIVATION

FIG. 132. — Kipp's hydrogen apparatus, (a) connected up to two washing
bottles containing (b) lead acetate 10 per cent, solution, to remove H2S
and (c) silver nitrate solution to remove AsH3. A third washing bottle
containing pyrogallic acid 10 per cent, solution, rendered alkaline, to
remove any trace of oxygen, is sometimes introduced.

FIG. 133. — Improved gas apparatus; the metal is contained in a perforated
glass tube which is submerged in acid when the triangular bottle is upright
(a), but is above the level of the liquid when the bottle is turned on its
side (6).

ANACROBIC CULTURES

243

(or the delivery tube of a Kipp's Fig. 132 or other
hydrogen apparatus, Fig. 133), 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
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.

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 (or metallic mercury).

METHOD. —

1 . Place the leaden cross inside the glass dish, resting
on the bottom.

2. Prepare the cultivations in the usual way.

3. Place the tube cultivations in a glass battery jar
(or the plate cultivations on a metal frame) , resting on
the centre of the leaden cross.

4. Cover the cultivations with the bell jar.

5. Adjust the U-shaped pieces of glass tubing in a
vertical position on opposite sides of the bell jar, one
arm of each inside the jar, the other outside. These
tubes are best held in position by embedding the U-

244

METHODS OF CULTIVATION

shaped bends in two lumps of plasterine stuck on the
bottom of the glass dish. Fix a short length of rubber
tubing clamped with a metal clip to each of the out-
side arms (Fig. 134).

6. Fill the glass dish with glycerine or metallic
mercury to a depth of about 5 cm.

FIG. 134. — Botkin's apparatus.

7. Connect up one U-shaped tube with the hydrogen
cylinder (or gas apparatus) 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. 135).
Or, jar for tube cultivations (Fig. 136).
Lubricant for stopper of jar.
Rubber tubing.
Cylinder of compressed hydrogen.

ANAEROBIC CULTURES

245

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.

FIG. 135. — Novy's jar for
plate cultivations.

FIG. 136. — Novy's jar for
tube cultivations.

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.

6. When the air is completely displaced by hydrogen,
turn the handle of the stopper at right angles to the
line of entry and exit tubes; this seals the orifice of
both tubes.

7. Disconnect the gas apparatus and incubate.

(E) Method XII (Bulloch's Method) .-

Apparatus Required. —

Bulloch'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.

246 METHODS OF CULTIVATION

Pyrogallic acid tablets.
Cylinder of compressed hydrogen.
Geryk or other air pump.
Rubber pressure tubing.
10 c.c. pipette.
Glass tubing.

Dry granulated caustic soda or compressed tablets each con-
taining 0.4 grammes sodic hydroxide.
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 sufficient number of pyrogallic acid
tablets at one side of the glass dish (i. e., i tablet for
each 100 cubic centimeters air capacity of the bell jar).
Place a small heap of dry granulated soda (or half a
dozen tablets of sodic hydroxide) by the side of the
pyro tablets.

4. Smear the flange of the bell jar with resin oint-
ment 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 tablets. Lubricate the
two stop-cocks with resin ointment (Fig. 137).

5. Connect up the short tube a with the gas-supply
by means of rubber pressure tubing and open both stop-
cocks.

6. Connect a long, straight piece of glass tubing to
the long tube b by means of a piece of rubber tubing
interposing a screw clamp : and collect samples of the
issuing gas from time to time and test.

7. When the air is displaced, shut off the stop-cock
of the entry tube, then that of the exit tube b. Screw
down the clamp and remove the glass tube from the
rubber connection 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 with two or three

ANAEROBIC CULTURES

247

strokes of the air pump, aspirate a small quanity of
gas, so creating a slight vacuum. Then shut off the
stop-cock and disconnect the air pump.

9. Fill the 10 c.c. bulb pipette with water; insert its
point into the rubber tubing on the long tube b as far as
the screw clamp. Open the screw clamp and run in
water until stopped by the internal pressure. Shut
off stop-cock. (The water dissolves the soda and

FIG. 137. — Bulloch's jar.

pyrogallic acid converting the latter into alkaline
pyro. and so bringing its latent capacity for oxygen
into action) .

10. Reverse the tubes from the tubulures so that
they meet, out of harm's way, over the top of the
bell glass ; again see that all joints are tight and trans-
fer the apparatus to the incubator.

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.

XV. 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
cultivations of various organisms previously isolated
and identified and supplied to the student in a state of
purity. A cultivation which comprises the prog-
eny of a single cell is termed a "pure culture"; one
which contains representatives of two or more species

FIG. 138. — Haematocytometer cell, showing, a, section through the centre of
the cell, and b, a magnified image of the cell rulings.

of bacteria is spoken of as an "impure, " or "mixed"
"cultivation," and it now becomes necessary to indi-
cate 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 laboratory
cultivation, or is contained in pus and other morbid
exudations, infected tissues, or water or food-stuffs.

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-

248

BIOLOGICAL DIFFERENTIATION 249

cytometer, Fig. 138), and diluting the fluid by the addi-
tion of sterile water or bouillon until a given volume
(usually i c.c.) of the dilution contained but one
organism. By planting this volume of the fluid into
several tubes or flasks of nutrient media, it occasion-
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,
more reliable and convenient, and in those methods
selected for description here, the segregation and isola-
tion of the required bacteria may be effected —

A. By Mechanical Separation.

i . By surface plate cultivation :

(a) Gelatine.

(b) Agar.

(c) Serum agar.

(d) Blood agar.

(e) Hanging-drop or block.
[2. By Esmarch's roll cultivation :

This archaic method (see page 226) is no longer
employed for the isolation of bacteria.]

3. By serial cultivation.

B. By Biological Differentiation.

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

250 METHODS OF ISOLATION

either the first or the third method will be found most
convenient, affording as each of them does an opportun-
ity for the simultaneous isolation of several or all
of the varieties of bacteria present in a mixture.

1. Surface Plate Cultivations. —

(a) Gelatine (vide page 164).

(b) A gar (vide page 167).

(c) Alkaline serum agar (vide page 211).

These plates are prepared in a manner precisely
similar to that adopted for nutrient gelatine and
agar surface plates (vide pages 231-233).

(d) Serum Agar. —

1. Melt three tubes of nutrient agar, label them i, 2,
and 3, and place them, with three tubes of sterile
fluid serum, also labelled la, 20,, and 30, 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. Take serum tube No. la 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.

3. Mix thoroughly and pour plate No. i secundum
artem.

4. Treat the remaining tube of agar and serum in a
similar fashion, and pour plates Nos. 2 and 3.

5 . Dry the serum agar plates in the incubator running
at 60° C. for one hour (see page 232).

6. Inoculate the plates in series as described for
gelatine surface plates (page 231).

(e) Blood Agar, Human —

i . Melt a tube of sterile agar and pour it into a sterile
plate; let it set.

SERIAL CULTIVATIONS 251

2. Collect a few drops of human blood, under all
aseptic conditions, in a sterile capillary teat pipette.

3. Raise the cover of the Petri dish very slightly,
insert the extremity of the capillary pipette, and deposit
the blood on the centre of the agar surface. Close the
dish.

4. Charge a platinum loop with a small quantity of
the inoculum. Raise the cover of the plate, introduce
the loop, mix its contents with the drop of blood,
remove the loop, close the dish and sterilise the loop.

5. Finally smear the mixture over the surface of the
agar with a sterilised L-shaped rod.

6. Label and incubate.

(If considered necessary, two, three, or more similar
plates may be inoculated in series.)

(/) Blood Agar, Animal. —

When preparing citrated blood agar (page 171) it is
always advisable to pour several blood agar tubes into
plates, which can be stored in the ice chest ready for
use at any moment for surface plate cultures.

(g) Hanging-drop or block culture, (vide page 233).

3. Serial Cultivations. — These are usually made upon
agar or blood-serum, although gelatine may also be used.
The method is as follows :

1. Take at least four " 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
smearing the contents of the loop over the surface of
the medium.

4. Without sterilising or recharging the loop, inocu-

252 METHODS OF ISOLATION

late tube No. 2, by making three parallel streaks from
end to end of the slanted surface.

5. Plant the remainder of the tubes in the series as
" smears" like tube No. i.

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. Toward 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 neighbouring colonies.

"Working" up Plates.—

Having succeeded in obtaining a plate (or tube
cultivation) in which the colonies are well grown and
sufficiently separated from each other, the process of
" working up," " pricking out," or " fishing " the colonies
in order to obtain subcultures in a state of purity from
each of the different bacteria present must now be
proceeded with.

Occasionally it happens that this is quite a simple
matter. For example, the original mixed cultivation
when examined microscopically was found to contain a
Gram positive micrococcus, a Gram positive straight
bacillus and a Gram negative short bacillus. The third
gelatine plate prepared from this mixture, on inspection
after four day's incubation, showed twenty-five colonies
— seven moist yellow colonies, each sinking into a shal-
low pit of liquefied gelatine, fourteen flat irridescent
filmy colonies, and four raised white slimy colonies.
A film preparation (stained Gram) from each variety ex-
amined microscopically showed that the yellow liquefy-
ing colony was composed of Gram positive micrococci ;
the flat colony of Gram positive bacilli and the white
colony of gram negative bacilli. One of each of these
varieties of colonies would be transferred by means of

FISHING COLONIES 253

the sterilised loop to a fresh gelatine culture tube, and
after incubation the growth in each subculture would
correspond culturally and microscopically with that of
the plate colony from which it was derived, — the object
aimed at would therefore be achieved.

Usually, however, the colonies cannot be thus readily
differentiated, and unless they are "worked up" in an
orderly and systematic manner much labour will be
vainly expended and valuable time wasted. The fol-
lowing method minimises the difficulties involved.

(A) Inspection.

a. Without opening the plate carefully study the
various colonies with the naked eye, with the assistance
of a watchmaker's lens or by inverting the plate on
the stage of the microscope and viewing with the
i -inch objective through the bottom of the plate and
the layer of medium.

b. If gross differences can be detected mark a small
circle on the bottom of the plate around the site of
each of the selected colonies, with the grease pencil.

c. If no obvious differences can be made out choose
nine colonies haphazard and indicate their positions by
pencil marks on the bottom of the plate.

(B) Fishing Colonies. —

a. Take a sterile Petri dish and invert it upon the
laboratory bench. Rule two parallel lines on the
bottom of the dish with a grease pencil, and two more
parallel lines at right angles to the first pair — so dividing
the area of the dish into nine portions. Number the
top right-hand portion i, and the central bottom por-
tion 8 (Fig. 139). Revert the dish. The numbers i
and 8 can be readily recognised through the glass and
by their positions enable any of the other divisions to
be localised by number. This is the stock dish.

b. Slightly raise the cover of the dish, and with a

254

METHODS OF ISOLATION

sterile teat-pipette deposit a small drop of sterile water
in the centre of each of the nine divisions.

c. With the sterilised platinum spatula raise one of
the marked colonies from the " plate 3 " and transfer it
to the first division in the ruled plate and emulsify it
in the drop of water awaiting it. Repeat this process
with the remaining colonies, emulsifying a separate
colony in each drop of water.

(C) Preliminary Differentiation of Bacteria. —

a. Prepare a cover-slip film preparation from each
drop of emulsion in the " stock dish" and number to

correspond to the division
from which it was taken.
Stain by Gram's method.

b. Examine microscop-
ically, using the oil immersion
lens and note the numbers of
those cover-slips which mor-
phologically and by Gram re-
sults appear to be composed
FIG. i39.-Diagram for stock of different species of bac-
Plate- teria.

(D) Preparing Isolation Subcultures. —

a. Inoculate an agar slope and a broth tube from the
emulsion in the stock dish corresponding to each of
these specially selected numbers.

b. Ascertain whether the cover-slips from the nine
emulsions in the stock dish include all the varieties
represented in the cover-slip film preparation made from
the original mixture before plating.

c. If some varieties are missing prepare a second
stock dish from other colonies on plate 3, and repeat
the process until each morphological form or tinctorial
variety has been secured in subculture.

d. Place the stock dishes in the ice chest to await the

DIFFERENTIAL INCUBATION 255

results of incubation. (If any of the subcultures fail,
further material can be obtained from the correspond-
ing emulsion ; or if it has dried, by moistening it with a
further drop of sterile distilled water.)

e: Incubate all the subcultures and identify the
organisms picked out.

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 should be employed when
pouring plates for the isolation of these organisms. To
obtain a pure cultivation of yeast from a mixture con-
taining bacteria as well, it is often 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 proc-
ess 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

256 METHODS OF ISOLATION

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 finds its chief
practical application in the differentiation of a spore-
bearing organism from one which does not form spores.
In this case the mixture 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 cul-
tivations made from the mixture will 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 construction,
designed by Balfour Stewart (Fig. 140) . It consists of a
double-walled copper vessel mounted on legs, and pro-
vided with a tubulure communicating with the space
between the walls. This space is nearly filled with
benzole (boiling-point 80° C. ; pure benzole, free from
thiophene must be employed for the purpose, otherwise
the boiling-point gradually and perceptibly rises in the
course of time), 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 (a tube half
this length if provided with a condensing bulb at

DIFFERENTIAL ATMOSPHERE CULTIVATION

257

the centre will be equally efficient). The interior
of the vessel is partly filled with water and covered with
a lid which is perforated for a thermometer. This
latter .dips into the water and records its temperature.
A very small Bunsen flame under the apparatus 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.

METHOD. — To use the apparatus.

1 . Place some of the mixture itself,
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 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
17

FIG. 140. — Benzole
bath.

258 METHODS OF ISOLATION

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 subcul-
tures, 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
Bulloch's or Novy's jar, and incubate at a statable
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 temperature
and atmosphere.

8. Animal Inoculation. — Finally, when dealing with
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.

XVI. METHODS OF IDENTIFICATION
AND STUDY.

IN order to identify an organism after isolation,
tube, plate, and other cultivations must be prepared,
incubated under suitable conditions as to temperature
and environment, and examined from time to time
(a) macroscopically, (b) by microscopical methods, (c)
by chemical methods, (d) by physical methods, (e)
by inoculation methods, and the results of these exami-
nations 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
observations.

In order to give to the recorded results their maxi-
mum value it is essential that they should be exact
and systematic, 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 (observer's name] in (date),
from (source of organism) .

1. Cultural Characters. — (Vide Macroscopical Ex-
amination of Cultivation, page 261.)
Gelatine plates,

Gelatine streak,

~ . , . . . at 20° C.

Gelatine stab,

Gelatine shake,

259

260 METHODS OF IDENTIFICATION AND STUDY

Agar plates,

Agar streak or smear,

Agar stab,

Inspissated blood-serum,

at2o°C. and 3 7° C.

Bouillon,
Litmus milk,
Potato,

Special media for the purpose of demonstrating
characteristic appearances.

2. Morphology. — (Vide Microscopical Examination of

Cultivations, page 272.)
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. Chemical Products of Growth. — (Vide Chemical

Examination of Cultivations, page 276.)
Chromogenesis.
Photogenesis.
Enzyme formation.

Fermentation of carbohydrates :
Acid formation.
Alkali formation.
Indol formation.
Phenol formation.
Reducing and oxidising substances.
Gas formation.

4. Biology. — ( Vide Physical Examination of Cultures,

page 295.)
Atmosphere.
Temperature.

PLATE CULTURES 26l

Reaction of nutrient media.
Resistance to lethal agents :
Physical :
Desiccation.
Light.
Colours.

Chemical germicides.
Vitality.
5. Pathogenicity :

Susceptible animals, subsequently arranged in

order of susceptibility.
Immune animals.

Experimental inoculation, symptoms of disease.
Post-mortem appearances.
Virulence :

Length of time maintained.
Optimum medium?
Minimal lethal dose.

Exaltation and attenuation of virulence?
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 269,
"stab" cultures).

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

262

METHODS OF IDENTIFICATION AND STUDY

water which has been expressed during the rapid solidi-
fication of the medium and has subsequently condensed.
Gelatine and Agar. — Examine the colonies at inter-
vals of twenty-four hours.

(a) With the naked eye.

(b) With a hand lens or watchmaker's glass.

(c) 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.

(4) Size. — The diameter in millimetres, at the various
ages.

(B) Shape.—

Punctiform: Dimensions too slight for defining form
by naked eye; minute, raised, hemispherical.

a b c

FIG. 141. — Types of colonies: a, Cochleate; b, amoeboid; c, mycelioid.

Round: Of a more or less circular outline.

Elliptical : Of a more or less oval outline.

Irregular: Outlines not conforming to any recognised
shape.

Fusiform: Spindle-shaped, tapering at each end.

Cochleate: Spiral or twisted like a snail shell (Fig.
,141, a).

SURFACE ELEVATIONS 263

Amoeboid: Very irregular, streaming (Fig. 141, b).

Mycelioid : A filamentous colony, with the radiate
character of a mould (Fig. 141, c).

Filamentous : An irregular mass of loosely woven
filaments (Fig. 142, a).

Floccose: Of a dense woolly structure.

Rhizoid: Of an irregular, branched, root-like char-
acter (Fig. 142, b).

Conglomerate: An aggregate of colonies of similar
size and form (Fig. 142, c).

a b c d

FIG. 142. — Types of colonies: a, Filamentous; b, rhizoid; c, conglomerate;

d, toruloid.

Toruloid : An aggregate of colonies, like the budding
of the yeast plant (Fig. 142, d).
Rosulate: Shaped like a rosette.
(C) Surface Elevation. —
i . General Character of Surface as a Whole:
Flat : Thin, leafy, spreading over the surface (Fig.

143, «)•

Effused: Spread over the surface as a thin, veily
layer, more delicate than the preceding.

Raised: Growth thick, with abrupt terraced edges
(Fig. 143, b).

Convex: Surface the segment of a circle, but very
flatly convex (Fig. 1 43 , c) .

264 METHODS OF IDENTIFICATION AND STUDY

Pulvinate: Surface the segment of a circle, but de-
cidedly convex (Fig. 143, d).

Capitate: Surface hemispherical (Fig. 143, e).
Umbilicate : Having a central pit or depression (Fig.

143, /)•

Conical: Cone with rounded apex (Fig. 143, g).

Umbonate: Having a central con-
vex nipple-like elevation (Fig. 143, h).

2 . Detailed Characters of Siirface:

Smooth : Surface even, without any
of the following distinctive characters.

Alveolate: Marked by depressions
separated by thin walls so as to re-
semble a honeycomb (Fig. 144).

Punctate: Dotted with punctures
like pin -pricks.

Bullate: Like a blistered surface,
rising in convex prominences, rather
coarse.

Vesicular: More or less covered

FIG. 143. FIG. 144.

FIG. 143. — Surface elevation of colonies: a, Flat; &, raised; c, convex;
d, pulvinate; e, capitate; /, umbilicate; g, conical; h, umbonate.
FIG. 144. — Types of colonies — alveolate.

with minute vesicles due to gas formation; more
minute than bullate.

Verrucose: Wart-like, bearing wart-like prominences.

Squamose: Scaly, covered with scales.

Echinate: Beset with pointed prominences.

Papillate: Beset with nipple or mamma-like proc-
esses.

INTERNAL STRUCTURE OF COLONY

265

Rugose: Short irregular folds, due to shrinkage of
surface growth.

Corrugated . In long folds, due to shrinkage.

Contoured: An irregular but smoothly undulating
surface, resembling 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 defined.

Refraction Strong: Outline and surface of relief

strongly defined; dense, not filamentous colonies.

a b c

FIG. 145. — Types of colonies: a, Grumose; &, moruloid; c, clouded.

1. General:

Amorphous : Without any definite structure, such as
is specified below.

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

266 METHODS OF IDENTIFICATION AND STUDY

appearance, and particles in clustered grains (Fig.

Moruloid: Having the character of a mulberry, seg-
mented, by which the colony is divided in more or
less regular segments (Fig. 145, b).

Clouded: Having a pale ground, with ill-defined
patches of a deeper tint (Fig. 145, c).

a b c

FIG. 146. — Types of colonies: a, Reticulate; b, gyrose; c, marmorated.

3. Colony Marking or Striping:

Reticulate: In the form of a network, like the veins
of a leaf (Fig. 146, a).

Areolate: Divided into rather irregular, or angular,
spaces by more or less definite boundaries.

Gyrose: Marked by wavy lines,
indefinitely placed (Fig. 146, b).

Marmorated: Showing faint, ir-
regular stripes, or traversed by
vein-like markings, as in marble
(Fig. 146, 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.

FIG. 147. — Types of
colonies — curled.

OPTICAL CHARACTERS 267

Curled: Filaments in parallel strands, like locks or
ringlets (Fig. 147).

(E) Edges of Colonies. —

Entire: Without toothing or division (Fig. 148, a).

Undulate: Wavy (Fig. 148, b).

Repand: Like the border of an open umbrella (Fig.
148, c).

Erose: As if gnawed, irregularly toothed (Fig. 148, d).

FIG. 148. — Edges of colonies: a, Entire; &, undulate; c, repand; d, erose.

Lobate.

Lobulate: Minutely lobate (Fig. 149, e).

Auriculate: With ear-like lobes (Fig. i49,/).

Lacerate: Irregularly cleft, as if torn (Fig. 149, g)

Fimbriate: Fringed (Fig. 149, h).

Ciliate: Hair-like extensions, radiately placed (Fig.

FlG. 149. — Edges of colonies: e, Lobar-lobulate; /, auriculate; g, lacerate;
h, fimbriate; i, ciliate.

Tufted.

Filamentous : As already defined.

Curled: As already defined.

(F) Optical Characters (after Shuttleworth) .—

i. General Characters:

Transparent : Transmitting light.

268 METHODS OF IDENTIFICATION AND STUDY

Vitreous : Transparent and colourless.

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. Chroma genicity:

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 Agar. — Note general points as indicated
tinder 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. —

i. Colonies Discrete: Size, shape, etc., as for plate
cultivations (vide page 261).

GELATINE STAB CULTURES 269

2. Colonies Confluent: Surface elevation and char-
acter of edge, as for plate cultivations (vide page 263).
Chromogenicity : As for plate cultures.

Gelatine Stab Cultures. —

(A) Surface Growth. — As for individual colonies in
plate cultures (vide page 261).

FIG. 150. — Stab cultivations — types of growth: a, Filiform; b, beaded;
c, echinate; d, villous; e, arborescent.

(B) Line of Puncture.—

Filiform: Uniform growth, without special char-
acters (Fig. 150, a).

Nodose: Consisting of closely aggregated colonies.

270

METHODS OF IDENTIFICATION AND STUDY

Beaded: Consisting of loosely placed or disjointed
colonies (Fig. 150, .6).

Papillate: Beset with papillate extensions.

Echinate: Beset with acicular extensions (Fig. 150, c).

Villous: Beset with short, undivided, hair-like
extensions (Fig. 150, d).

Plumose : A delicate feathery growth.

FIG. 151.— Stab cultivations— types of growth:/, Crateriform; g, saccate;
h, infundibuliform; j, napiform; k, fusiform; I, stratiform.

Arborescent: Branched or tree-like, beset with
branched hair-like extensions (Fig. 150, e).
(C) Area of Liquefaction (if present) . —
Crateriform: A saucer-shaped liquefaction of the
gelatine (Fig. 151, /).

FLUID MEDIA 271

Saccate: Shape of an elongated sack, tubular cylin-
drical (Fig. 151, g).

Infundibuliform : Shape of a funnel, conical (Fig.

IS1**)-

Napiform : Shape of a turnip (Fig. 151, /) .

Fusiform : Outline of a parsnip, narrow at either end,
broadest below the surface (Fig. 151, k).

Stratiform: Liquefaction extending to the walls of
the tube and downward horizontally (Fig. 151, /).

(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.

272 METHODS OF IDENTIFICATION AND STUDY

Amount.
Colour.

Carbohydrate Media. —

Growth.
Reaction.
Gas formation.

Coagulation or not of serum albumen (when serum
water media are employed).

Litmus Milk Cultivations.—

Unaltered.

i. Reaction:

Acid.

Alkaline.

2. Odour.

3. Formation of gas.

Unaltered.

4. Consistency:

Peptonised (character of solution) .
Coagulated.

f hard: solid.

soft : floculent.
Clot: Character -, -, , , ,

ragged and broken up by gas

bubbles.

(a) Coagulum undissolved.

(b) Coagulum finally peptonised, completely : in-

completely.
Resulting solution, clear: turbid.

Abundant.
Scanty.

6. Whey: Clear.
Turbid.
Coagulated by boiling, or not.

BY MICROSCOPICAL METHODS.

As a council of perfection preparations must be
made from pure cultivations 4, 6, 8, 12, 18, and 24

LITMUS MILK CULTIVATIONS 273

hours; and subsequently at intervals of, say, twenty-
four hours, during the entire 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.,
when examined in an unsuitable medium, atmosphere,
temperature, 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 con-
ditions 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 or the eikonometer (vide page 63), and average the
results.

If the organism is one which forms spores, observe —

(a) Spore Formation. — Prepare hanging-drop culti-
vations (vide page 78) from vegetative forms of the
organism, adding a trace of magenta solution (0.5 per
cent.) or other intra vitam stain (see page 77) 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 -j^-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. — Prepare hanging-drop culti-
vations from old cultivations in which no living vegeta-

18

274 METHODS OF IDENTIFICATION AND STUDY

tive forms are present, and observe the process of
germination in. a similar manner.

The comfort of the microscopist is largely enhanced
in those cases where the period of observation is at all
lengthy, by use of some form of eye screen before the
unemployed eye, such as is figured on page 58 (Fig. 49).

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 ; then examine.

(b) Spore Germination. — Expose a thick emulsion of
the spores to a temperature of 80° C. for ten minutes
in the differential steriliser (vide page 257).

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., wet, as under (a), and examine.

(B) Fixed. — 2. In stained preparations.

(a) To determine points in morphology:

Shape (vide classification, page 131).

Size:

(a) Prepare cover-slip film preparations at the
various ages, and fix by exposure to a tem-
perature of 115° C. for twenty minutes in hot-
air oven.

(b) Stain the preparations by Gram's method
(if applicable) or with dilute carbol-fuchsin,
and mount in the usual way.

LITMUS MILK CULTIVATIONS 275

(c) Measure (vide page 66) some twety-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.

At what period of development.
(6) To demonstrate details of structure:
Flagella: If noted, record-
Method of staining (vide page 101).

Position and arrangement (vide page 136).

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 139).

Optimum medium and temperature.

Age of cultivation.

Conditions of environment as to temperature,
atmosphere.

Method of germination (vide page 140).
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.

276 METHODS OF IDENTIFICATION AND STUDY

3. Staining Reactions. —

1. Gram's Method. — Positive or negative.

2. Neisser's Method. — If granules are noted, record —

1. Position.

2. Number.

3 . Ziehl-Neelsen's Method. — Acid-fast or decolourised.

4. Simple Aniline Dyes. — (Noting those giving the
best results, with details of staining processes.)

Methylene-blue

Fuchsin

0 , . . ! , \ and their modifications,

Gentian violet

Thionine blue

BY BIOCHEMICAL METHODS.

Test cultivations of the organism for the presence of —

Soluble enzymes — proteolytic, diastatic, invertase.

Organic acids — (a) quantitatively — i. e., estimate
the total acid production; (b) qualitatively for formic,
acetic, propionic, butyric, lactic.

Ammonia.

Neutral volatile substances — ethyl alcohol, aldehyde,
acetone.

Aromatic products — indol, phenol.

Soluble pigments.

Test the power of reducing (a) colouring matters,
(b) nitrates to nitrites.

Investigate the gas production — H2S, CO2,H2. 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 investi-
gate, as to

(a) Reaction of medium ;

(b) Incubation temperature;

(c) Atmospheric environment;

ENZYME PRODUCTION 277

and keep careful records of these points, and also of the
age of the cultivation used in the final examination.
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 from the same batch, kept for
a similar period under identical conditions.

' If the results are negative, test further cultivations
at three days, five days, and ten days.

1. Enzyme Production.—

(A) Proteolytic Enzymes. — (Convert proteins into
proteose, peptone and further products of hydrolysis;
e. g., B. pyocyaneus.)

Media Required:

Blood-serum and milk-serum which have been carefully filtered
through a porcelain candle.
Reagents Required:

Ammonium sulphate.

Thirty per cent, caustic soda solution.

Copper sulphate, 0.5 per cent, aqueous solution.

One per cent, acetic acid solution.

Millon's reagent.

Glyoxylic acid solution.

Concentrated sulphuric acid.

METHOD.—

1. Prepare cultivations in bulk (50 c.c.) in a flask
and incubate.

2 . Make the liquid faintly acid with acetic acid, then
boil. (This precipitates the unaltered proteins.)

3. Filter.

4. Take 10 c.c. of the filtrate in a test-tube and add
i c.c. of the caustic soda, then add the copper sulphate
drop by drop.

Pink colour which becomes violet with more
copper sulphate = proteose and peptone.

5. Saturate the rest of the filtrate with ammonium
sulphate.

Precipitate = proteose.

278 METHODS OF IDENTIFICATION AND STUDY

6. Filter and divide the filtrate into three parts a, b
and c.

a. Repeat the copper sulphate test, using excess of
caustic soda to displace the ammonia from the am-
monium sulphate.

Pink colour = peptone.

b. Boil with Millon's reagent.
Red colour = tyrosine.

c. Add glyoxylic acid solution and run in concen-
trated sulphuric acid.

Violet ring at upper level of acid = tryptophane.

Both the tyrosine and tryptophane may be either
in the free state or in combination as polypeptid or
peptone.

(B) Diastase. — (Converts starch into sugar; e.g., B.
subtilis.)

Medium Required:

Inosite-free bouillon.
Reagents Required:

Starch.

Thymol.

Fehling's soltuion.

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 nitrate for sugar.

Boil some of the Fehling's solution in a test-tube.

Add the nitrate drop by drop until, if necessary, a
quantity has been added equal in amount to the Feh-
ling's solution employed, keeping the mixture at the
boiling-point during the process.

Yellow or orange precipitate = sugar.

FERMENTATION REACTION 279

(C) Invertase. — (Convert saccharose into a mixture of
dextrose and laevulose e.g., B. fluorescens liquefaciens.)

Medium Required:

Inosite-free bouillon.
Reagents Required:

Cane sugar, 2 per cent, aqueous solution.

Carbolic acid.

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 filtrate for reducing sugar as in the pre-
ceding section.

(D) Rennin 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 rennin ferment formation.

Fermentation Reactions.

As tested upon carbohydrate substances and organic
salts.

280 METHODS OF IDENTIFICATION AND STUDY

Media Required:

Peptone water containing various percentages (gen-
erally 2 per cent.) of each of the substances referred
to under "sugar" media (page 177), also tubes of
peptone water containing i per cent, respectively of
each of the following:

Organic salts: Sodium citrate, formate, lactate, malate,

tartrate.

METHOD. —

1. Prepare tube cultivations in each of the above
media.

2. Observe from day to day up to the expiration of
ten days if necessary.

3. Note growth, reaction, gas production.

2. Acid Production.

(a) Quantitative. —
Medium Required:

Sugar (glucose) bouillon of known "optimum" reaction.
Apparatus and Reagents Required:

As for estimating reaction of media (vide page 150).

METHOD.—

1. Prepare cultivation in bulk (100 c.c.) in a flask;
also "control" flask of medium from same batch.

2. After suitable incubation, heat both flasks in the
steamer at 100° C. for thirty minutes to sterilise.

3 . Determine the litre of the medium in " inoculated ' '
and "control" flasks as described in the preparation of
nutrient media (vide page 151).

4. The difference between the titre of the medium
in the two flasks gives the total acid production of the
bacterium under observation 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 employed in the titration.

ACID PRODUCTION 28 1

(5) Qualitative (of all the organic acids present). —
Medium Required:

Sugar (glucose or lactose) bouillon as in quantitative examina-
tion.
Reagents Required:

Hydrochloric acid, concentrated.

Hydrochloric acid, 25 per cent.

Sulphuric acid, concentrated (pure).

Phosphoric acid, concentrated solution.

Ammonia.

Ammonium sulphate.

Baryta water.

Sodium carbonate, saturated aqueous solution.

Absolute alcohol.

Ether.

Calcium chloride.

Calcium chloride solution.

Zinc carbonate.

Copper sulphate saturated aqueous solution.

Alcoholic thiophene solution (0.15 c.c. in 100 c.c.).

Animal charcoal.

Five per cent, sodium nitroprusside solution.

Potassium bichromate.

Schiff's reagent.

Arsenious oxide.

Ferric chloride, 4 per cent, aqueous solution.

Silver nitrate, i per cent, aqueous solution.

Lugol's iodine.

Ten per cent, caustic soda 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.

The paraffin, which liquefies and forms a thin layer
on the surface of the fluid, is necessary to prevent
the cultivation frothing up and running unaltered
through the condenser during the subsequent process
of distillation.

3. Distill over 200 to 300 c.c.

282

METHODS OF IDENTIFICATION AND STUDY

Use a rose-top burner to minimise the danger of
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 285) ; the residue "a" will contain the volatile
and fixed acids.

4. Disconnect the flask and filter. The residue "a"
then = filtrate B and residue b.

FIG. 152. — Arrangement of distillation apparatus for acids, etc.

5 . Residue b. Wash the residue from the filter paper,
dissolve by heating with dilute hydrochloric acid, and
add calcium chloride solution and ammonia until
alkaline.

White precipitate insoluble in acetic acid = oxalic
acid.

6. Make up filtrate B to 500 c.c. with distilled water
and divide into two parts.

ACID PRODUCTION

283

7. Acidify 250 c.c. with 20 c.c. concentrated phos-
phoric acid (this liberates the volatile acids) and distil
to small bulk.

The distillate "B" may contain formic, acetic,
propionic, butyric and benzoic acids.

DISTILLATE " B."
(Volatile Acids.)

i. Add baryta water till alkaline,
and evaporate to dryness.

2. Add 50 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. Acidify with phosphoric acid and dis-

til.

4. Saturate distillate with calcium chlo-

ride and distill over a few c.c.

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
cannot be distinguished from buty-
ric acid by tests within the scope
of the bacteriological laboratory.

RESIDUE

may contain barium acetate,
barium formate, barium benzoate.

1. Evaporate off alcohol and dissolve

up the residue on the filter in hot
water and neutralise.

2. Divide the solution into four por-

tions:

(a) Add ferric chloride solution.
Brown colour = acetic or for-
mic acids.
Buff ppt. = benzoic acid (see

ether soluble acids).
(6) Add silver nitrate solution;
then add one drop am-
monia water, and boil.
Black precipitate of me-
tallic silver = formic acid.

(c) Evaporate to dryness; mix

with 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 mercurors
chloride which is slowly
reduced to mercury =•
formic acid.

284 METHODS OF IDENTIFICATION AND STUDY

8. If the distillation of " B" is continued as long as
acid conies over (distilled water being occasionally
added to the distilling flask) the distillate can be
measured and 50 c.c. used for titration. This will
give the amount of volatile acid formation.

9. The second part of the filtrate "B" (see page
282) should be examined for lactic, oxalic, succinic,
benzoic, salicylic, gallic and tannic acids, as follows :

Ether Soluble Acids.—

1. Evaporate to a thin syrup, acidify strongly with
phosphoric acid.

2. Extract with five times its volume of ether by
agitation in a separatory funnel.

3. Evaporate the ethereal extract to a thin syrup.

4. Add 100 c.c. water and mix thoroughly.

5. To a small portion of this solution add slight ex-
cess of sodium carbonate, evaporate to dryness on the
water-bath, dissolve in 5-10 c.c. pure sulphuric acid, add
2 drops saturated copper sulphate solution, place in a
test-tube and heat in a boiling water- bath for 2 minutes,
cool, add 2 or 3 drops of the alcoholic thiophene and
warm gently.

Cherry red colour = lactic acid.

If a brown colour is produced on the addition of
sulphuric acid, another sample should be taken and
boiled with animal charcoal before evaporating.

6. If lactic acid is definitely present, prepare zinc
lactate by boiling part of the solution of the ether extract
with excess of zinc carbonate, filtering and evaporating
to crystallise. The crystals so obtained have a char-
acteristic form, and if dried at 110° C., should contain
26.87 per cent, of zinc.

7 . Test a portion of the rest of the solution of the ether
extract for oxalic acid (page 282, step 5). Carefully
neutralise the remainder and add ferric chloride solution.

Red brown gelatinous precipitate = succinic acid.

ALCOHOL PRODUCTION 285

Buff precipitate = benzoic acid, and other acids re-
lated to benzoic acid.

Violet colour = salicylic acid.

Inky black colour or precipitate = gallic acid or tannic
acid.

For further identification the melting-points of the
crystalline acids, and the percentage of silver in their
silver salts should be determined.

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 dis,til.

3 . Collect 50 c.c. distillate, from each, in a Nessler glass.

4. Add i c.c. Nessler reagent to each glass by means
of a clean pipette.

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 282, step 3) into four portions and test for
the production of alcohol, acetaldehyde, acetone.

i. Add Lugol's iodine, then a little NaOH solution,
and stir with a glass rod till the colour of the iodine
disappears.

Pale-yellow crystalline precipitate of iodoform, with
its characteristic smell, appearing in the cold, indicates
acetaldehyde, or acetone; appearing only on warming
indicates alcohol.

286 METHODS OF IDENTIFICATION AND STUDY

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 2.5 c.c., 25 per cent,
sulphuric acid, and a crystal or two of potassium
bichromate and distil. Reduction of the bichromate
to a green colour and a distillate, which smells of acet-
aldehyde and reacts with Schiff's reagent, shows the
presence of alcohol in the original liquid.

4. Add a few drops of sodium nitroprusside solution,
make alkaline with ammonia, then saturate with
ammonium sulphate crystals. Acetone gives little
colour on the addition of ammonia, but after the addi-
tion of ammonium sulphate a deep permanganate
colour, which takes ten minutes to reach its full inten-
sity. Aldehyde gives a carmine red unaltered by
ammonium sulphate.

5. Indol Production. —

Media Required:

Inosite-free bouillon (vide page 183).
Or peptone water (vide page 177).
Reagents Required:

Potassium persulphate, saturated aqueous solution.
Paradimethylamino-benz aldehyde solution. This is prepared by
mixing:

Paradimethylamino-benzaldehyde ... 4 grammes

Absolute alcohol 380 c.c.

Hydrochloric acid, concentrated .... 80 c.c.

METHOD. —

Prepare several test-tube cultivations of the
organism to be tested, and incubate.

Test for indol by means of the Rosindol reaction in
the following manner. (If the culture has been incu-
bated at 37° C., it must be allowed to cool to the room
temperature before applying the test.)

i. Remove 2 c.c. of the cultivation by means of a
sterile pipette and transfer to a clean tube, then,

PHENOL PRODUCTION 287

2. Add 2 c.c. paradimethylamino-benzaldehyde
solution.

3. Add 2 c.c. potassium persulphate solution.

The presence of indol is indicated by the appearance
of a delicate rose-pink colour throughout the mixture
which deepens slightly on standing.

Indol is tested for in many laboratories by the ordinary
nitrosoindol reaction which, however, is not so delicate a method
as that above described. The test is carried out as follows:

1. Remove the cotton- wool plug from the tube, and run in
i c.c. pure concentrated sulphuric acid down the side of the tube
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 at the junction of the two liquids =
indol (plus a nitrite).

2. If the colour of the medium remains unaltered, add 2 c.c. of
a o.o i per cent, aqueous solution sodium nitrite, 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.

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., 25 per cent, sulphuric acid to the
cultivation and connect up the flask with a condenser.

3. Distil over 15 to 20 c.c. Divide the distillate
into three portions a, b and c.

4. Add to (a) 0.5 c.c. Millon's reagent and boil.
Red colour = phenol.

288 METHODS OF IDENTIFICATION AND STUDY

5. Add to (b) about 0.5 c.c. ferric chloride solution.
Violet colour = phenol.

(If the distillate be acid the reaction will be nega-
tive.)

6. Add to (c) bromine water. Crystalline white ppt.
of tribromo -phenol = phenol.

NOTE. — If both indol and phenol appear to be present in culti-
vations 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. Render definitely acid by the addition of acetic
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).

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 tempera-
ture (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.

GAS PRODUCTION 289

4. Note spectroscopic reactions.

7. Reducing Agent Formation. —

(a) Colour Destruction. —

1 . Prepare tube cultivations in nutrient bouillon tin-
ted 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 185).

Or nitrate peptone solution (vide page 186).
Reagents Required:

Sulphuric acid (25 per cent.).

Metaphenylene diamine, 5 per cent, aqueous solution.

METHOD. —

1. Prepare tube cultivations and incubate together
with control tubes (i. e., uninoculated tubes of the
same medium, placed under identical conditions as to
environment) .

This precaution is necessary as the medium is liable
to take up nitrites from the atmosphere, and an
orjfinion as to the absence of nitrites in the cultivation
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.

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 161) containing sugar bouillon

2QO METHODS OF IDENTIFICATION AND STUDY

(glucose, lactose, etc.). The medium should be prepared from
inosite-free bouillon (vide page 183).
Reagent Required:

- caustic soda.

2

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 C02 and H2, proceed as follows :

Fill the bulb of the fermentation tube with caustic
soda solution.

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:

Iron peptone solution (vide page 185).
Lead peptone 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-

GAS PRODUCTION

291

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

FIG. 153. — Gas-collecting apparatus.

with a perforated rubber stopper, carrying 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 contained. 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 in. This
rises vertically 5 cm., and is then bent at right angles, the horizon-
tal arm being 10 cm. in length. A three-way tap is let hori-
zontally into the vertical tube just above its junction with the
bell jar.

An iron cylinder just large enough to contain the bell jar.

About 1 5 kilos of metallic mercury.

Melted paraffin.

2p2 METHODS OF IDENTIFICATION AND STUDY

An Orsat-Lunge working with mercury instead of water,
provided with two gas tubes of extra length (capacity 120 and
60 c.c. respectively and graduated throughout, both being water-
jacketed) or other gas analysis apparatus, capable of dealing with
C02, 02, H2, and N2.

METHOD.—

i. Inoculate the medium in the flask in the usual
manner, by means of a platinum needle, taking care
that the neck of the flask and the rubber stopper are

FIG. 154.— Orsat-Lunge gas analysis apparatus.

thoroughly flamed before and after the operation.

2. Fill the iron cylinder with mercury.

3. Place the bell jar mouth downward 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

GAS PRODUCTION 293

pressure tubing (after removing the cotton-wool plug
from the rubber tube), as shown in Fig. 153.

6. Give the three-way tap half turn to open com-
munication between flask and receiver, and seal all
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
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
receiver.

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

294 METHODS OF IDENTIFICATION AND STUDY

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 contained
in the culture flask) of the organism it is desired to
investigate.

6. Slightly release the clamp on the pressure tub-
ing 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
flask containing recently boiled and quickly cooled dis-
tilled water.

8. Release the clamp again arid 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 re-
ceiver with hot water.

ATMOSPHERE

295

11. Connect up the culture flask with the horizontal
arm of the gas receiver.

1 2 . 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 its growth and development under the following
headings :
Atmosphere :

(a) In the presence of oxygen.

(b) In the absence of oxygen.

(c) In the presence of gases other than oxygen.
Temperature :

(a) Range.

(b) Optimum.

(c) Thermal death-point:
Moist: Vegetative forms.

Spores.
Dry: Vegetative forms.

Spores.

Reaction of medium.
Resistance to lethal agents:

(a) Desiccation.

(b) Light: Diffuse.

Direct.
Primary colours.

(c) Heat.

(d) Chemical antiseptics and disinfectants.
Vitality in artificial cultures.

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

296 METHODS OF IDENTIFICATION AND STUDY

in the fermentation tubes. Obvious growth in the
closed branch as well as in the bulb or in the inverted
gas tube as well as in the bulk of the medium will
indicate that it is a facultative anaerobe ; whilst growth
only occurring in the bulb or in the closed branch
shows that it is an obligate aerobe or anaerobe respec-
tively. 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) Sloped glucose formate agar, and incubate
aerobically at 3 7° C.

Sloped glucose formate gelatine, and incubate aero-
bically at 20° C.

(B) Sloped glucose agar to incubate anaerobically
at37°C.

Sloped glucose formate gelatine to incubate
anaerobically at 20° C.

(C) Sloped glucose formate agar to incubate anaero-
bically at 37° C.

Glucose formate bouillon to incubate anaerobi-
cally at 37° C.

(D) Sloped glucose formate gelatine to incubate
anaerobically at 20° C.

Glucose formate bouillon to incubate anaerobi-
cally at 20° C.

ATMOSPHERE 297

2. Seal the cultures forming set B in Buchner's
tubes (vide page 239).

3 . Seal the cultures forming set C in Bulloch's appara-
tus; 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 in solu-
tion (vide page 245). Treat set D in the same way.

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 40).

Gasometer containing the gas it is desired to test (SO2, N2O,
NO, CO2, etc.) or gas generator for its production.

METHOD. —

1. Prepare at least seven tube cultivations upon
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 as shown by the result of
analyses of samples collected from the exit tube.

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 appara-
tus 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.

298 METHODS OF IDENTIFICATION AND STUDY

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-
tures, varying 5°C. and including temperatures between
5° C. and 50° C.

(In the absence of a sufficient number of incubators
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 (Optimum temperature).

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 — if considered necessary
arranging the series of incubators to include each degree
centigrade for five degrees beyond each of the extremes
previously noted.

(B) Optimum. —

1. Prepare a second series of ten tube cultivations
under similar conditions as to reaction of medium.

2. Incubate in a series of incubators in which the
temperature is regulated at intervals of i° C. for five
degrees on either side of optimum temperature observed
in the previous experiment (A, 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.

(C) Thermal Death-point (t. d. p.) —
Moist — Vegetative Forms :

The /. d. p. here is that temperature which with

TEMPERATURE

299

certainty kills a watery suspension of the organisms in
question after an exposure of 10 minutes.

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 means of a capsule regulator

FIG. 155. — Hearson's water-bath.

that can be adjusted 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 current of air may be blown
through the water while the bath is in use, and thus ensure a
uniform temperature of its contents. Through a second hole is
suspended a certified centigrade thermometer, the bulb of which
although completely immersed in the water is raised at least 2 cm.
above the floor of the bath.

Sterile glass capsules.

Flask containing 250 c.c. sterile normal saline solution.

Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).

Special platinum loop.

Test-tubes, 18 by 1.5 cm., of thin German glass.

Case of sterile petri dishes.

Tubes of agar or gelatine.

300 METHODS OF IDENTIFICATION AND STUDY

METHOD.—

1. Prepare tube cultivations on solid media of opti-
mum reaction; incubate forty-eight hours under opti-
mum 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 surface growth
(using a special platinum loop, vide page 316) in the
normal saline solution by emulcifying evenly against
the moist walls of each capsule.

5. Transfer emulsion from each capsule to sterile
250 c.c. flask, and mix.

6. Pipette 5 c.c. emulsion into each of twelve sterile
test-tubes numbered consecutively.

7. 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.

8. Arrange a control test-tube containing 5 c.c. sterile
saline solution 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.

9. Close the unoccupied perforations in the lid of
the water-bath by means of glass balls.

10. 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-
pension, and cool rapidly by immersing its lower end in
a stream of running water.

11. Pour three gelatine (or agar) plates containing
respectively 0.2, 0.3, and 0.5 c.c. of the suspension,
and incubate.

TEMPERATURE 301

12. Pipette the remaining 4 c.c. of the suspension
into a culture flask containing 250 c.c. of nutrient
bouillon, and incubate.

13. Observe these cultivations from day to day.
"No growth" must not be recorded as final until after
the completion of seven days' incubation.

14. 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.

15. Note that temperature, after exposure to which
no growth takes place up to the end of seven days'
incubation, = the thermal death-point.

1 6. 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 fixed temperature of
100° C. necessary to effect the death of all the spores
present in a suspension.

NOTE. — If it is desired to retain the time constant 10 minutes

and investigate the temperature necessary to destroy the spores,
varying amounts of calcium chloride must be added to the. water
in the bath, when the boiling-point will be raised above 100° C.
according to the percentage of calcium in solution. In such case
use the bath figured on page 227 the bath figured on page 299 can
only be used if the capsule; is first removed.

It is determined in the following manner

Apparatus Required:

Steam-can fitted with a delivery tube and a large bore safety-
valve tube.

Water-bath at 100° C.

Erlenmeyer 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:

302

METHODS OF IDENTIFICATION AND STUDY

(a)

Thermometer to 120° C., its bulb immersed in the normal
saline.

Straight entry tube, reaching to the bottom of the flask,
the upper end plugged with cotton-wool.
Bent syphon tube, with pipette nozzle attached by means
of rubber tubing and fitted with pinch-cock.
The nozzle is protected from accidental contamination by
passing it through the cotton-wool plug of a small test-tube.

(c)

FIG. 156. — 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 contain 5 c.c.
Media Required:
Gelatine or agar.
Culture flasks containing 200 c.c. nutrient bouillon.

METHOD. —

i. Prepare twelve tube cultivations upon the sur-
face (or two cultures in large flat culture bottles —

TEMPERATURE 303

vide page 5) 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., open the
spring clip on the syphon, discard the first cubic centi-
meter of suspension that syphons over (i.e., the con-
tents of the syphon tube) ; collect the next 5 c.c. of
the suspension in the sterile graduated test-tube and
pour plates and prepare flask cultures therefrom as in
the previous experiments.

7. Repeat this process at intervals of twenty-five
minutes' steaming.

8. Observe the inoculated plates and flasks up to
the completion, if necessary, of seven days' incubation.

9. Control these experiments, but in this instance
syphon off portions of the suspension at intervals of
one-half to one minute during the five or ten minutes
preceding the previously determined death-point.

Thermal Death-point. —

Dry — Vegetative Forms: The thermal death-point
in this case is that temperature which with certainty
kills a thin film of the organism in question after a
time exposure of ten minutes.

Apparatus Required:

Hot-air oven, provided with thermo-regulator.

304 METHODS OF IDENTIFICATION AND STUDY

Sterile cover-slips.

Flask containing 250 c.c. sterile normal saline solution.

Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).

Case of sterile capsules.

Crucible tongs.

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 accidental
contamination has taken place but that the organism
originally spread on the film is responsible for the
growth.

7. Control the result of these experiments.

Dry — Spores : The thermal death-point in this case
is that temperature which with certainty kills the spores
of the organism in question when present in a thin film
after a time exposure of 10 minutes.

Apparatus Required:
As for vegetative forms.

METHOD. —

i. Prepare a sloped agar tube cultivation and incu-
bate under optimum conditions as to spore formations.

REACTION OF MEDIUM 305

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.

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 ster-
ile capsule; add 9.9 c.c. sterile bouillon and mix
thoroughly.

3. Prepare a series of tubes of nutrient bouillon of
varying reactions, from +25 to —30 (vide page 155),
viz.: +25, +20, +15, +10, +5, neutral, -5, -10,

-15. -20» ~25> -3°-

4. Inoculate each of the bouillon tubes with o.i c.c.

of the diluted cultivation by means of a sterile gradu-
ated pipette and incubate under optimum 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, if
necessary, of seven days. 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

20

306 METHODS OF IDENTIFICATION AND STUDY

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 represent smaller variations in reaction
than those previously employed (say, i instead of 5)
for five points on either side of the previously observed
optimum. For example, the optimum reaction ob-
served in the set of experiments to determine the
range was + 10. Now plant tubes having reactions

of +15, +14, +13, +I2> +IJ> +I0» +9> +8> +7»
+ 6, +5, and observe as before.

IV. Resistance to Lethal Agents. —

(A) Desiccation. —

Apparatus Required:

Mueller'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 the top of which pure sulphuric acid
is poured whilst the material to be dried is placed within its walls
on a glass shelf (6) . The air is exhausted from the interior and
the acid rapidly converts the clay vessel into a large absorbing
surface (Fig. 157).

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. 1 58) .

METHOD. —

1. Prepare a sunace cultivation on nutrient agar in
a culture bottle and incubate under optimum condi-
tions for forty-eight hours.

2 . Examine preparations from the cultivation, micro-
scopically, to determine the absence of spores.

3. Pipette 5 c.c. sterile normal saline solution into
the flask and suspend the entire growth in it.

RESISTANCE TO LETHAL AGENTS

307

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.

•w

FIG.. 157. — Mueller's desiccator.

6. Place the Petri dish inside the Mueller's desiccator;
fill the upper chamber with pure sulphuric acid, cover
with the bell jar, and exhaust the air from its interior.

FIG. 158. — Petri dish for drying cultivations.

Ten minutes later connect up the desiccator to a
sulphuric acid wash-bottle interposing an air filter so
that only dry sterile air enters.

308 METHODS OF IDENTIFICATION AND STUDY

7. At intervals of five hours open the apparatus,
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 again exhaust, and subsequently
admit dry sterile air as before.

8. Incubate the culture flask under optimum condi-
tions until the completion of seven days, if necessary;
and determine the time exposure at which death occurs.

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 :

i. Prepare a tube cultivation in nutrient bouillon,
and incubate under optimum conditions, for forty-eight
hours.

FIG. 159. — Plate with star for testing effect of light.

2. Pour twenty plate cultivations, ten of nutrient
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. 159).

RESISTANCE TO LETHAL AGENTS 309

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 under optimum
conditions.

7. Examine the plate cultivations after twenty-four
and forty-eight hours' incubation, 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 incubation and daily obser-
vation 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.

5. After exposures for periods similar to those em-
ployed in the preceding experiment, incubate and
complete the observation as above.

(c) Primary Colours: Each colour — violet, blue,
green and red — must be tested separately.

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.

310 METHODS OF IDENTIFICATION AND STUDY

Chrysoidin (for red) .
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. The bactericidal action of light appears to
depend upon the more refrangible rays of the violet
end of the spectrum and is noted whether the red yellow
rays are transmitted or not.

5. Control the results.

NOTE. — The ultra-violet rays obtained from a quartz mercury
vapour lamp destroy bacterial life with great rapidity under labo-
ratory conditions.

(C) Heat. — (Vide Thermal Death-point, page 298.)

(D) Antiseptics and Disinfectants. — The resistance
exhibited by any given bacterium toward any specified
disinfectant or germicide should be investigated with
reference to the following points:

(A) Inhibition coefficient — i. e., that percentage of
the disinfectant present in the nutrient medium which
is sufficient to prevent the growth and multiplication
of the bacterium.

(B) Inferior lethal coefficient — i. e., the time expo-
sure necessary to kill vegetative forms of the bacterium
suspended in water at 20° to 25° C., in which the disin-
fectant is present in medium concentration (concen-
tration insufficient to cause plasmolysis) . And if the
bacterium is one which forms spores,

(C) Superior lethal coefficient — i. e., the time expo-
sure necessary to kill the spores of the bacterium under
conditions similar to those obtaining in B.

The example here detailed only specifically refers
to certain of the disinfectants :

viz:- Bichloride of mercury;

INHIBITION COEFFICIENT 311

Formaldehyde ;

Carbolic acid ;

investigated with regard to B. anthracis, but the tech-
nique is practically similar for all other chemical
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 each containing a measured 10 c.c.
of medium.

Twenty-four-hour-old agar culture of a recently isolated B.
anthracis

Germicides:

1. Five per cent, aqueous solution of carbolic acid.

2. One per cent, aqueous solution of perchloride of mercury.

3. One-tenth per cent, aqueous solution of formaldehyde.

METHOD. —

1. Number six bouillon tubes consecutively i to 6.
Inoculate each from the stock cultivation of B. anthra-
cis and at once add varying quantities1 of the carbolic
acid solution, viz. :

To tube i add 2 . o c.c. ( = i : 100)
To tube 2 add i . o c.c. ( = i : 200)
To tube 3 add 0.6 c.c. ( = i 1300)
To tube 4 add 0.5 c.c. ( = i : 400)
To tube 5 add 0.4 c.c. ( = i : 500)
To tube 6 add o . 2 c.c. ( = i : i ,000)

2. Prepare a similar series of tube cultivations
numbered consecutively 7 to 12 and add varying
quantities of the mercuric perchloride solution, viz. :

To tube 7 add o.i .( =
To tube 8 add 0.05 ( =
To tube 9 add o . 03 ( =
To tube 10 add 0.025 ( =
To tube ii add 0.02 ( =
To tube 1 2 add o . o i ( =

:i,ooo)
: 2,000)
.•3,000)
14,000)
.•5,000)
: 10,000)

1 The quantities here given are not absolutely correct. If exactitude is
essential the student must calculate the amount required by the aid of the
Percentage Formula, Appendix, page 496.

3I2

METHODS OF IDENTIFICATION AND STUDY

3. Prepare a similar series of tube cultivations
numbered consecutively 13 to 18 and add varying
quantities of the formaldehyde solution, viz. :

To tube No. 13 add i.o c.c. (=1:1,000)
To tube No. 14 add 0.4 c.c. (= 1 12,500)
To tube No. 15 add 0.2 c.c. (= 1 15,000)
To tube No. 16 add o.i c.c. (=1:10,000)
To tube No. 17 add 0.075 c.c. (= i : 15,000)
To tube No. 18 add 0.05 c.c. (=1:20,000)

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.

6. From such tubes as show growth prepare sub-
cultivations upon suitable media, and ascertain that
the organism causing the growth is the one orig-
inally employed in the test and not an accidental
contamination.

Inferior Lethal Coefficient.—

Apparatus Required:

Highly concentrated solutions of the disinfectants.

Sterile test-tubes in which to make dilutions from the concen-
trated solutions of the disinfectants.

Hanging-drop slides.

Cover-slips.

Erlenmeyer flask containing 100 c.c. sterile distilled water.

Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).

Case of sterile pipettes, i c.c. (in tenths of a cubic centimetre).

METHOD. —

1. Prepare a surface cultivation of the "test"
organism B. anthracis upon nutrient agar in a culture
bottle and incubate under optimum conditions for
twenty-four 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

SUPERIOR LETHAL COEFFICIENT 313

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 18° C.

10. Add to each of the remaining tubes a sufficient
quantity r of a concentrated solution of the disinfectant
to produce the percentage previously determined upon
(vide step 5).

11. Incubate the tubes at 18° C. to 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.

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. —

i. Prepare surface cultivations of the "test" organ-
isms upon nutrient agar in a culture bottle, and incu-

1 See Percentage Formula, Appendix, page 496.

314 METHODS OF IDENTIFICATION AND STUDY

bate under optimum conditions, for three days, for
the formation of their spores.

2. Transfer the emulsion to a sterile test-tube and
heat in the differential steriliser for ten minutes at
80° C. to destroy all vegetative forms.

3 . Employing that percentage solution of the disinfec-
tant determined in the previous experiment, and com-
plete the investigations as detailed therein, steps 7 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 disin-
fectant from the bacteria 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. Consequently it is essential in all
gerrnicidal experiments to determine first of all the inhibition
coefficient of the germicide employed. Under the circumstances
referred to above it is usually sufficient to prepare the subcultures
in such a volume of fluid nutrient medium as would suffice to
reduce the concentration of the germicide to about one hun-
dredth of the inhibition percentage, assuming that the entire
bulk of inoculum was made up of that strength of germicide
employed in the test. In some cases it is a simple matter to neu-
tralise the germicide and render it inert by washing the organisms
in some non-germicidal solution (such for example as ammonium
sulphide when using mercurial salts as the germicide). When,
however, it is desired to remove the last traces of germicide proceed
as follows:

1. Transfer the suspension of bacteria to sterile centrifugal
tubes; add the required amount of disinfectant, and allow it to
remain in contact with the bacteria for the necessary period.

2. Centrifugalise thoroughly, pipette off the supernatant fluid;
fill the tube with sterile water and distribute the deposit evenly
throughout the fluid.

3. Centrifugalise again, pipette off the supernatant fluid; fill
the tube with sterile water; distribute the deposit evenly through-
out 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.

PATHOGENETIC PROPERTIES 315

PATHOGENESIS.

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 healthy frog, into the reptile's dorsal
lymph sac..

3. Observe until death takes place, or, in the event
of a negative result, until the completion of twenty-
eight days (vide Chapter XVIII).

4. If, and when, death occurs, make a careful post-
mortem examination (vide Chapter XIX) .

(b) Mesophilic Bacteria: When the organism grows
at 3 5° to 3 7° C.,

1. Prepare cultivations in nutrient broth and incu-
bate under optimum conditions for forty-eight hours.

2. Select two white mice, as nearly as possible of
the same age, size, and weight.

3. Inoculate the first mouse, subcutaneously at
the root of the tail, with an amount of cultivation
equivalent to i per cent, of its body- weight.

4. Inoculate the second mouse intraperitoneally
with a similar dose.

5. Observe carefully until death occurs, or until the
lapse of twenty-eight days.

6. 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 (vide infra) of the
growth upon solid media for the frog or white mouse

316 METHODS OF IDENTIFICATION AND STUDY

respectively. Other experimental animals — e. g., the
white rat, guinea-pig, and rabbit — should next be
tested in a similar manner.

7. If the inoculated mice are unaffected, test the
action of the organism in question upon white rats,
guinea-pigs, rabbits, etc.

Minimal Lethal Dose (m. I. d.); If the purpose of the
inoculation is to determine the minimal lethal dose, a
slightly different procedure must be followed. For this
and other exact experiments a special platinum loop is
manufactured, some 2.5 mm. by 0.75 mm., with parallel
sides, and calibrated by careful weighing, to determine
approximately the amount of moist bacterial growth
the loop will hold when filled.

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.

MINIMAL LETHAL DOSE 317

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).

The dilutions thus prepared may be summarised in
a table ;

Capsule I=i loopful+io c.c. water .'. i c.c. = o.i loop.

Capsule II = o.i c.c. capsule I +9.9 c.c. water .'. i c.c. = o.ooi loop.

Capsule III = o.i c.c. capsule II +9.9 c.c. water .'. i c.c. = 0.00001 loop.

Capsule IV = o.i c.c. capsule III + 9.9 c.c. water .'. i c.c. = 0.0000001 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 are small in bulk, add
the necessary quantity of sterile bouillon to make up
to i c.c.

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.

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 inoculated animal is noted as the minimal
lethal dose.

318

METHODS OF IDENTIFICATION AND STUDY

Toxins. —

Prepare flask cultivations of the organism under
observation in glucose formate broth, and incubate for
fourteen days under optimum conditions.

(a) Intracellular or Insoluble Toxins :

1. Heat the fluid culture in a water-bath at 60° C.
for thirty minutes. (The resulting sterile, turbid fluid
is often spoken of as " killed" culture,)

2. Inoculate a tube of sterile bouillon with a similar
quantity, and incubate under optimum conditions.
This "control" then serves to demonstrate the freedom
of the toxin from living bacteria.

FIG. 160. — Apparatus arrange for toxin filtration.

3. Inject intraveneously 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.

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 :

MINIMAL LETHAL DOSE 319

1. Filter the cultivation through a porcelain filter
candle (Berkfeld) into a sterile filter flask, arranging
the apparatus as in the accompanying figure (Fig. 160).

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, to determine the freedom
of the filtered toxin from living bacteria.

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.

The estimation of the m. I. d. of a toxin is carried
out on lines similar to those laid down for living bac-
teria (vide page 316) merely substituting i c.c. of toxin
as the unit in place of the unit "loopful" of living
culture.

It frequently happens, during the course of casual in-
vestigations that a bouillon-tube culture is available
for a toxin test whilst a flask cultivation is not. In
such cases, Martin's small filter candle and tube (Fig.
161) specially designed for the filtration of small quan-
tities of fluid, is invaluable. This consists of a narrow
filter flask just large enough to accommodate an ordi-
nary 18X2 cm. test-tube. The mouth of the tubular
Chamberland candle 15X1.5 cm. is closed by a perfor-
ated rubber cork into which fits the end of the stem
of a thistle headed funnel, whilst immediately below
the butt of the funnel is situated a rubber cork to close
the mouth of the filter flask. When the apparatus is
fixed in position and connected to an exhaust pump,
the cultivation is poured into the head of the funnel
and owing to the relatively large filtering surface the

320

METHODS OF IDENTIFICATION AND STUDY

germ free filtrate is rapidly drawn through into the
test-tube receiver.

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
each individual case. Among the
most important are the following :

1 . Passage of Virus. — The inocula-
tion of pure cultivations of the organ-
ism into highly susceptible animals,
and passing it as rapidly as possible
from animal to animal, always select-
ing that method of inoculation —
e. g., intraperitoneal — which places
the organism under the most favor-
able conditions for its growth and
multiplication.

2. Virus Plus Virulent Organisms.
—The inoculation of pure cultiva-
tions of the organism together with
pure cultivations of some other mi-
crobe which in itself is sufficiently
virulent to ensure the death of the

experimental animal, either into the
.bio. 161— -Martin s ., • .

filtering apparatus same situation or into some other

oTfluidf11 quantities part of the body. By this associa-
tion the organism of low virulence
will frequently acquire a higher degree of virulence,
which may be still further raised by means of "pas-
sages" (vide supra).

3.^ Virus Plus Toxins.— ^hQ 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

ATTENUATING THE VIRULENCE OF AN ORGANISM 321

of the feeble virus. By this means the natural .resist-
ance of the animal is lowered, and the organism inocu-
lated 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 varying the environment of the cultivations, as
for example:

1. Cultivating in such media as are unsuitable by
reason of their (a) composition or (b) reaction.

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.

Attenuation of the virus can also be secured by

5. Passage through naturally resistant animals.

6. Exposure to desiccation.

7. Exposure to gaseous disinfectants.

8. By a combination of two or more of the above
methods.

IMMUNISATION.

The further study of the pathogenetic powers of any
particular bacterium involves the active immunisation
of one or more previously normal animals. This end may
be attained by various means; but it must be remem-
bered that immunisation is not carried out by any hard
and fast rule or by one method alone, but usually by a
combination of methods adapted to the exigencies of
each particular case. The ordinary methods include :

A. Active Immunisation.

I. By inoculation with dead bacteria (i. e.,

322 METHODS OF IDENTIFICATION AND STUDY

bacteria killed by heat; the action of
ultra-violet rays, of chemical germicides,
or by autolysis).

II. By the inoculation of attenuated strains
of bacteria.

III. By the inoculation of living virulent
bacteria (exalted in virulence if neces-
sary).

B. Combined Active and Passive Immunisation :

IV. By the inoculation of toxin-antitoxin
mixtures.

ACTIVE IMMUNISATION.

The immunisation of the rabbit against the Diplococ-
cus pneumonias may be instanced as an example of the
general methods of immunisation of laboratory animals.

1. Take a full grown rabbit weighing not less than
1200 to 1500 grammes (large rabbits of 2000 grammes
and over are the most suitable for immunising experi-
ments). Observe weight and temperature carefully
during the few days occupied in the following steps.

2. Inoculate a small rabbit intraperitoneally with
one or two loopfuls of a twenty-four-hour-old blood
agar cultivation of a virulent strain of Diplococcus
pneumoniae.

Death should follow within twenty-four hours, and in
any case will not be delayed beyond forty-eight hours.

3. Under aseptic precautions, at the post-mortem,
transfer a loopful of heart blood to an Erlenmeyer flask
containing 50 c.c. sterile nutrient broth. Incubate at
3 7° C. for twenty-four hours.

4. Prepare also several blood agar cultures from the
heart blood of the rabbit, label them all O.C. (original
culture) . After twenty-four hours incubation at 3 7° C.
place an india-rubber cap over the plugged mouth of
the tube of all but one of these cultures and paint the
cap with Canada balsam or shellac varnish, dry, and
replace in the hot incubator.

ACTIVE IMMUNISATION 323

This will prevent evaporation, and cultures thus
sealed will remain unaltered in virulence for a consider-
able time.

5. Make a fresh subcultivation on blood agar from
the uncapped O. C. cultivation and after twenty-four
hours incubation at 37° C. determine the minimal
lethal dose of this strain upon a series of mice (see
page 3 1 6).

6. Suspend the flask containing the twenty-four-
hour-old broth culture (step 3) in the water-bath at
60° C. for one hour. Cool the flask rapidly under a
stream of cold water.

7. Determine the sterility of this (?) killed cultiva-
tion by transferring one cubic centimetre to each of
several tubes of nutrient broth, and incubate at 37° C.
for twenty -four hours. If growth of Diplococcus
pneumonias occurs, again heat culture in water- bath
at 60° C. for one hour and again test for sterility.

8. Inject the selected rabbit intravenously (see page
363) with 2 c.c. of the killed cultivation, and inject a
further 10 c.c. into the peritoneal cavity.

During the next few days the animal will lose some
weight and perhaps show a certain amount of pyrexia.

9. When the temperature and weight have again
returned to normal — generally about seven days after
the inoculation — again inject killed cultivation, this
time giving a dose of 5 c.c. intravenously and 20 c.c.
intraperitoneally. A temperature and weight reaction
similar to, but less marked than that following the
first injection will probably be observed, but after
about a week's interval the animal will be ready for the
next injection.

10. When ready to give the third injection prepare
a fresh blood agar subculture from another O. C. tube
and after twenty-four hours incubation prepare a
minimal lethal dose (as determined in 5) and inject it
subcutaneously into the rabbit's abdominal wall.

324

METHODS OF IDENTIFICATION AND STUDY

A slight local reaction will probably be observed as
well as the weight and temperature reactions.

1 1. A week to ten days later inject a similar minimal
lethal dose into the peritoneal cavity.

12. Observe the weight and temperature of the
rabbit very carefully, and regulating the dates of
inoculation by the animal's general condition, continue
to inject living cultivations of the pneumococcus
into the peritoneal cavity, gradually increasing the
dose by multiples of ten.

13. At intervals of two months samples of blood may
be collected from the posterior auricular vein and the
serum tested for specific anti-bodies.

14. Under favourable conditions it will be found
after some six months steady work that the rabbit
may be injected intraperitoneally with an entire blood
agar cultivation without any ill effects being apparent ;
and this characteristic — resistance to the lethal effects
of large doses of the virus — is the sole criterion of
immunity. Further, the serum separated from blood
withdrawn from the animal about a week after an
injection, if used in doses of .01 c.c., will protect a
mouse against the lethal effects of at least ten minimal
lethal doses of living pneumococci.

In the foregoing illustration it has been assumed that
complete acquired active immunity has been conferred
upon the experimental rabbit in consequence of the for-
mation of antibody, specific to the diplococcus pneu-
moniac, sufficient in amount to ensure the destruction
of enormous doses of the living cocci — the antigen (that
is the substance injected in response to which antibody
has been elaborated) in this particular case being the
bacterial protoplasm of the pneumococcus with its
endo- toxins.

But provided death does not immediately follow the
injection of the antigen, specific antibody is always
formed in greater or lesser amount; and in experi-

ACTIVE IMMUNISATION 325

mental work a sufficient amount of any required anti-
body can often be obtained without carrying the process
of immunisation to its logical termination.

For instance, if the immunisation of a rabbit toward
Bacillus typhosus is commenced on the lines already
set out it will often be found, after a few injections of
"killed" cultivation that the blood serum of the animal
(even when diluted with several hundred times its
volume of normal saline) contains specific agglutinin
for B. typhosus — and if the sole object of the experi-
ment has been the preparation of agglutinin the inocu-
lations may well be stopped at this point, although the
animal is not yet immune in the strict meaning of the
word.

Again, antibodies may be formed in response to
antigens other than infective particles — thus the injec-
tion into suitable animals of foreign proteins such as
egg albumin, heterologous blood sera or red blood discs
from a different species of animal, will result in the
formation of specific antibodies possessing definite
affinities for their respective antigens.

The most important antibody of this latter type is
Haemolysin, a substance that makes its appearance in
the blood serum of an animal previously injected with
washed blood cells from an animal of a different species.
The serum from such an animal possesses the power of
disintegrating red blood discs of the variety employed
as antigen and causing the discharge of their contained
haemoglobin, and is specific in its action to the extent
of failing to exert any injurious effect upon the red
blood cells of any other species of animal.

The action of this serum is due to the presence of
two distinct bodies, complement and haemolysin.

Complement (or alexine) is a thermo-labile readily
oxidised body present in variable but unalterable
amount in the normal serum of every animal. It is
a substance which exerts a lytic effect upon all foreign

326 METHODS OF IDENTIFICATION AND STUDY

matter introduced into the blood or tissues; but by
itself is a comparatively inert body, and is only capable
of exerting its maximum lytic effect in the presence of
and in combination with a specific antibody, or immune
body.

Complement is obtained (unmixed with antibody) by
collecting fresh blood serum from any healthy normal
(that is uninoculated) animal. Guinea-pigs' serum is
that most frequently employed for experimental work.

H&molysin (immune body, copula, sensitising body,
amboceptor) is a thermostable antibody formed in
response to the injection of red cells which although
in itself inert is capable of linking up complement
present in the normal serum to the red cells of the
variety used as antigen — a combination resulting in
haemolysis.

Haemolysin is obtained by collecting fresh blood serum
from a suitably inoculated animal and exposing it to a
temperature of 56° C. (to destroy the thermo-labile
complement) for 15 to 30 minutes before use. It is
then referred to as inactivated, and is reactivated by
the addition of fresh normal serum — that is serum
containing complement.

Haemolysin is of importance academically owing to
the fact that many of the problems of immunity have
been elucidated by its aid; but its present practical
importance lies in the application of the hcemolytic
system (that is haemolysin, corresponding erythrocyte
solution and complement) to certain laboratory
methods having for their object either ^the identifi-
cation of the infective entity or the diagnosis of the
existence of infection.

For use in these laboratory methods of diagnosis it is
most convenient to prepare haemolytic serum specific for
human blood — whether the laboratory is isolated or
attached to a large hospital. Ox blood, sheep blood or
goat blood if readily obtainable, may however be used

HJEMOLYTIC SERUM 327

instead, and although the following method is directed
to the preparation of human haemolysin the same
procedure serves in all cases.

THE PREPARATION OF H^MOLYTIC SERUM.

Apparatus Required:

Small centrifuge, preferably electrically driven, with two recep-
tacles for tubes, and enclosed in a safety shield (Fig. 162).

Sterile centrifuge tubes (10 c.c. capacity), Fig. 163.

FIG. 162. — Small electrical centrifuge.

Sterile pipettes (10 c.c. graduated) in case.

Sterile glass capsules (in case). ^^

Sterile test-tubes. ^ugliube™'

Sterile all glass syringe (5 c.c. or 10 c.c. capa-
city) and needle.
Reagents Required:

Normal saline solution.

10 per cent, sodium citrate solution in normal saline.

Human blood (vide infra}.

METHOD. —

1 . Select a healthy full-grown rabbit of not less than
2500 grammes weight in accordance with the directions
already given (page 322) and prepare it for intraperi-
toneal inoculation.

2. Measure out 2 c.c. citrated human blood (collected
at a surgical operation or a venesection, or withdrawn
by venipuncture from the median basilic or median
cephalic vein of a normal adult) into a centrifuge tube
and centrifugalise thoroughly.

328 METHODS OF IDENTIFICATION AND STUDY

3. Wash with three changes of normal saline (vide
also page 388).

4. Transfer the washed cells to a sterile capsule by
means of a sterile pipette. Add 5 c.c. of normal saline
and mix thoroughly.

5. Take up the mixture of cells and saline in the all-
glass syringe and inject into the peritoneal cavity of the
rabbit.

6. Seven days later inject intraperitoneally the
washed cells from 5 c.c. human blood mixed with 5 c.c.
normal saline.

7. Seven days later inject the washed cells from 10
c.c. human blood mixed with 5 c.c. normal saline.

8. After a further interval of seven days repeat the
injection of washed cells from 10 c.c. human blood
mixed with 5 c.c. normal saline.

NOTE. — Better results are obtained if the second and subsequent
injections are made intravenously, even when smaller quantities
of washed red cells are employed. If, however, the intravenous
route is selected exceeding great care must be exercised to avoid
the introduction of air into the vein — an accident which is fol-
lowed, within a few minutes, by the death of the rabbit from
pulmonary embolism.

9. Allow five days to elapse, then collect a prelimi-
nary sample of blood, say about 2 c.c., from the rabbit's
ear. Allow it to clot, separate off the serum and trans-
fer to a sterile test-tube. Place the test-tube in a
water-bath at 56° C. for fifteen minutes (to inactivate)
and test the serum quantitatively for haemolytic prop-
erties in the following manner:

THE TITRATION OF HAEMOLYTIC SERUM.

Apparatus Required,
Electrical centrifuge.
Sterile centrifuge tubes.
Water-bath regulated at 56° C.
Sterilised pipettes 10 c.c. graduated in tenths.
Sterilised pipettes i c.c. graduated in tenths.

TITRATING H^EMOLYSIN 329

Sterile test-tubes, 16X2 cm.
Small sterile test-tubes, 9X1 cm.
Small test-tube rack, or roll of plasticine.
Capillary teat pipettes.

Stout rubber band or length of small rubber tubing.
Reagents Required and Method of Preparation:

1. Normal saline solution.

2. Haemolytic serum inactivated by preliminary heating to
56° C. for 15 minutes (vide supra) in test-tube labelled H. S.

3. Complement. Fresh guinea-pig serum in test-tube labelled C.

Kill a normal guinea-pig with chloroform vapour.

Open the thorax with all aseptic precautions, and collect

as much blood as possible from the heart with a sterile

Pasteur pipette.
Transfer it to a sterile centrifuge tube and place the tube

in the incubator at 37° C. Two hours later separate

the clot from the sides of the tube, and centrifugalise

thoroughly.
Pipette off the clear serum to a clean sterilised test-tube.

4. Erythrocyte solution, in test-tube labelled E.

Collect and wash human red blood cells (see page 388, 1-8).
Measure the volume of red cells available and prepare a 2
per cent, suspension in normal saline solution.

METHOD. —

1. Take two test-tubes and number them i and 2,
and pipette into each 9 c.c. of normal saline solution.

2. Add i c.c. of haemolytic rabbit serum to tube No. i
and mix thoroughly: take up i c.c. of the mixture and
add it to tube No. 2 ; mix thoroughly.

3 . • Set up ten small test-tubes in test-tube rack or in
roll of plasticine, and number i to 10.

4. Pipette into tube No. i 0.5 c.c. =0.5 c.c. 1

haemolytic serum [ From tube

Pipette into tube No. 2 o.i c.c. = 0.1 c.c. { H. S.
haemolytic serum

Pipette into tube No. 3 0.5 c.c. = 0.05 c.c.

hasmolytic serum
Pipette into tube No. 4 0.3 c.c. = 0.03 c.c.

haemolytic serum
Pipette into tube No. 5 0.2 c.c. =0.02 c.c.

haemolytic serum
Pipette into tube No. 6 o.i c.c. = 0.01 c.c.

haemolytic serum

From
tube i.

330 METHODS OF IDENTIFICATION AND STUDY

Pipette into tube No. 7 0.5 c.c. = 0.005 c-c-

haemolytic serum
Pipette into tube No. 8 0.3 c.c. =0.003 c-c-

haemolytic serum [ From

Pipette into tube No. 9 0.2 c.c. =0.002 c.c. f tube 2.

haemolytic serum
Pipette into tube No. 10 o.i c.c. =0.001 c.c.

hsemolytic serum

5. To each tube add i c.c. of erythrocyte solution.

6. When necessary (that is to say in tubes 2, 4, 5, 6,
8, 9 and 10) add normal saline solution to the mixture
in the test-tubes till the column of fluid in each reaches
to the same level.

7. Shake each tube in turn, so as to thoroughly mix
its contents. Plug the mouth of each tube with cotton
wool, and place entire set in the incubator at 37° C.
for one hour.

8. Remove the tubes from the incubator and into
each tube pipette o.i c.c. complement (guinea-pig's
serum) and replace tubes in incubator at 3 7° C. for
further period of one hour.

9. Remove the tubes from the incubator, and if com-
plete haemolysis has not taken place in every tube,
stand on one side, preferably in the ice chest, for an
hour.

10. Then examine the tubes.

Complete haemolysis is indicated by a clear red
solution, with no deposit of red cells at the
bottom of the test-tube.

Absence of haemolysis is indicated by a clear or
turbid colourless fluid, with a deposit of red
cells at the bottom of the test-tubes.

The smallest amount of haemolytic serum that has
caused complete haemolysis is known as the minimal
haemolytic dose (M. H. D.) and if haemolysis has
occurred in all the tubes down to No. 7 — the m. h. d.
of this particular serum is .005 c.c. = 200 minimal

STORAGE OF H^EMOLYSIN 33!

haemolytic doses per cubic centimetre. Such a serum
is strong enough for experimental work; indeed, for
many purposes, complete haemolysis down to tube 6
will indicate a serum sufficiently strong ( = 100 m. h. d.
per cubic centimetre) . If, however, only the first one
or two tubes are completely haemolysed, this is an
indication that the rabbit should receive further in-
jections in order to raise the haemolytic power to a
sufficiently high level.

STORAGE OF H^EMOLYSIN.

If, and when the haemolysin content of the rabbit's
serum is found to be sufficient, destroy the animal by
chloroform vapour.

Remove as much of its blood as possible from the
heart under aseptic precautions into sterilized centri-
fuge tubes.

Transfer the tubes of blood to the incubator at
37° C. for two hours — then centrifugalize thoroughly.

Pipette off the clear serum, and fill in quantities of
i c.c., into small glass ampoules or pipettes, and her-
metically seal in the blow-pipe flame, care being taken
to avoid scorching the serum.

Place the ampoules when filled with serum and sealed,
in a water-bath at 56° C. for 30 minutes. This destroys
the complement, i. e., inactivates the serum, and at the
same time, provided the various operations have been
carried out under aseptic precautions, ensures its
sterility. A longer exposure reduces the haemolytic
power.

Place the ampoules in a closed metal box and store
in the ice chest for future use.

XVII. EXPERIMENTAL INOCULATION OF
ANIMALS.

The use of living animals for inoculation experiments
may become a necessary procedure in the Bacterio-
logical Laboratory for some one or more of the follow-
ing reasons :

A. Determination of Pathogenetic Properties of Bac=
teria already Isolated in Pure Culture (see page 315).

The exact study of the conditions influencing the
virulence (including its maintenance, exaltation and
attenuation) of an organism, and precise observations
upon the pathogenic effects produced by its entrance
into, and multiplication within the body tissues can
obviously only be carried out by means of experimental
inoculation; whilst many points relating to vitality,
longevity, etc., can be most readily elucidated by such
experiments.

B. Isolation of Pathogenetic Bacteria.

Certain highly parasitic bacteria (which grow with
difficulty upon the artificial media of the laboratory)
can only be isolated with considerable difficulty from
associated saprophytic bacteria when cultural methods
alone are employed ; but if the mixture of parasite and
saprophytes is injected into an animal susceptible to
the action of the former, the pathogenic organism can
readily be isolated from the tissues of the infected
animal. The pneumococcus for example occurs in
the sputum of patients suffering from acute lobar
pneumonia, but usually in association with various
saprophytes derived from the mouth and pharynx.
The optimum medium for the growth of the pneumo-
coccus, blood agar, is also an excellent pabulum for the

332

STUDY OF THE PROBLEMS OF IMMUNITY 333

saprophytes of the mouth, and plate cultures are
rapidly overgrown by them to the destruction of the
more delicate pneumococcus. But inoculate some of
the sputum under the skin of a mouse and three or
four days later the pneumococcus will have entered
the blood stream (leaving the saprophytes at the seat
of inoculation) and killed the animal. Cultivations
made at the post-mortem (see page 398) from the
mouse's heart blood will yield a pure growth of the
pneumococcus .

C. Identification of Pathogenetic Bacteria.

The resemblances, morphological and cultural, exist-
ing between certain pathogenetic bacteria are in some
cases so great as to completely overwhelm the differ-
ences; again the same bacterium may under varying
conditions assume appearances so different from those
regarded as typical or normal as to throw doubt on its
identity. In each case a simple inoculation experiment
may decide the point at once. As a concrete example
may be instanced an autopsy on an animal dead from
an unknown infection. Cultivations from the heart
blood gave a pure growth of a typical (capsulated)
pneumococcus. Cultivations from the liver gave a
pure growth of what appeared to be a typical (non-
capsulated) Streptococcus pyogenes longus. The latter
inoculated into a rabbit caused the death of the animal
from pneumococcic septicaemia, and cultures from the
rabbit's blood gave a pure growth of a typical (capsu-
lated) pneumococcus.

D. Study of the Problems of Immunity.

It is only by a careful and elaborate study of the
behaviour of the animal cell and the body fluids vis-a-
vis with the infecting bacterium that it becomes pos-
sible to throw light upon the complex problem whereby
the cell opposes successful resistance to the diffusion
of the invading microbe, or succeeds in driving out

334 EXPERIMENTAL INOCULATION OF ANIMALS

the microbe subsequently to the occurrence of that
diffusion.

At the moment, however, our attention is directed
to the first of these broad headings, for it is by the
application of the knowledge acquired in its pursuit
that we are able to deal with problems arising under
any of the remainder.

For whatever purpose the inoculation is performed,
it is essential that the experiment should be planned to
secure the maximum amount of information and the
minimum of discomfort to the animal used. Every
care therefore must be taken to ensure that the virus is
introduced into the exact tissue or organ selected ; and
the operation itself must be carried out with skill and
expedition, and under strictly aseptic conditions.

In the course of inoculation studies many instances of
natural immunity, both racial and individual, will be
met with; but it must be recollected that natural im-
munity is relative only and never absolute, and care
be taken not to label an organism as non-pathogenic
until many different methods of inoculation have been
performed upon different species of animals, combined
when necessary with various procedures calculated to
overcome any apparent immunity, and have invariably
given negative results.

In some countries experiments upon animals are
only permitted under direct license from the Govern-
ment, and then only within premises specially licensed
for the purpose. In England this license is in the
grant of the Home Secretary, and confers the permis-
sion to experiment upon animals under general anaes-
thesia, provided that after the experiment is completed
the animal must be destroyed before regaining con-
sciousness. If it is intended to carry out simple
hypodermic inoculations and superficial venesections,
Certificate A, granting this specific permission and
dispensing with the necessity for general anaesthesia

PREPARATION 335

must be obtained in addition to the license; whilst if
the inoculation entails more extensive operative pro-
cedures, and it is necessary to observe the subsequent
course of the infection, should such occur, the license
must be coupled with Certificate B — since this certifi-
cate removes the compulsion to destroy the animal
whilst under the anaesthetic. Further special certifi-
cates and combinations of certificates are required if
cats, dogs, horses, asses or cattle are to be the subjects
of experiment. Under every certificate it is expressly
stipulated that if the animal shows signs of pain it must
be destroyed immediately.

The animals generally employed in the study of the
pathogenic properties of the various micro-organisms
are:

Cold Blooded Warm Blooded. Hot Blooded.

Frog. Mouse. Fowl.

Toad. Rat. Pigeon.

Lizard. Guinea pig.

Rabbit.

Monkey.

Preparation. — Before inoculation, the experimental
animals should be carefully examined, to avoid the
risk of employing such as are 'already diseased: since
it must be remembered that in a state of nature, as
well as in captivity, the animals employed for labora-
tory inoculations are subject to infection by various
animal and vegetable parasites, and in some instances
such infection presents no symptoms which are ob-
vious to the casual examination; the sex should be
noted, the weight recorded, and the rectal tempera-
ture taken. The remaining items of importance are
the time of the inoculation, the material that is inocu-
lated, and the method of inoculation, and finally under
what authority the experiment is performed. In the
author's laboratory these data are entered upon a pink
card which forms part of a card index system. The

336 EXPERIMENTAL INOCULATION OF ANIMALS

card further provides space for notes on the course of
the resulting infection, and carries on the reverse the
weight and temperature chart (Figs. 164 and 165).

Preliminary Inspection and Examination. — The pre-
liminary examination should comprise observation

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of the animal at rest and in motion; the appearance
of the fur, feathers or scales, inspection of the eyes,
and of external orifices of the body; tactile examina-
tion of the body and limbs, and palpation of the groins

THE RABBIT

337

and abdomen; and in many cases the microscopical
examination of fresh and stained blood-films.

Some of the commoner forms of naturally acquired
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touching upon the various fleas, lice and ticks which
at times infect the ordinary laboratory animals.

The Rabbit, particularly in captivity, is subject to

338 EXPERIMENTAL INOCULATION OF ANIMALS

attacks of Psoric Acari, and the infection is readily
transmitted to rabbits in neighbouring cages and also
to guinea pigs, but not to rats and mice. One species
(Sarcoptes minor var. cunicuU) gives rise to the ordi-
nary mange. The infection first shows itself as thick
yellowish scales and crusts around the nose, mouth
and eyes, spreads to the bases and outer surfaces of
the ears (never to the inside of the concha), to the
fore and hind legs and into the groins and around
the genitals. The acari can be readily demonstrated
microscopically in scrapings of the skin, treated with
liquor potassae. Another form of scabies (due to
Psoroptes communis cuniculi) commences at the bottom
of the concha, which is filled with whitish-yellow masses
consisting of dried crusts, scales, faeces, and dead acari.
The base of the ear is hard and swollen, and lifting the
animal by the ears — as is usually done — gives rise to
considerable pain; indeed this symptom may be the
one which first attracts attention to an infection, which
causes progressive wasting and terminates in death.
A mixed infection — sarcoptic plus psorotic acariasis — •
is sometimes seen.

If it is decided to try and save animals suffering from
infection by these parasites, they must be segregated,
the scabs carefully cleaned from the infected areas and
the denuded surfaces washed with 5 per cent, solution
of Potassium persulphate (a few drops being allowed
to run into the concha) , or with a preparation contain-
ing equal parts of soft paraffin and vaseline with a few
drops of lysol. This treatment should be repeated
daily until the acarus is destroyed and the animal
has regained its normal condition. The cages should
be disinfected and all neighbouring animals carefully
examined, and any which show signs of infection
should be treated in a similar manner. Favus also
attacks the rabbit, and the typical spots are first noted
around the base of the ear.

THE MONKEY 339

Infection by Coccidium oviforme is very common,
without however presenting any symptoms by which
the infection may be recognised. . Usually the condi-
tion is only noted post-mortem, when the liver is found
to be studded with numerous caseating tubercles,
which on examination prove to be cystic areas crowded
with coccidia. Sometimes too the liver of a rabbit
dead from some intentional or accidental bacterial
infection is found at the post-mortem to be marked by
fine yellowish streaks and small tubercles due to the
embryos of Tcenia serrata, while the cystic form
(Cysticercus pisiformis) is often noted free in the
peritoneal cavity, or invading the mesentery.

Abscess formation from infection with ordinary
pyogenic bacteria occurs naturally in the rabbit, and fre-
quently the animal house of a laboratory is decimated
by an infective septicaemia due to B. cuniculicida.

The Mouse and Rat suffer from septicaemia, and from
the cysticercus form of Tcunia murina; the cystic form
(Cysticercus fasciolaris) of T. crassicollis has its habitat
in their livers. These small rodents are frequently
infected with scabies, but if freely provided with clean
straw will clean themselves by rubbing through it.
The mouse is also attacked by favus, and the rat is
often infected with Trypanosoma Lewisi.

The Guinea pig, like the rabbit, suffers from scabies
and coccidiosis. In addition it is often naturally
infected with B. tuberculosis, and it is a wise precaution
to test animals as soon as they reach the laboratory by
injecting Koch's Old Tuberculin — 0.5 c.c. causing death
in the tuberculous cavy within 48 hours.

The Monkey is naturally prone to tuberculosis, and
should be injected with i c.c. Old Tuberculin on
arrival in the laboratory. The tissues of the monkey
also serve as the habitat for a Nematode worm para-
sitic in cattle (CEsophagostoma inflatuni) resembling the
Anchylostomum, and this parasite frequently bores

340

EXPERIMENTAL INOCULATION OF ANIMALS

through the intestinal wall, and provokes the forma-
tion of small cysts in the immediately adjacent mesen-
tery. The presence of these cysts may give rise to
considerable speculation at the post-mortem.

The Pigeon may be infected by H&mosporidia, and
its blood show the presence of halteridia. This bird
may also be the subject of a bacterial infection known
as pigeon diphtheria; while the fowl may be subject
to scabies and ringworm, or suffer from fowl cholera
or fowl septicaemia — infections due to members of
the haemorrhagic septicaemia group.

Weighing. — The larger animals are most conveniently
weighed in a decimal scale provided with a metal cage
for their reception instead of the ordinary pan (Fig.
1 66). Mice and rats are weighed in a modification

FIG. 166.— Rabbit scales.

of the letter balance, weighing to 250 grammes, which
has a conical wire cage, (carefully counterpoised)
substituted for its original pan (Fig. 167) .

Temperature.— To take the rectal temperature of
any of the laboratory animals, the animal should be
carefully and firmly held by an assistant. Introduce
the bulb of an ordinary clinical thermometer, well
greased with vaseline, just within the sphincter ani.
Allow it to remain in this position for a few seconds,

CAGES

341

and then push it on gently and steadily until the en-
tire bulb and part of the stem, as far as the constric-
tion, have passed into the rectum. Three to five min-
utes later, the time varying of course with the sensi-
bility of the thermometer used, withdraw the instru-
ment and take the reading. The thermometers em-

FIG. 167. — Mouse scales

ployed for recording temperature should be verified
from time to time by comparision with a standard
Kew certified Thermometer kept in the laboratory
for that purpose.

Cages. — During the period which elapses between
inoculation and death, or complete recovery, the ex-
perimental animals must be kept in suitable recep-
tacles which can easily be kept clean and readily
disinfected.

The mouse is usually stored in a glass jar (Fig. 168)

342

EXPERIMENTAL INOCULATION OF ANIMALS

ii cm. high and n cm. in diameter, closed by a wire
gauze cover which is weighted with lead or fastened
to the mouth of the jar by a bayonet catch. A small
oblong label, 5 cm. by 2.5 cm., sand-blasted on the
side of the cylinder, is a very convenient device as
notes made upon this with an ordinary lead pencil
show up well and only require the use of a damp cloth
to remove them (Fig. 168).

The rat is kept under observation in a glass jar simi-
lar, but larger, to that used for the mouse.

FIG. 1 68. — Mouse jar.

FIG. 169. — Tripod.

A layer of sawdust at the bottom of the jar absorbs
any moisture and cotton- wool or paper shavings should
be provided for bedding. The food should consist of
bran and oats with an occasional feed of bread-and-
milk sop.

The use of a metal tripod, on the platform of which
are soldered two small cups for the reception of the
food, inside the cage, prevents waste of food or its con-
tamination with excreta (Fig. 169).

After use the jars and tripods are sterilised 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. 170). The

CAGES

343

sides and top and bottom are of woven wire work;
beneath the cage is a movable metal tray filled with saw-
dust, for the reception of the excreta. The cage as a
whole is raised from the ground on short legs . The sides,
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 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 cm. by 24 cm., for it to
stand upon.

FIG, 170. — Metal rabbit rage.

A plentiful supply of straw should be provided
for bedding and the. food should consist of fresh vege-
tables, cabbage leaves, carrot and turnip tops and the
like for the morning meal and broken animal biscuits
for the evening meal. Occasionally a little water may
be placed in the cage in an earthenware dish.

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.

These cages are sterilised after use either by auto-
claving or spraying with formalin.

344 EXPERIMENTAL INOCULATION OF ANIMALS

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 inoculations
given below comprises practically everything needed
for any inoculation. Needless to remark, all the appa-
ratus will never be required for any one inoculation.

Apparatus Required for Animal Inoculation :

i. Water steriliser (vide page 33) . 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.

0»

FIG. 171. — Hypodermic syringe with finger rests.

2. Injection syringe. The best form is one of the ordinary
hypodermic pattern, i c.c. capacity graduated in twentieths of a
cubic centimeter (0.05 c.c.), fitted with finger rests, but with the
leather washers and the packing of the piston replaced by those
made of asbestos (Fig. 171). The instrument must be easily taken
to pieces, and spare parts should be kept on hand to replace acci-
dental breakage or loss. Other useful syringes are those of 2 c.c.,
5 c.c., 10 c.c., and 20 c.c. capacity. 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. Operating table.

4- Surgical instruments. Sterilise these before use by boiling,
and disinfect them after use by the same means. Wipe perfectly
dry immediately the disinfection is completed.

APPARATUS REQUIRED FOR ANIMAL INOCULATION 345

Scissors, probe and sharp-pointed.

Dissecting forceps of various patterns.

Pressure forceps.

Retractors (small self retaining Fig. 172).

Aneurism needles, sharp and blunt.

Scalpels, j

Keratomes, > with metal handles.

Trephines, J

Michel's steel clips and special forceps for applying the same.

These small steel clips enable the operator to easily and

rapidly close skin incisions and are most satisfactory for

animal operations.
Surgical needles.
Needle holder.

Soft rubber catheters, various sizes.
Gum elastic cesophageal bougies with con- FIG. 172. Small

nection to fit syringe. self retaining re-

5. Anesthetic.

(a) General: The safest general anaesthetic for animals is an
A. C. E. mixture, freshly prepared, 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, or from a saturated cotton -wool
pad packed into the bottom of a small beaker.

(6) Local:

1. Cocaine hydrochloride, 2 per cent, in adrenalin i per
mille solution.

2. Beta-eucaine, 2 per cent, in adrenalin, i per mille
solution.

3. Ethyl chloride jet.

6. Sterile glass capsules of various sizes.

10 c.c. (in tenths of a cubic

._ . centimetre).

7. Cases of sterile pipettes ' ^, , , .

i c.c. (in hundredths of a cubic

centimetre) .

8. Flasks (75 c.c.) containing sterilised normal saline solution
(or sterile bouillon) .

9. Sterilised cotton-wool. Cotton-wool (absorbent) is packed
loosely in a copper cylinder similar to that used for storing capsules,
and sterilised in the hot-air oven.

10. Sterilised gauze. Gauze is sterilised in the same way as
cotton- wool.

11. Sterilised silk and catgut for sutures. These are sterilised,
as required, by boiling for some ten minutes in the water steriliser.

12. Flexible collodion (or compound tincture of benzoin).

13. Grease pencil.

14. Tie-on celluloid labels, to affix to the cages.

346 EXPERIMENTAL INOCULATION OF ANIMALS

15. Razor.

1 6. Small pot of warm water.

17. Liquid soap. Liquid soap is prepared as follows: Meas-
ure 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."

1 8. In place of the liquid soap and razor it is sometimes con-
venient to use a Depilatory powder.

Barium sulphide i part

Rice starch 3 parts

Dust the powder thickly over the aiea to be denuded of hair,
spi inkle with water and mix into a thin paste in situ; allow the
paste to act for three minutes, then scrape off with a bone spatula
— the hair comes away with the paste and leaves a perfectly bare
patch. This process is preferably carried out, the day previous to
the operation.

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.

MATERIAL UTILISED FOR INOCULATION

347

(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 ;
or take up the required quantity of the cultivation
directly into the graduated syringe from the tube or
flask. -

If it is necessary to introduce a large bulk of fluid
into the animal, the cultivation should be transferred

FIG. 173. — Conical separately funnel, fitted for injection of fluid cultivations.

with aseptic precautions, to a sterile separatory funnel,
preferably of the shape shown in figure 173, 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 piece of
sterilised rubber tubing of suitable length, fitted with an
injection needle and provided with a screw clamp, is
now attached to the nozzle of the funnel and the opera-

348

EXPERIMENTAL INOCULATION OF ANIMALS

tion completed according to the requirements of the
particular case.

This method is quite satisfactory when the injection
is made into the pleural or abdominal cavities or directly
into a vein but 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 hand bellows to the air inlet tube (interposing an
air filter) and attach the tubing with the injection
needle to the outlet tube (Fig. 174). By careful use
sufficient force can be obtained to drive the injec-
tion in.

(b) Cultivations on Solid Media (e. g., Sloped Agar) . —
i. By means of a sterile graduated pipette introduce

FIG. 174. — Arrangement of pressure injection apparatus.

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,
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.

METHODS OF SECURING ANIMALS 349

(c) Toxins. — Prepared by previously described
methods (vide page 318), 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 treated as fluid cultivations ; but if the material 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 agate mortar (using an
agate pestle, with a small quantity of sterile bouillon,
and the syringe filled from the resulting emulsion.

FIG. 175. — Holding rabbit 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 wound by
means of Michel's steel clips and 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. 175).

If working single handed Voge's holder for guinea-
pigs, is a useful piece of apparatus the method of using
which is readily seen from the accompanying figures
(Figs. 176, 177).

The instrument itself consists of a hollow copper

350

EXPERIMENTAL INOCULATION OF ANIMALS

cylinder, one end of which is turned over a ring of stout
copper wire, and from this open end a slot is cut ex-
tending 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 resist-
ing attempts to remove it from
the holder backwards, this cap
is taken off and the holder
placed on the table and the
guinea-pig allowed to walk out.

To provide for different-sized
animals, two sizes of this holder
will be found useful :

1. Length, 16 cm.; breadth, 6
cm. ; size of slot, 8 cm. by 2.5 cm.

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 178, the tail being securely held by
the spring clip. Needless to say, the holder should

FIG. 176. — Taking guinea-
pig's temperature.

FIG. 177. — Voge's holder.

be entirely of metal, and the wire cage detachable and
easily renewed.

When the animal is anaesthetised, it is more conven-
ient to secure it firmly to some simple form of operat-
ing table, such as Tatin's (Fig. 179), which will accom-

OPERATION TABLE 351

modate rabbits, guinea-pigs, and rats: or to the more
elaborate table devised by the author (Fig. 180).

Operation Table. — This is a table of the "aseptic"
type, composed of steel tubing, nickel-plated or enam-
elled. The table-top frame is sufficiently large to
accommodate rabbits, dogs and monkeys; and is sup-

FIG. 178. — Mouse holder.

ported upon telescopic uprights, so that it is adjustable
as to height ; in its long axis it can be inclined (at either
end) to 45° from the horizontal. Further it can be
completely rotated about its long axis. The table-
top itself is composed of a sheet of copper wire gauze

FIG. 179. — Taten's operation table.

loosely suspended from the long sides of the tubular
frame. The slackness of the gauze bed permits of an
india rubber hot water bottle, or an electrotherm being
placed under the animal, and if during the course of
an experiment it is necessary to reverse the animal,

352

EXPERIMENTAL INOCULATION OF ANIMALS

the table-top frame is completely rotated, the device
adopted for suspending the gauze is detached and the
gauze reversed also, so that it again supports the animal
from below

FIG. 180. — Author's operating table1.
METHODS OF INOCULATION.

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. — (Anesthetic, 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

1 This table is made by Messrs. Down Bros., St. Thomas's Street, London,
S. E.

SUBCUTANEOUS 353

selected for inoculation, with a wad of cotton- wool,
and lather freely by the aid of warm water; shave
carefully and thoroughly; or apply the depilatory
powder.

3. Wash the denuded area of skin thoroughly with
2 per cent, lysol solution.

4. Wash off the lysol with ether and allow the latter
to evaporate.

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.

(c) Particulars as to source and dose of inoculum.

(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 right hand, enter the needle into
a ridge of skin raised by the left finger and thumb,
and push it steadily onward 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 a wad of cotton wool, to prevent
the escape of any of the inoculum. The injected fluid,

23

354 EXPERIMENTAL INOCULATION OF ANIMALS

unless large in amount, will be absorbed within a very
short time.

7. Label, etc.

(6) Solid Inoculum. — (Anesthetic, none; or Ethyl
chloride spray.}

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 or with the point of a
scalpel.

6. Insert a probe through the opening and push it
steadily onward 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 toward 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.

FIG. 181. — Glass tube syringe for subcutaneous "solid" inoculation.

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
the end of the tube, and withdraw the apparatus,
leaving the tissue behind in the wound.

8. Close the wound in the skin with Michel's clips
and a dressing of gauze sealed with collodion (or Tinct.
benzoin) .

9. Label, etc.

INTRAPERITONEAL 355

3, Intramuscular.—

(a) Fluid Inoculum. — (Ancesihetic, 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.

(6) 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 incision.

6. Close the wound in the muscle with buried sutures
and the cutaneous wound with either continuous or
interrupted sutures or with Michel's steel clips.

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. — (Anesthetic, 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
apposition) one over the other, ascertain that no coils
of intestine are included in the fold.

356 EXPERIMENTAL INOCULATION OF ANIMALS

6. Take the syringe in the right hand and with the
needle transfix the fold near its base (Fig. 182) .

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 (see Fig. 183).

FIG. 182. — Intraperitoneal inoculation — fluid.

8. Inject the fluid from the syringe.

9. Label, etc.
Second Method:

Steps 1-4. As in the first method.

5. Anaesthetise a small selected
area of skin by spraying it with
ethyl chloride.

6. Heat platinum searing wire
(0.5 mm. wire, twisted to the

FIG. 183.— Section of ab- shape indicated in figure 184,

dommal wall, etc., showing ., . <. . ^ .

point of needle lying free mounted in an aluminum handle)

t0 redneSS' and With it bum a
hole through the anaesthetic area

of skin and abdominal muscle down to, but not through,
the visceral peritoneum.

7. 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.

8. Inject the fluid from the syringe.

Ibo

COLLODION SACS 357

9. 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.

FIG. 184. — Platinum wire for burning hole through parietes.

(6) Solid Inoculum (or the implantation of capsules
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 Lembert's su-
tures.

8. Close the skin and aponeurosis incisions together
with interrupted sutures or Michel's steel clips, and
apply a sealed dressing.

9. Release the animal from the operating table.

10. Label, etc.

Suitable sacs may be readily prepared by either of
the following methods*

A. Collodion Sacs.

i. Dip a small test-tube (5 by 0.5 cm.), bottom
downward, into a beaker of collodion, and dry in the
air; repeat this process three or four times.

358 EXPERIMENTAL INOCULATION OF ANIMALS

2. Dip the tube, with its coating of collodion, alter-
nately 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
steam steriliser 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.

B. Celloidin Sacs (Harris).
Materials Required.

Quill glass tubing.

Gelatine capsules such as pharmacists prepare for the exhibition
of bulky powders.

Various grades of celloidin, thick and thin, in wide-mouthed
bottles.

1. Take a piece of quill glass tubing some 4 cm. long
by 5 mm. diameter ; heat one end in the bunsen flame.

2. Thrust the heated end of the tube just through

CELLOIDIN SACS 359

one end of a gelatine capsule and allow it to cool
(Fig. 185).

3 . Remove any gelatine from the lumen of the tube
with a heated platinum needle; paint the joint between
capsule and tube with moderately thick celloidin and
allow to dry.

4. Dip the capsule into a beaker containing thin
celloidin, beyond the junction with the glass and after
removal rotate it in front of the blow-
pipe air blast to dry it evenly. Repeat

these manoeuvres until a sufficiently thick
coating is obtained.

5. Apply thick celloidin to the tube-
capsule joint, the opposite end of the cap-
sule, and the line of junction of the cap-
sule with its cap ; dry thoroughly.

6. With a teat pipette fill the capsule
(through the attached tube) with hot
water, and stand the capsule in a beaker
of boiling water for a few minutes to

melt the gelatine. Making celioidin

7 . Remove the solution of gelatine from capsules.
the interior of the celloidin case with a pipette.

8. Fill the sac with nutrient broth and place it,
glass tube downward, in a tube containing sufficient
sterile nutrient broth to cover the sac to the depth
of i cm. Plug the tube and sterilise in the steamer
in the usual manner.

9. To prepare the sac for use, empty it out of the
broth tube into a sterile glass dish.

10. Grasp the tube near its junction with the sac
in the jaws of sterile forceps, and with a teat pipette
remove sufficient of the contained broth to leave a
small space in the sac. Introduce the inoculum in the
form of an emulsion by means of another pipette.

11. Still holding the tube in the forceps, draw it
out and seal off near the sac in the blowpipe flame.

360 EXPERIMENTAL INOCULATION OF ANIMALS

12. When cool wash the sac in sterile water, then
transfer to a tube of nutrient broth and incubate
over night to determine its impermeability to bacteria.

13. If the broth outside the sac remains sterile,
insert the sac in the peritoneal cavity of the experi-
mental animal.

5. Intracranial. — (Anesthetic, A. C. E.)
Trephines and Surgical Engine. — The most useful
instrument for intracranial operations upon animals
is the small nasal trephine (Curtis) having a tooth cut-

FIG. 1 86.— Guarded trephine.

ting circle of 7 mm. The addition of an adjustable
collar guard — secured by a screw — prevents acciden-
tal laceration of the dura mater or brain substance1
(Fig. 1 8 6) . This size is suitable for monkeys, dogs, cats
and large rabbits. Other smaller sizes which will be
found useful for guinea pigs and other small animals
cut circles of 6 and 4 mm. ; for very small animals —
young guinea pigs and rats — a small dental drill or
screw will make a sufficiently large hole to admit the
syringe needle. The trephine can be set in ordinary
metal handles and rotated by hand, but a surgical
engine of some kind is much preferable on the score
of rapidity and safety to the animal. The Guy's elec-
trical Dental engine2 (Fig. 187) which can be connected
to a lamp socket or wall plug, and is operated by a foot
switch, although inexpensive is eminently satisfactory.

NOTE. — A fine dental drill attached to the dental engine renders
the manufacture of aluminium handles needles (see page 71)
quite an easy matter.

1 This modification is made for the author by Messrs. Down Bros., St.
Thomas's Street, London, S. E.

2 Manufactured by Messrs. Francis Lepper, 56, Great Marlborough
Street, London, W.

INTRACRANIAL 361

(a) Subdural.

1. Anaesthetise the animal and secure it to the oper-
ating table, dorsum uppermost.

2. Shave a portion of the scalp immediately in front
of the ears.

3. Mark out with a sharp scalpel a crescentic flap

FIG. 187. — Guy's electrical dental engine.

of skin muscle, etc., convexity forward, 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. Make a corresponding incision through the perios-
teum and raise it with a blunt dissector.

5. With a small trephine (diameter 6 mm.) remove
a circular piece of bone from the parietal segment.
The centre of the trephine hole should be at the inter-
section of the median line and a line joining the pos-
terior canthi (Fig. 1 88).

6. Introduce the inoculum by means of a hypo-
dermic syringe, perforating the dura mater with the
needle and depositing the material immediately below
this membrane, at the same time taking care to avoid
injuring the sinuses.

362

EXPERIMENTAL INOCULATION OF ANIMALS

7. Turn back the flap of skin and secure it in position
with Michel's steel clips.

8. Dress with sterile gauze and wool and seal the
dressing with collodion.

9. Label, etc.

(b) Intracerebral. — This inoculation is performed
precisely as for subdural save in step
6 the needle after perforating the dura
mater is pushed onward into the sub-
stance of one or other cerebral hemis-
pheres before the contents are ejected.

6. Intraocular. —

(a) Fluid Inoculum. — (Anesthetic,
cocaine.)

1. Instil a few drops of a sterile solu-
tion of cocaine, and repeat the instilla-
tion in two minutes.

2. Five minutes later have the animal
firmly held by an assistant as in intra-
venous injection (see Fig. 189) the head
being steadied by the assistant's hands.

3. Select two needles to accurately fit
the same syringe and sterilise.

4. Attach one needle to the syringe
and take up the required dose of inocu-
lum and remove the needle.

5. Steady the eye with fixation forceps; then pierce
the cornea with the other syringe needle and allow the
aqueous to escape through the needle.

6. Without removing the needle from the cornea
attach the syringe and make the injection into the
anterior chamber.

7. Irrigate the conjunctival sac with sterile saline
solution.

8. Label, etc.

(b) Solid Inoculum. — (Anesthetic, A. C. E.)

FIG. 188.—
tracranial inocula-
t i o n of rabbit.

of the
hole.

trephine

INTRAVENOUS 363

1. Anaesthetise the animal and secure it firmly to
the operating table.

2. Irrigate the conjunct! val 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. Separate the lips of the corneal wound with a
flexible silver spatula; 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. — (Anesthetic, 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.)

The site selected for the injection in the rabbit is the
posterior auricular vein (see Fig. 192). 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 general anaes-

364 EXPERIMENTAL INOCULATION OF ANIMALS

thetic must be administered to the animal. In the
monkey or the dog, the internal saphenous vein is the
most convenient and before puncturing should be dis-
tended or rendered prominent by compressing the vein
above the selected site.)

Preparation of the Inoculum. — 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. A piece of sterile filter paper
is then impaled on the needle and the piston of the
syringe pressed upward until all the air is expelled from
the barrel and needle. Should any drops of the inocu-
lum be forced out, they will fall on the filter paper,
which should be immediately burned.

1. Have the animal firmly held by an assistant.
The selected ear is grasped at its root and stretched
forward toward 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 ether 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.

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. 189) .
Now press it onward in the direction of the blood
stream — i. e., toward the body of the animal.

6. Direct the assistant to cease compressing the

INHALATION 365

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 at a spot closer to the root of the ear or
at some point on the corresponding vein on the opposite
ear.) -

7 . Withdraw the needle and press a pledget of cotton-
wool over the puncture to ensure closure of the aperture
in the vein wall.

8. Label, etc.

FIG. 189. — Intravenous inoculation.

9. Inhalation. —

(a) Fluid Inoculum. — (Anesthetic, 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.

366 EXPERIMENTAL INOCULATION OF ANIMALS

6. Label, etc.

7. Thoroughly disinfect the inhalation chamber.

(6) Fluid or Powdered Inoculum. — A n&sthetic, A.C.E.
i. Anaesthetise the animal and secure it firmly to
the operating table.

FIG. 190. — Gag for rabbits.

2. Prop open the mouth by means of some form of
gag ; seize the tongue with a pair of forceps and draw it
forward.

The most convenient form of gag for the rabbit or
cat is that shown in Fig. 190. It is simply a strip of
hard wood shaped at the middle and provided with a
square orifice through which a tracheal or cesophageal
tube can be passed.

3. Pass a previously sterilised glass tube (17 cm.
long, 0.5 cm. diameter, with its terminal 2 cm. slightly
curved) down through the larynx into the trachea.

4. 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.

5. Allow the fluid inoculum to run into the lungs by
gravity, or blow in the powdered inoculum by means
of a rubber-ball bellows.

6. Remove the intratracheal tube ; release the animal
from the table.

7. 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.

INTRAGASTRIC 367

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 or even a teat pipette.

10. Intragastric Inoculation. — Fluid or semifluid in-
oculum. (Anaesthetic none.)

The method of performing the operation is varied
slightly according to the size of the experimental animal.

A. Monkey, Rabbit, Guinea-pig.

1. Secure the animal to the operating table ventral
surface uppermost.

2 . Prop the mouth open with a gag ; draw the tongue
forward with forceps.

3. Sterilise a soft rubber catheter (No. 10 or 8 Eng-
lish scale, or No. 1 8 or 15 French) and lubricate it with
sterile glycerine.

4. Pass it to the back of the pharynx, keeping the
end in the middle line.

5 . Gently assist the progress of the catheter down the
oesophagus until it passes the cardiac orifice of the
stomach. Do not use any force.

6. Take up the required dose of inoculum into a
sterilised pipette. Insert the point of the pipette
into the open end of the catheter and allow the fluid
to run down into the stomach. Remove the pipette
and drop it into a jar of lysol.

7. -With another sterile pipette run one cubic cen-
timetre of sterile saline solution through the catheter
to wash out the last, traces of the inoculum.

8. Withdraw the catheter.

9. Label, etc.

B. Rats and Mice (Mark's Method).

•i. Secure the animal in the vertical position.

(a) Rat. — Take a pair of catch sinus forceps about
22 cm. in length and seize the animal by the loose skin
of the head as far forward as possible — fix the forceps,
and holding the instrument vertically upward, transfer

368

EXPERIMENTAL INOCULATION OF ANIMALS

to the left hand of an assistant who secures the animal's
tail between the fingers grasping the handle of the for-
ceps. (See Fig. 191.)

(b) Mouse. — An assistant grasps the loose skin
between the ears as far forwards as possible between
the fore-finger and thumb of the left hand. He now

FIG. 191. — Intragastric inoculation of rat.

grasps the tail with the right hand, draws the mouse
straight and passes the tail between the fourth and little
fingers of the left hand and secures it there.

2. The assistant takes a closed pair of thin-bladed
forceps in his right hand, passes the ends into the
animal's mouth, then allows the blades to separate.
This opens the animal's jaw and serves as a gag.

FEEDING 369

3 . Moisten the sterilised oesophageal tube with sterile
water. (This tube is of silk rubber, 6.5 cm. in length,
with the distal end rounded, the proximal end mounted
in a syringe needle head, which fits the nozzles of the
two sterile syringes to be used.)

4. Grasp the tube about its middle and pass it into
the animal's mouth, downwards and a little to one side
or the other until its length is lost in the digestive tract
and mouth. Gentle guidance is alone necessary. Do
not use any force.

5. Take up the required dose of inoculum into the
syringe ; insert the nozzle of the syringe into the needle-
mount, and force the piston down.

6. Steadying the needle-mount with the left hand,
detach the syringe.

7. Draw up some sterile water in the second (sterile)
syringe, and inserting its nozzle into the needle-mount
force a few drops of water through the tube to wash it
out.

8. With one quick upward movement remove the
tube from the animal's mouth.

9. Label, etc.

One other method of inoculation remains to be
described, which does not require operative inter-
ference.

11. 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.

XVIII. THE STUDY OF EXPERIMENTAL
INFECTIONS DURING LIFE.

The possession of pathogenetic properties by an
organism under study is indicated by the " infection"
of the experimental animal — a term which is employed
to summarise the condition resulting from the success-
ful invasion of the tissues of the experimental animal
by the micro-organisms inoculated and by their multi-
plication therein. Infection is considered to have
taken place :

1. When the death of the animal is produced as a
direct consequence of the inoculation.

2. When without necessarily producing death the
inoculation causes local or general changes of a patho-
logical character.

3. When either with or without death, or local or
general changes occurring, certain substances make
their appearance in the body fluids, which can be
shown (in vitro or in vivo) to exert some profound and
specific effect when brought into contact with sub-
cultivations of the organism originally inoculated.

The important factors in the production of infection
are:

A. Seed. Virulence of organism.

Dose of organism.

B. Soil. Resistance offered by the cells of the

experimental animal.

The first two factors, although variable, are to a
certain extent under the control of the experimenter.
Thus by suitable means the virulence of an organism

37°

GENERAL OBSERVATIONS 37!

can be exalted or attenuated, whilst the size of the dose
may be increased or diminished. The third factor
also varies, not only amongst different species of
animals, but also amongst different individuals of the
same species. The essential causes of this variation
are not so obvious, so that beyond selecting the animals
intended for similar experiments with regard to such
points as age, size or sex, but little can be done to
standardise cell resistance.

Immediately an animal has been inoculated a period
of clinical observation must be entered upon, which
should only terminate with the death of the animal.
The general observations should at first and if the
infection is an acute one, be made daily — later, and if
the animal appears to be unaffected or if the infection
is chronic, both general and special observations
should be carried out at weekly intervals. If the ani-
mal appears to be still unaffected, it should be killed
with chloroform vapour at the end of two or three
months and a complete post-mortem carried out.

A. The general observations should take cognisance
of:

1. General appearance. The experimental animal
should be inspected daily, not only with a view to
detecting symptoms due to the experimental infection,
but also to prevent any intercurrent infection, natur-
ally acquired, from escaping notice (vide page 337).

2. The weight of the inoculated animal should be
observed and recorded each day during the course
of an experimental infection at precisely the same hour,
preferably just before the morning feed.

3. The temperature should similarly be recorded
daily, if not more frequently, during the whole period
the animal is under observation, and carefully charted
—individual variations will at once become apparent.
It should be borne in mind that the temperature re-
garded as normal for man (37.5° C.) is not the normal

372

EXPERIMENTAL INFECTIONS DURING LIFE

average temperature of any of the lower animals save
the rat and mouse. The accompanying table of nor-
mal averages for the animals usually employed in bac-
teriological research may be of use in preventing the
erroneous assumption that pyrexia is present in an ani-
mal, which merely shows its own normal temperature.

NORMAL AVERAGES.

Rectal

Pulse.

Respirations.

Temp. °C.

Rate pe

r minute.

Frog

8 . Q— 17 . 2

80

12

Mouse

37.4

I2O

Rat

37 "\

2 IO

Guinea pig

38.6

I SO

80

Rabbit

38.7

17 tr

etr

Cat . .

38 7

I3O

24

Do?

38 6

O ^

I E;

Goat

40 . o

7cr

*«9

16

Ox

38.8

4C

Horse . . .

37 0

38

ii

Monkey (Rhesus) . .
Pigeon

38.4
4O O

IOO

136

19

3O

Fowl

41.6

140

ow

12

B. Special observations comprise some or all of the
following, according to the method of inoculation and
the character of the virus.

1. The site of inoculation should be minutely exam-
ined at least at weekly intervals, and the neighbouring
lymphatic glands palpated.

2. Any local reaction at the site of inoculation and
any other readily accessible lesion should be carefully
investigated. Any suppurative process which may
occur, whether in the subcutaneous tissues or in
joints, should be explored and the pus carefully ex-
amined both microscopically and culturally.

SPECIAL OBSERVATIONS 373

Fluid secretions and excretions, such as pus or
serous exudates when accessible are collected direct
from the body in sterile capillary pipettes (vide Fig.
130,) in the following manner:

i. 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. Attach a rubber teat (vide page 10) to the plugged
end of the pipette and use the teat as the handle of the
pipette.

3. Pass the entire length of the pipette twice or
thrice through the flame of the Bunsen burner.

4. Snap off the sealed end of the pipette with a pair
of sterile forceps.

5. Compress the india-rubber teat, thrust the point
of the pipette into the secretion ; now relax the pressure
on the teat and allow the pipette to fill.

6. Remove the point of the pipette from the secretion,
allow the fluid to run a short distance up the capillary
stem and seal the point of the pipette in the flame. (If
using a pipette with a constriction below the plugged
mouthpiece (Fig 136), this portion of the pipette may
also be sealed in the flame.)

When ready to examine the morbid material snap
off the sealed end of the pipette with sterile forceps and
eject the contents of the pipette into a sterile capsule.
The material can now be utilized for coverslip prep-
arations, cultivations and inoculation experiment.

3. The peripheral blood should be examined from
time to time for from this tissue is often obtained
the fullest information as to the course and progress
of an infection.

a. The histological examination of the blood should
be directed chiefly to observations on the number and
kind of white cells; and since but few bacteriologists
are at the same time expert comparative haematolo-
gists, some notes on the normal characters of the blood

374

EXPERIMENTAL INFECTIONS DURING LIFE

of the commoner laboratory animals, contrasted with
those of man, are inserted for reference. These have
been very kindly compiled for me by my friend and one
time colleague Dr. Cecil Price Jones.

COMPARATIVE H^EMOCYTOLOGY OF LABORATORY
ANIMALS.

Totals

Percentages

Animal

Lympho-

Large

Poly-

Eosin-

Mast

Red cells

White
cells

Hb, per-
cent.

cytes,
per

monos,
per

morph,
per

oph,
per

cells,
per

cent.

cent.

cent.

cent.

cent.

Frog

490,000

8,000

58

40

10. 0

22 .0

15

13

Mouse. . .

8,700,000

8,000

78

60

21.5

17.0

i .4

O. I

Rat

9,000,000

9,000

85

54

7.0

37-5

i -3

0. 2

Guinea-

pig

5,700,000

10,000

99

55

9.o

32.8

3-o

O.2

Rabbit....

6,000,000

7,000

70

So

2 .0

46.0

0.6

1.4

Rhesus...

4,500,000

13,000

77

43

5-0

50.0

1-3

0.7

Goat

14,600,000

15,000

58

35

6.3

56.7

i -25

0.75

Fowl

3,500,000

30,000

100

49

3-0

42 .0

I .0

5-o

Pigeon. . .

3,500,000

20,000

101

43

9-o

43 -o

3-0

2 .0

Man

(adult)..

5,000,000

7,500

100

25

5-5

65

4.0

0.5

Normal

(4-5-5)

(7-9)

(95-101)

(20-30)

(4-8)

(55-68)

(3-5)

(0.5-2)

limits.

millions.

thou-

sands.

The above table represents in each case the average
of a large number of counts.

REMARKS.

Frog. — The red cells are large oval nucleated (20-25^
by 12-15/4 discs, the nucleus relatively small and
irregularly elongated or oval, about IO/JL in length.
Many primitive and developing forms are usually ob-
served— also free nuclei and many cells in various stages
of degeneration. Haemoglobin estimation is difficult
owing to turbidity of the blood after dilution with
water. The polymorphonuclear leucocytes are large

COMPARATIVE H^EMATOLOGY 375

cells, about 20^; no definite granules can be observed.
The eosinophile cells contain large deeply staining
coccal-shaped granules.

Mouse. — The granules of the polymorphonuclear
leucocytes are usually not stained, or only very faintly
so. The nucleus of the eosinophile cell is ring-shaped
or much divided, and the granules are coccal and stain
oxyphile. The remarkable character of the blood is
the high percentage of large mononuclear cells.

Rat. — The fine rod-shaped granules of the poly-
morphonuclear leucocytes are usually very faintly
stained. The granules of eosinophile cells are well
stained and coccal-shaped, the nucleus is often ring
shaped. The basophile granular cells are few — but the
granules are large, and stain deeply basophile.

Guinea-pig. — Polychromasia and punctate baso-
philia of red cells are very commonly observed—
nucleated red cells are also frequent. The large mono-
nuclear cells often contain vacuoles — " Kurlow cells" —
possibly of a parasitic nature.

Rabbit. — It is not uncommon to find nucleated
red cells in films from quite healthy animals. The
granules of the polymorphonuclear leucocytes stain
oxyphile. The coarse granules of the eosinophile cells
appear to stain less deeply oxyphile, probably owing to
the basophile staining of the cytoplasm.

Rhesus monkey. — The blood cells resemble those
met with in human blood. The minute neutrophile
granules of the polymorphonuclear leucocytes are often
very scanty, and sometimes apparently absent. The
eosinophile cells are not so densely packed with coarse
oxpyhile granules as in the human eosinophile, and the
nuclei of these cells are usually much divided, or
polymorphous .

Goat. — The red cells are small, nonnucleated discs,
only about 4.5^ diameter, not much more than half
that of the human red cell. The polymorphonuclear

376 EXPERIMENTAL INFECTIONS DURING LIFE

leucocytes have only a few very minute coccal-shaped
oxyphile granules, the nucleus is polymorphous. The
eosinophile cells are large cells up to 20^, the cyto-
plasm is basophile and contains coarse coccal-shaped
oxyphile granules, and the nucleus is often much
divided.

Fowl. — The red cells are oval nucleated discs about
I2/JL by 6/*, the nucleus being relatively small (about
4fi long), irregularly elongated or oval; round, more
deeply stained cells with round or diffuse nuclei, also
free nuclei and degenerated forms of red cells are often
present. The granules of the cells corresponding to
the polymorphonuclear leucocytes are rod-shaped, often
beaded or with clubbed ends. The nucleus is not
polymorphous, but usually divided into two, though
it may be single. The cells probably corresponding to
eosinophile leucocytes have fine coccal-shaped granules,
faintly staining eosinophile or neutrophile. The baso-
phile granules of the "mast" cells are coccal-shaped, of
various size — often quite powdery.

Pigeon. — Red cells resemble those of the fowl, and
similar varieties of appearance may be noted. The
granules of those cells which correspond to polymorpho-
nuclear leucocytes are rod-shaped, but smaller and
finer than in the fowl, and do not show clubbed ap-
pearances. The nucleus is not polymorphous, and only
occasionally divided. The coccal-shaped granules of
the eosinophile cells are stained more deeply oxyphile
than those of the corresponding cells of the fowl.

The preparation of dried films for this histological
examination of the blood is carried out as follows :

i. Small samples of blood for the preparation of
blood films are most conveniently obtained from the
veins of the ear in most of the ordinary laboratory
animals, viz., monkey, goat, dog, cat, rabbit, guinea-
pig ; in the pigeon and fowl the axillary vein should be
punctured; in the rat and mouse either a vein in the

EXAMINATION OF BLOOD 377

ear or preferably by wounding the tip of the tail ; in the
frog, the web of the foot should be selected.

2. Puncture the selected vein with a sharp needle.
A flat Hagedorn needle (size No. 8) with a cutting
edge is the most useful for this purpose. If the vein
cannot be distended by proximal compression, vigourous
friction with a piece of dry lint may have the desired
effect — or a test-tube full of water at about 40° C.
may be placed close to the vein. Failing these methods,
a drop or two of xylol may be dropped on the skin just
over the vein, left on for a few seconds and then wiped
off with a piece of dry lint.

3. One of the short ends of a 3 by i glass slip is
brought into contact with the exuding drop of blood,
so that it picks up a small drop.

4. The slide is then lowered transversely on to the
surface of a second 3 by i slip, which rests on the
bench near to one end at an angle of about 45°, and
retained in this position for a few seconds, while the
drop of blood spreads along the whole of the line of
contact (see also Fig. 69).

5. Draw the first slide firmly and evenly along the
entire length of the lower slide, leaving a thin regular
film which will probably show the blood cells only one
layer thick.

6. Allow the film to dry in the air.

7. Stain with one of the polychrome blood stains
(seepage 97).

8. Examine microscopically.

b. The bacteriological examination of the blood is
directed solely to the demonstration of the presence in
the circulating blood of the organisms previously in-
jected into the animal. For this purpose several cubic
centimetres of blood should be taken in an all-glass
syringe from an accessible vein corresponding to one of
those suggested as the site of intravenous inoculation —
and under similar aseptic precautions.

378 EXPERIMENTAL INFECTIONS DURING LIFE

1. Sterilise an all-glass syringe of suitable size, and
when cool draw into the syringe some sterile sodium
citrate solution and moisten the whole of the interior of
the barrel ; then eject all the citrate solution if less than
5 c.c. blood are to be withdrawn ; if more than 5 c.c. are
required retain about half a cubic centimetre of the fluid
in the syringe. This prevents coagulation of the blood.

The sodium citrate solution is prepared by dissolving :

Sodium citrate 10 gramme.

Sodium chloride °-75 grammes.

In distilled water 100 c.c.

Sterilise by boiling.

2. Prepare the animal as for intravenous inoculation
(see page 363) and introduce the syringe needle into the
lumen of the selected vein.

3. Slowly withdraw the piston of the syringe. When
sufficient blood has been collected direct the assistant
to release the proximal compression of the vein; and
withdraw the needle.

4. Remove the needle from the nozzle of the syringe
and deliver the citrated blood into a small Ehlenmeyer
flask containing about 250 c.c. of nutrient broth.

5. Label, incubate and examine daily until growth
occurs or until the expiration of ten days.

c. The serological examination of the blood is directed
to the demonstration of the presence of certain specific
antibodies in the sera of experimentally infected an-
imals, and within certain limits to an estimation of
their amounts.

The chief of these bodies are :

Antitoxin.

Agglutinin.

Precipitin.

Opsonin.

Immune body or Bacteriolysin.

None of these substances are capable of isolation in

COLLECTION OF SERUM 379

a state of purity apart from the blood serum, conse-
quently special methods have been elaborated to per-
mit of their recognition. In every instance the be-
haviour of serum from the experimental animal,
which may be termed "specific" serum, is studied in
comparison with that of serum from an uninoculated
animal of the same species, and which is termed " nor-
mal" serum. In view of minor differences in constitu-
tion exhibited by the serum of various individuals
of the same series, it is usual to employ a mixture of
sera obtained from several different normal animals
of the same species as the inoculated animal, under the
term "pooled serum." The method of collecting blood
(e. g., from the rabbit) for serological tests is as follows :

Collection of Serum.

Apparatus required:

Razor.

Liquid soap.

Cotton-wool.

Lysol 2 per cent, solution, in drop bottle.

Ether in diop bottle.

Flat Hagedorn needles.

Blood pipettes (Fig. 16, page 12).

Centrifugal machine.

Centrifuge tubes.

Glass cutting knife.

Bunsen flame.

Writing diamond or grease pencil.

METHOD. —

1. Shave the dorsal surface of the ear over the
course of the posterior auricular vein (see Fig. 192).

2 . Sterilise the skin by washing with lysol.

The lysol should be applied with sterile cotton-wool
and the ear vigourously rubbed, not only to remove
superficial scales of epithelium, but also to render the
ear hypersemic and the vein prominent.

3. Remove the lysol with ether dropped from a
drop bottle, and allow the ether to evaporate.

380 EXPERIMENTAL INFECTIONS DURING LIFE

4. Puncture the vein with a sterile Hagedorn needle.

5. Take a small blood-collecting pipette (Fig. 161)
and hold it at an angle to the ear, one end touching the
issuing drop of blood, the other depressed.

The blood will now enter the pipette at first by
capillarity; afterward gravity will also come into
play and the pipette can be two-thirds filled without
difficulty.

6. Hold the tube by the end containing the blood,
the clean end pointing obliquely upward — warm this

FIG. 192. — Collecting blood from rabbit.

end at the bunsen flame to expel some of the contained
air ; then seal the clean point in the flame.

7. Place the pipette down on a cool surface (e. g., a
glass slide) . The rapid cooling of the air in the clean
end of the pipette creates a negative pressure, and the
blood is sucked back into the pipette, leaving the
soiled end free from blood. Seal this end in the bunsen
flame.

8. Mark the distinctive title of the specimen (e. g.,
animal's number) upon the pipette with a writing
diamond or grease pencil.

9. When the sealed ends are cold and the blood has
clotted, place the pipette on the centrifuge, clean end
downward ; counterpoise and centrifugalise thoroughly.
On removing the pipette from the centrifuge, the red
cells will be collected in a firm mass at one end, and
above them will appear the clear serum.

DILUTION OF THE SPECIFIC SERUM 381

10. By marking the blood pipette above the level
of the serum with the glass cutting knife and snapping
the tube at that point, the blood-serum becomes
readily accessible for testing purposes.

If larger quantities of blood are required, the animal,
after puncturing the vein, should be inverted, an
assistant holding it up by the legs. Blood to the
volume of several cubic centimetres will now drop from
the punctured vein, and should be caught in a tapering
centrifuge tube, the tube transferred to the incubator
at 37° C. for two hours, then placed in the centrifugal
machine, counterpoised and centrifugalised thoroughly.
The three most important of the antibodies referred to
which can be demonstrated with a certain amount of
facility are agglutinin, opsonin and bacteriolysin ; and
the methods of testing for these bodies will now be
considered.

AGGLUTININ.

Agglutinin is the name given to a substance present
in the blood-serum of an animal that has successfully
resisted inoculation with a certain micro-organism.
This substance possesses the power of collecting
together in clumps and masses, or agglutinating watery
suspensions of that particular microbe.

Dilution of the Specific Serum :

Apparatus required:

Sterile gA aduated capillary pipettes to contain 10 c. mm. (Fig. 1 7).

Sterile graduated capillary pipettes to contain 90 c. mm. (Fig. 17).

Small sterile test-tubes 5X0.5 cm.

Normal saline solution in flask or test-tube.

Pipette of specific serum.

Glass cutting knife, or three-square file.

Glass capsule, nearly full of dry silver sand, or roll of plasticine.

Grease pencil.

382 EXPERIMENTAL INFECTIONS DURING LIFE

METHOD.—

1. Take three sterile test-tubes and number them i,
2 and 3.

2. Pipette 0.9 c.c. sterile normal saline solution into
each tube, and stand tubes upright in the sand in the
capsule, or in the plasticine block.

3 . Make a scratch with the glass cutting knife on the
blood pipette above the upper level of the clear serum,
and snap off and discard the empty portion of the tube.

4. Remove o.i c.c. of the serum from the blood
pipette tube, and mix it thoroughly with the fluid in
tube No. i ; and label s.s., (specific serum), 10 per cent.

5. Remove o.i c.c. of the solution from tube No. i
by means of a fresh pipette, and mix it with the con-
tents of tube No. 2 ; and label s.s., i per cent.

6. Remove o.i c.c. of the solution from tube No. 2
by means of a fresh pipette, and mix it with the con-
tents of tube No. 3 ; and label s.s., o.i per cent.

When the yield of serum from the specimen of blood
which has been collected, or is available, is small, the
above method of diluting is not practicable, and the
dilution should be carried out by Wright's method
in a capillary teat pipette.

Dilution of Serum by Means of a Teat Pipette.

Materials required:

Blood pipette containing sample of specific serum after centrif-
ugalisation.

Capsule of diluting fluid — normal saline solution.

Supply of Pasteur pipettes (Fig. 130).

India-rubber teats.

Small test-tubes.

A block of plasticine to act as a test-tube stand.

Grease pencil.

METHOD:

i . Mark three small test-tubes 10 per cent., i per cent,
and o.i per cent, respectively, and stand them upright
in the plasticine block.

DILUTION OF SERUM BY MEANS OF A TEAT PIPETTE 383

2. Take a Pasteur pipette, nick the capillary stem
just above the sealed end with a glass cutting knife, and
snap off the sealed end with a quick movement so that
the fracture is clean cut and at right angles to the long
axis of the capillary stem — cut "square", in fact.
Prepare several, say a dozen, in this manner.

3. Fit a rubber teat to the barrel of each of the
pipettes.

4. Make a mark with the grease pencil on the stem
of one of the pipettes about 2 or 3 cm. from the open
extremity.

FIG. 193. — Filling the capillary teat pipette.

5. Compress the teat between the finger and thumb
(Fig. 193) to such an extent as to drive out the greater
part of the contained air.

6. Maintaining the pressure on the teat pass the
stem of the pipette into the capsule holding the
saline solution, until the open end of the pipette is
below the level of the fluid.

7. Now cautiously relax the pressure on the teat
and let the fluid enter the pipette and rise in the stem
until it reaches the level of the grease pencil mark. As
soon as this point is reached, check the movement of

384 EXPERIMENTAL INFECTIONS DURING LIFE

the column of fluid by maintaining the pressure on the
teat, neither relaxing nor increasing it.

8. Withdraw the point of the pipette clear of the
fluid, and again relax the pressure on the teat very
slightly. The column of saline solution rises higher in
the stem, and a column of air will now enter the pipette
and serve as an index to separate the first volume of
fluid drawn into the stem from the next succeeding one.

9. Again introduce the end of the pipette into the
fluid and draw up a second volume of saline to the level
of the grease pencil mark, and follow this with a second
air index.

10. In like manner take up seven more equal volumes
of saline solution and their following air bubbles.
There are now nine equal volumes of normal saline in
the pipette.

1 1 . Now pass the point of the pipette into the blood
tube and dip the open end below the surface of the
serum. Proceeding as before, aspirate a volume of
serum into the capillary stem up to the level of the
pencil mark.

12. Eject the contents of the pipette into the small
tube marked 10 per cent, by compressing the rubber
teat between thumb and finger.

13. Mix the one volume of serum with the nine
volumes of saline solution very thoroughly by re-
peatedly drawing up the whole of the fluid into the
pipette and driving it out again into the test-tube.

14. Now take a clean pipette and proceed precisely
as before, 4 to 10.

15. Having aspirated nine equal volumes of saline
into this second pipette, now take up one similar
volume of the fluid in the " 10 per cent, tube."

1 6. Eject the contents of this pipette into the second
tube marked i per cent, and mix thoroughly as before.

17. In similar fashion make the o.i per cent, solution
and transfer to the third tube.

TEE MICROSCOPICAL REACTION 385

18. Further dilutions in multiples of ten can be pre-
pared in the same way, and by varying the number
of volumes of diluting fluid or serum any required
dilution can be made (see Appendix, Dilution Tables) .

NOTE.— The saline diluting fluid must always be taken into the
pipette first, otherwise if the serum contains a very large amount
of agglutinin the traces of this serum added to the saline solu-
tion may be sufficient to entirely vitiate the subsequent observa-
tions— whilst if more than one sample of serum is diluted from the
same saline solution serious errors may be introduced into the
experiments.

The Microscopical Reaction :

Apparatus Required:

Five hanging-drop slides (or preferably two slides, with two cells
mounted side by side on each (Fig. 62, 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 (e.g., Bacillus typhi abdominalis)

Pipette end with the remainder of the specific serum labelled s.s.

Tubes containing the three solutions of the specific serum, 10,
i, and o.i per cent, respectively.

Pipette end with pooled normal serum labelled p.s.

METHOD. —

i. Make five hanging-drop preparations, thus:

(a) One loopful of bouillon cultivation + one loopful
pooled serum; label "Control."

(b) One loopful culture + one loopful undiluted
specific serum; label 50 per cent.

Mount these two cover-slips on a double-celled slide.

(c) One loopful bouillon culture + one loopful 10
per cent, serum; label 5 per cent.

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.

25

386 EXPERIMENTAL INFECTIONS DURING LIFE

Mount these two cover-slips on a double-celled slide.

2 . Note the time : Examine the control to determine
that the bacilli are motile and uniformly scattered over
the field — not collected into masses.

3. Next examine the 50 per cent, serum preparation.
If agglutinin is present and 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 owing to the viscosity
of the concentrated serum.

4. Observe the 5 per cent, preparation microscopic-
ally.

If the bacilli are aggregated into clumps, positive
reaction.

If the bacilli are not aggregated into clumps, observe
until thirty minutes from the time of preparation before
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, preparation
is positive, according to the potency of the specific
serum ; and by the examination of a series of dilutions
a quantitative comparison of the valency of specific
sera from different sources, or of serum from the same
animal at different periods during the course of active
immunisation may be obtained.

NOTE. — The graduated pipettes supplied with Thoma's haema-
tocytometer (intended for the collection of the specimen of blood
required for the enumeration 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 diameter to per-
mit of their use.

The Macroscopical Reaction :

Sterile graduated capillary pipettes to contain 90 c. mm.
Eighteen to twenty-four-hours-old bouillon cultivation of the
organism to be tested.

THE MACROSCOPICAL REACTION 387

Three test-tubes containing the 10, i, and o.i per cent, solutions
of specific serum (about 90 c. mm. remaining in each) .
Tube containing 50 per cent, solution of pooled serum.
Sedimentation pipettes (vide page 1 7) or teat pipettes.

METHOD.—

1. Pipette 90 c. mm. of the bouillon culture into each
of the tubes containing the diluted serum; and the
same quanitity into the tube containing the pooled
serum.

2. Fill a sedimentation tube (by aspirating) or a teat
pipette 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 as compared with the
appearances in the control pooled serum.

OPSONIN.

Opsonin is the term applied by Wright to a substance,
present in the serum of an inoculated animal, which is
able to act upon or sensitise bacteria of the species
originally injected, so as to render them an easy prey
to the phagocytic activity of polymorphonuclear leuco-
cytes. In the method for demonstrating opsonin about
to be described, a comparison is made between the
opsonic " power" of the pooled serum and the specific
serum.

Apparatus:

Small centrifuge and tubes for same (made from the barrels
of broken capillary pipettes by sealing the conical ends in the
bunsen flame).

Capillary Pasteur pipettes.

India-rubber teats.

388 EXPERIMENTAL INFECTIONS DURING LIFE

Grease pencil.

Bunsen burner with peep flame.

Electrical signal clock (see page 39) stop watch, or watch.

Rectangular glass box or tray to hold pipettes.

Incubator regulated at 37° C.

3X1 slides.

Piece of light rubber tubing.

Retangular block of plasticine.

Flask of normal saline solution.

Flask of sodium citrate (1.5 per cent.) in normal saline solution.
Materials required, and their preparation :

Small tube of "washed cells" (red blood discs and leucocytes);
human cells are used in estimating the opsoiiising power of the
serum of experimental animals.

Small tube of emulsion of bacteria of the species responsible
for the infection of the experimental animal.

Blood pipette containing specific serum.

Blood pipette containing "pooled" serum.

Washed Cells. —

1. Take a small centrifuge tube and half fill it with
sodium citrate solution. Mark with the grease pencil
the upper limit of the fluid.

2 . Cleanse the skin of the distal phalanx of the second
finger of the left hand above the root of the nail with
lint and ether. Wind the rubber tubing tightly round
the second phalanx ; puncture with a sterile Hagedorn
needle through the cleansed area of skin.

3 . Take up a sufficiency of the issuing blood (more or
less according to the number of tests to be performed)
with a teat pipette, transfer it to the tube of citrate
solution and mix thoroughly. Make a second mark on
the tube at the upper level of the mixed citrate solution
and blood.

4. Place the tube in the centrifuge, counterpoise
accurately and centrifugalise until the blood cells are
thrown down in a compact mass occupying approxi-
mately the same volume as is included between the
two pencil marks.

The column of fluid in the tube now shows clear
supernatant fluid (citrate solution and blood plasma)

ESTIMATION OF OPSONIN 389

separated from the sharp cut upper surface of the red
deposit of corpuscles by a narrow greyish layer of
leucocytes.

5. Remove the supernatant column of citrate solution
by means of a teat pipette, fill normal saline solution
into the tube up to the upper pencil mark, and dis-
tribute the blood cells throughout the saline by means
of the teat pipette. Centrifugalise as before.

6. Again remove the supernatant fluid and fill in a
fresh supply of saline solution and centrifugalis once
more.

7. Remove the supernatant saline solution as nearly
down to the level of the leucocytes as can be safely
done without removing any of the leucocytes.

8. Next distribute the leucocytes evenly throughout
the mass of red cells by rotating the tube between the
palms of the hands — just as is done with a tube of
liquefied medium prior to pouring a plate.

9. Set the tube upright in the plasticine block neat
to one end.

Bacterial Emulsion. —

1. Take an 18- to 24-hour culture of the required
bacterium (e. g.-, 'Diplococcus pneumonias) grown upon
sloped blood agar at 37° C. Pour over the surface of
the medium some 5 c.c. of normal saline solution.

2. With a platinum loop emulsify the growth from
the surface of the medium as evenly as possible in the
saline solution.

3 . Allow the tube to stand for a few minutes so that
the large masses of growth may settle down ; transfer the
upper portion of the saline suspension to a centrifuge
tube and centrifugalise thoroughly.

4. Examine a drop of the supernatant opalescent
emulsion microscopically to determine its freedom from
clumps and masses. If unsatisfactory prepare another
emulsion, this time scraping up the surface growth with

390

EXPERIMENTAL INFECTIONS DURING LIFE

a platinum spatula, transferring it to an agate mortar
and grinding it up with successive small quantities of
normal saline. If satisfactory insert the tube in the
plasticine block next to that containing the washed cells.

Specific Serum. —
Pooled Serum. —

These sera are collected and treat-
ed as already described (see page
379), and the portions of the blood
pipettes containing them are ar-
t_,FIG' -1,94' P1^icine ranged in the remaining space in

block with materials ar- ° .

ranged for opsonin esti- plasticine block.

The plasticine block now presents
the appearances shown in Fig. 194.

METHOD FOR DETERMINING THE OPSONIC INDEX. —

1. Take a capillary pipette fitted with a teat, cut the
distal end square and make a pencil mark about 2 cm.
from the end.

2. Aspirate into the pipette one volume of washed
cells, air index, one volume of bacterial emulsion, air
index, and one volume of specific serum (see Fig. 195).

Serum

Bacterial Washed cells
emulsion

FIG. 195. Opsonin pipette.

3. Mix thoroughly on a 3 by i slide by compressing
the teat and ejecting the contents of the pipette on
to the surface of the slide, relaxing the pressure and
so drawing the fluid up into the pipette again. These
two processes should be repeated several times ; finally
take up the mixture in an unbroken column to the cen-
tral portion of the capillary stem.

4. Seal the point of the pipette in the peep flame of
the bunsen burner and remove teat.

ESTIMATION OF OPSONIN 39 1

5. Mark the pipette (with the grease pencil) with the
distinctive number of the serum and place it in the
glass box or tray.

6. Take another similarly prepared pipette and aspi-
rate into it equal volumes of washed cells, bacterial
emulsion and pooled serum. Treat precisely as in 3
and 4, label it "control" or "N.S." (normal serum)
and place in the box by the side of the specific serum
preparation.

7. Place the box with the pipettes in the incubator
and set the signal clock to ring at 1 5 minutes (or start
the stop watch).

6. At the expiration of the incubation time remove
the pipettes from the incubator.

9. Cut off the sealed end of the specific serum prepa-
ration. Mix its contents thoroughly as in step 3, and
then divide the mixture between two 3 by i slips and
carefully spread a blood film (vide page 376) on each
in such a way that only one-half of the surface of each
slide is covered with blood — the free edge of the blood
film approximating to the longitudinal axis of the slide.

Allow films to dry and label the slides with writing
diamond.

10. Treat the contents of the control pipette in
similar fashion.

11. Select the better film from each pair for fixing
and staining.

12. Fixing and staining must be carried out under
strictly comparable conditions, and to this end the
slides are best handled by placing in a glass staining
rack which can be lowered in turn into each of a series
of glass troughs containing the various reagents (Fig.
196) . Place the rack in the first trough which contains
the alcoholic solution of Leishman's stain for two min-
utes to fix.

Transfer to the second trough containing the diluted
stain for ten minutes.

392 EXPERIMENTAL INFECTIONS DURING LIFE

Transfer to the third trough containing distilled
water, and holding the trough over a sink, run in a
stream of distilled water until washing is complete.
Remove slides from the rack and dry.

Leishman's stain is the best for routine work for all
bacteria other than B. tuberculosis. Films containing
tubercle bacilli must of course be stained by the Ziehl
Neelsen method.

FIG. 196. Glass staining trough for blood films.

1 3 . Examine specific serum slide microscopically with
i/ 12 inch oil immersion. Find the edge of the blood
film — along this the bulk of the leucocytes will be
collected. Starting at one end of the film move the
slide slowly across the microscope stage and as each
leucocyte comes into view count and record the number
of ingested bacteria. The sum of the contents of the
first 50 consecutive polymorphonuclears that are en-
countered is marked down. (The average number of
bacilli ingested per leucocyte = the " phagocytic index.")

14. In precisely similar manner enumerate the bac-
teria present in the first 50 cells of the control prepa-
ration. This number is recorded as the denominator

COMPLEMENT FIXATION 393

of a vulgar fraction of which the numerator is the
number recorded for the specific serum. This fraction,
expressed as a percentage of unity = the opsonic index.

IMMUNE BODY.

Immune body or amboceptor is the name given to a
substance present in the serum of an infected animal
that has successfully resisted inoculation with some par-
ticular micro-organism, and which possesses the power
of linking the complement normally present in the
serum to bacteria of the species used as antigen in such
a manner that the micro-organisms are rendered in-
nocuous, and ultimately destroyed. The presence of
the immune body in the serum can be demonstrated
in vitro by the reaction elaborated by Bordet and Gen-
gou, known as the complement fixation test, the exist-
ence or the absence of the phenomenon of complement
fixation being rendered obvious macroscopically by the
absence or presence of haemolysis on the subsequent
addition of "sensitised" red blood corpuscles, (e.g., a
mixture of erythrocyte solution and the appropriate
haemolysin — two of the three essentials in the haemoly-
tic system, vide page 326).

Apparatus Required:

Sterile pipettes i c.c., (graduated in tenths).

16X2 cm. test-tubes.

9X1 cm. test-tubes.

Test-tube racks for each size of test-tube.
Reagents Required:

Normal saline solution.

Erythrocyte solution (human red cells, page 329) =E.

Haemolytic serum (for human cells) = H.S.

Complement (fresh guinea-pig serum) =C.

Specific serum from inoculated animal, inactivated = S.S.

Control pooled serum from normal animals of same species,
inactivated = P.S.

Antigen (cultivation upon solid medium of the organism (e.g., B.
typhosus) which has already served as antigen in the inoculation
of the experimental animal) =A

394 EXPERIMENTAL INFECTIONS DURING LIFE

To prepare the antigen for use, emulsify the whole of
the bacterial growth in 5 c.c. normal saline solution.

Shake the emulsion in a test-tube with some sterilised
glass beads to ensure a homogenous emulsion, and ster-
ilise by heating to 60° C. in a water-bath for one hour.

METHOD. —

1. Take five small test-tubes, and number them i to 5
with a grease pencil.

2. Into tubes Nos. i, 3, 4 and 5 pipette o.i c.c. of
complement.

3. Into tubes Nos. i and 2 pipette 0.2 c.c. of the
serum to be tested.

4. Into tube No. 4 pipette 0.2 c.c. of control serum.

5. Into tubes Nos. i, 2, 3 and 4 pipette i c.c. of the
bacterial emulsion which forms the antigen.

6. Place the whole set of tubes in the incubator at
3 7° C. for a period of one hour.

7. Remove the tubes from the incubator and pipette
i c.c. erythrocyte solution and 4 minimal hsemolytic
doses of the corresponding hsemolysin into each tube.

8. Mix thoroughly and return the tubes to the incu-
bator at 37° C. for further period of one hour.

9. At the expiration of that time transfer the tubes
to the ice chest, and allow them to stand for three
hours.

10. Examine the tubes.

Tubes 3, 4 and 5 should show complete haemolysis;
tube 2 should give no evidence whatever of haemolysis.

These tubes form the controls to the first tube, which
contains the serum to be tested.

In tube No. i the absence of haemolysis would indicate
the presence in the serum of the inoculated animal of a
specific antibody to the micro-organism used in the
inoculations; since it shows that the complement has
been bound by the immune body to the bacterial
antigen, and none has been left free to enter into the

COMPLEMENT FIXATION 395

haemolytic system ; on the other hand the presence of
haemolysis would show that no appreciable amount of
antibody has yet been formed in response to the inocu-
lations. In other words, there is an absence of infec-
tion, since the complement remained unfixed at the
time of the addition of the erythrocyte solution and
haemolytic serum, and was ready to combine with those
reagents to complete the haemolytic system.

The method may be shown diagramatically as under
using the symbols already indicated •

Test-tubes.

©

©

®

© ©

o.i c.c. C

o.i c.c. C.

o.i c.c. C. o.i c.c. C.

0.2 C.C. S

,S. 0.2 C.C. S.S.

0.2 C.C. P.S

A.

A.

A.

A.

Incubate

at 37° C. for

one hour.

i c.c. E.

i. c.c. E.

i c.c. E.

i c.c. E. i c.c. E.

H.S.*

H.S.*

H.S.*

H.S.4 H.S.*

Incubate

at 37° C. for

one hour.

(?)

No haemolysis.

v

Haemolysis.

NOTE. — It is sometimes more convenient to sensitise the erythro-
cytes just before they are needed. This is done forty-five minutes
after the experiment has been started (page 394, step 6), that is to
say, before the completion of the first period of incubation, thus :

1. Measure out into a sterile test-tube (or flask) five c.c. of
erythrocyte solution.

2. Measure out twenty minimal haemolytic doses of haemolysin,
add to the erythrocyte solution on the test-tube.

3. Allow the erythrocyte and hsemolysin to remain in contact for
fifteen minutes at room temperature. The red cells are then sensi-
tised and ready for use.

4. When the tubes are removed from the incubator at the end of
the first hour (i.e., step 7) add i c.c. sensitised red cells to each
tube by means of a graduated pipette.

5. Mix thoroughly, return the tubes to the incubator at 37° C.
and complete the experiment as previously described (steps 8
onward) .

XIX. 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 examination
will rarely need to be carried out in its entirety.

NOTE. — Throughout the autopsy 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 contaminated and a fresh instrument taken
for the next step.

Apparatus Required :

Water steriliser.

f Scalpels.

o • i • Scissors.

Surgical instruments: < ~

Forceps.

I Bone forceps.

Spear-headed platinum spatula (Fig. 199).
Searing irons (Fig. 198).
Tubes of media — bouillon and sloped agar.

Surface plates in petri dishes (of agar or one of its derivatives).
Platinum loop.
Aluminium "spreader."
Grease pencil.

Sterile capillary pipettes (Fig. 13, a).
Sterile glass capsules, large and small.
Cover-slips or slides.

Bottles of fixing fluid (vide page 114) for pieces of tissue intended
for sectioning.

396

APPARATUS REQUIRED

397

1. Place the various instruments, forceps, scissors,
scalpels, etc., needed for the autopsy inside the steriliser
and sterilise by boiling for ten minutes ; then open the
steriliser, raise the tray from the interior and rest it
crosswise on the edges.

2 . Heat the searing irons to redness in a separate gas
stove.

3. Drench the fur (or feathers) with lysol solution,
2 per cent. This serves the twofold purpose of pre-

FiG. 197. — Apparatus for post-mortem examination, animal on board.

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.

4. Examine the cadaver carefully. Recollect that
laboratory animals are not always hardy ; death may be

FIG. 198. — Searing iron.

due to exposure to heat or cold, to starvation or over-
or improper feeding or to the attack of rats — and not
to the bacterial infection.

5. Fasten the body of the animal, ventral surface
upward (unless there is some special reason for having

398 POST-MORTEM EXAMINATIONS

the dorsum exposed) , out on a board by means of copper
nails driven through the extremities.

6. 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.)

Seat of Inoculation.—

• 7. Inspect the seat of inoculation. If any local les-
ion is visible, sear its exposed surface and with the plat-
inum loop, remove material from the deeper parts to
make tube and surface plate cultivations and cover-slip
preparations

Collect specimens of pus or other exudation in
capillary pipettes for subsequent examination.

8. Inspect the neighbouring lymphatic glands and
endeavour to trace the path of the virus.

9. Sear the whole of the exposed surface of the
thorax with the searing irons.

Pleural Cavity.—

10. 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, exposing
the heart. Place the infected instruments by the side
of the first set.

11. Observe the condition of the anterior mediastinal
glands, the thymus and the lungs. Collect a quantity
of pleuritic effusion, if such is present, in a pipette for
further examination later.

1 2 . Raise the pericardial sac in a fresh pair of forceps
and burn through this structure with a searing iron.

Collect a sample of pericardial fluid in a pipette for
microscopical and cultural examination.

PERITONEAL CAVITY 399

13. Grasp the apex of the heart in the forceps and
sear the surface of the right ventricle.

14. 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.

15. Collect a further sample of blood or serum for
subsequent investigation as to the presence of anti-
bodies.

Peritoneal Cavity.—

1 6. Sear a broad track in the middle line of the abdom-
inal wall; open the peritoneal cavity by an incision
in the centre of the seared line. Observe the condition
of the omentum, the mesentery, the viscera and the
peritoneal surface of the intestines.

17. Collect a specimen of the peritoneal fluid (or pus,
if present) in a capillary pipette. Make cultivations,
tube and surface plate, and cover-slip preparations
from this situation.

1 8 . Collect a specimen of the urine from the distended
bladder in a large pipette (in the manner indicated for
heart blood) , for further examination, by cultivations,
microscopical preparations, and chemical analysis.

19. Collect a specimen of bile from the gall bladder
in similar manner.

20. Excise the spleen and place it in a sterile cap-
sule. Later, sear the surface of this organ; plunge the
spear-headed spatula through the centre of the seared
area, twist is round between the finger .and thumb, and
remove 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.

21. Seize one end of the spleen with sterile forceps.
Sear a narrow band of tissue, right around the organ

400 POST-MORTEM EXAMINATIONS

and divide the spleen in this situation with a pair of
scissors. Holding the piece of spleen in the forceps,
dab the cut surface on to a surface plate in a number of
different spots.

22. In like manner examine the other organs — liver,
lungs, kidneys, lymphatic glands (mesenteric, hepatic,
lumbar, etc), etc. Prepare cultivations and cover-slip
preparations.

23. Dissect out a long bone from one upper and
one lower limb and one of the largest ribs. Prepare
cultures from the bone marrow in each case. Set aside
these bones for the subsequent preparation of marrow
films.

24. Film preparations of bone marrow are best made
by the Price- Jones method. Seize the bone in a pair
of pliers and squeeze out some of the marrow ; receive
it in a platinum loop, and transfer to a watch glass of
dissociating fluid and emulsify. The dissociating fluid
is a neutral 10 per cent, solution of glycerine prepared
as follows : —

*

Measure out 10 c.c. Price's best glycerine and 90 c.c. sterile
ammonia-free distilled water. Mix. Titrate against — sodic hy-
drate solution using phenolphthalein as the indicator. The initial
reaction is usually + o.i to + 0.5; add the calculated amount of
— sodic hydrate solution to neutralise.

25. Place a loopful of fresh desiccating fluid on a
3X1 glass slide ; add a similar loopful of the marrow
emulsion, and spread very gently over the surface of
the slip.

26. Allow film to dry in the air (protected from
dust) without heating.

27. Stain with Jenner's polychrome stain (page 97)
for two and a half minutes.

28. Wash with ammonia-free distilled water, dry
thoroughly and mount in xylol balsam.

CRANIAL AND SPINAL CAVITIES 401

Cranial and Spinal Cavities. —

29. In some instances it may be necessary (e. g.,
experimental inoculation of rabies) to examine the
cranial cavity or to remove the spinal cord. Return
the viscera to the adbominal cavity; draw the flaps of
skin together and secure with Michel's steel clips.
Draw the copper nails securing the limbs to the board,
reverse the animal and again nail the limbs down — the
body now being dorsum uppermost.

30. Make a longitudinal incision in the mesial line
from snout to root of tail, and four transverse incisions
— one joining the roots of the two ears, one across the
body at the level of the spinis of the scapulae, another
at the level of the costal margin and the last across the
upper level of the pelvis. Reflect these flaps of skin.

3 1 . With forceps and scalpel dissect out the muscles
lying in the furrow on either side of the spinal processes.

32. Cut through the bases of the transverse processes
with bone forceps. Cut away the vault of the skull,
cut through the roots of the nerves and remove the brain
and spinal cord, place in a large glass dish for examina-
tion. Prepare cultivations from the cerebro-spinal
fluid. The removal of the brain and cord is a tedious
process and during the dissection it is difficult to avoid
injury to these structures.

The operation is, however, carried out very
expeditiously and neatly with the aid of the surgical
engine (vide page 361). A small circular saw is fitted
to the hand piece. The bones of the skull are cut
through and the whole of the vault removed, exposing
the entire vertex of the brain. Similarly all the spinous
processes can be removed in one string by running the
saw down first one side of the spinal column and then
the other. In this way ample space for the removal
of the nervous tissues is obtained with a minimum
of labour.
26

402 POST-MORTEM EXAMINATIONS

33. Having completed the preparation of cultures
remove small portions of various organs at leisure and
place each in separate bottles of fixing fluid for future
sectioning. Affix to each bottle a label bearing all
necessary details as to its contents.

34. If necessary, remove portions of the organs for
preservation and display as museum speciments (vide
page 404).

35. Gather up all the infected instruments, return

FIG. 199. — Spear-headed platinum spatula (actual size.)

them to the steriliser, and disinfect by boiling for ten
minutes.

36. Sprinkle dry saw-dust into the exposed body
cavities to absorb blood and fluid. Cover the body
with blotting or filter paper, moistened with 2 per cent,
lysol solution. Place in a galvanised iron pail, provided
with a lid, ready for transport to the crematorium.

37. Cremate the cadaver together with the board
upon which it is fixed.

38. Stain the cover-slip preparations by suitable
methods and examine microscopically.

39. Incubate the cultivations and examine carefully
from day to day.

40. 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
microscopical and cultural investigation when
available.

As part of the card index system in use in the author's
laboratory already referred to (vide page 335) there is a
special yellow card for P-M notes. On the face of the
card are printed headings for various data — some of
which are sometimes unintentionally omitted — and
on the reverse is a schematic figure which can be utilised

AUTOPSY RECORDS

403

for indicating the position of the chief lesions in the
cadaver of any of the laboratory animals.

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or organisms isolated may be correlated with the
symptoms observed during life and the observations
summarised under the following headings:

404

POST-MORTEM EXAMINATIONS

Tissue changes:

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 1 Acute

reactions. j- or

(b) Degeneration and necrosis. J chronic.

2. General (i. e., produced at a distance from the bacteria, by

absorption of toxins) :

FIG. 201. — Back of post-mortem card.

(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. Tissues. — The naked-eye appearances of morbid
tissues may be preserved by the following method :

i. Remove the tissue or organ from the cadaver as
soon after death as possible, using great care to avoid
distortion or injury.

PERMANENT PREPARATIONS 405

2. Place it in a wide-mouthed stoppered jar, large
enough to hold it conveniently, 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 fixing solution, and
stopper the jar; allow the tissues to remain in this
solution for from forty-eight hours to seven days
(according to size) to fix. Make any necessary sections.

Kaiserling modified solution is prepared as follows :
Weigh out

Potassium acetate 30 grammes.

Potassium nitrate 15 grammes.

and dissolve in

Distilled water 1000 c.c.

then add

Formalin 150 c.c.

Filter.

This fixing solution can be used repeatedly so long
as it remains clear. Even when it has become turbid,
if simple filtration is sufficient to render it clear, the
filtrate may be used again.

4. Transfer the tissue to a bath of methylated spirit
(95 per cent.) for thirty minutes to one hour.

5. Remove to a fresh bath of spirit and watch care-
fully. When the natural co Jours show in their original
tints, average time three to six hours, remove the tis-
sues from the spirit bath, dry off the spirit from the
cut surfaces by mopping with a soft cloth, then transfer
to the mounting solution.

Jore 's mounting solution (modified) consists of

Glycerine 500 c.c.

Distilled water 750 c.c.

Formalin 2 c.c.

406 POST-MORTEM EXAMINATIONS

Equally good but .much cheaper is Frost's mounting
solution :

Potassium acetate 160 grammes.

Sodium fluoride 80 grammes.

Chloral hydi ate 80 grammes.

Cane sugar (Tate's cubes) . . . . 3,500 grammes.
Saturated thymol water 8,000 c. c.

6. After twenty-four hours in this solution, or as
soon as the tissue sinks, transfer to a museum jar, fill
with fresh mounting solution, and seal.

6a. Or transfer to museum jar and fill with liquefied
gelatine, to which has been added i per cent, formalin.
Cover the jar and allow the gelatine to set. When
solid, seal the cover of the jar in place.

7. To seal the museum preparation first warm the
glass plate which forms the cover. This is most
conveniently done by placing the cleaned and polished
cover-plate upon a piece of asbestos millboard over a
bunsen flame turned low.

8. Smear an even layer of hot cement over the flange
of the jar. The cement is prepared as follows :

Weigh out and mix in an iron ladle

Gutta percha (pure) 4 parts.

Asphaltum 5 parts.

and melt together over a bunsen flame, stirring with an
iron rod until solution is complete.

9. Invert the glass plate over the jar and press down
firmly into the cement. Place a piece of asbestos
board on the top and on that rest a suitable weight
until the cement is cold and has thoroughly set.

10. Trim off any projecting pieces of cement with
an old knife, burr over the joint between jar and cover-
plate with a hot smooth piece of metal (e. g., the sear-
ing iron) .

1 1 . Paint a narrow band of Japan black to finish off,
round the joint, overlapping on to the cover-plate.

PERMANENT PREPARATIONS 407

//. 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 thick 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
upward, inside the jar. (If water of con-
densation is present in any of the culture
tubes, it should be removed by means of
a capillary pipette before placing the tubes
in the formalin chamber.)

5. Adjust the glass disc, ground side
downward, over the mouth of the jar and
secure it by pressing it firmly down into
the ointment, with a rotary movement.

6. Remove the tubes from the formalin

. FIG. 202.— Bul-

chamber after the lapse of a week, and loch's tubes,
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
employ Bulloch 's tubes. These are slightly longer than
the ordinary tubes, and are provided with a constriction
some 2 cm. below the mouth (Fig. 202) — a feature which
renders sealing in the blowpipe flame an easy matter.

XX. THE STUDY OF THE PATHOGENIC
BACTERIA.

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 de-
scriptions 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.

During the course of this work attention must also be
directed, as occasion arises, to such other bacteria,
pathogenic or saprophytic, as are allied to the particular
organisms under observation, or so resemble them as to
become possible sources of error, by working them
through on parallel lines — in other words the various
bacteria should be studied in " groups." In the follow-
ing pages the grouping in use in the author's elemen-
tary classes for medical and dental students and for
candidates for the Public Health service is adopted,
since a fairly long experience has completely vindicated
the value and utility of this arrangement, and by its
means a fund of information is obtained with regard
to the resemblances and differences, morphological and
cultural, of a large number of bacteria. The fact that
some bacteria appear in more than one of these groups,
so far from being a disadvantage, is a positive gain to
the student, since with repetition alone will the neces-

408

STUDY OF PATHOGENIC BACTERIA 409

sary familiarity with the cultural characters of impor-
tant bacteria be acquired. The study of the various
groups will of course vary in detail with individual
demonstrators, and with the student's requirements —
the general line it should take is indicated briefly in
connection with the first group only (pages 410-411).
This section should be carefully worked through before
the student proceeds to the study of bacterioscopical
analysis.

It is customary to commence the study of the patho-
genic bacteria with the Organisms of Suppuration.
This is a large group, for all the pathogenic bacteria
possess the power, under certain conditions, of initiat-
ing purely pyogenic processes in place of or in addition
to their specific lesions, (e. g., Bacillus tuberculosis,
Streptococcus lanceolatus, Bacillus typhosus, etc.).
There are, however, 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 in this group are :

1. Staphlococcus pyogenes albus.

2. Staphylococcus pyogenes aureus.

3. Staphylococcus pyogenes citreus.

4. Streptococcus pyogenes longus.

5. Micrococcus tetragenus.

6. Bacillus pyocyaneus.

7. Bacillus pneumoniae.
and in certain special tissues

8. Micrococcus gonorrhoeas.

9. Microcoecus intracellularis meningitidis (Menin-
gococcus) .

10. Micrococcus catarrhalis.

11. Bacillus aegypticus (Koch- Weeks Bacillus).

The group may with advantage be subdivided as
indicated in the following pages :

41 0 STUDY OF PATHOGENIC BACTERIA

I. Pyo genie cocci.

Staphylococcus pyogenes albus.
Staphylococcus pyogenes aureus.
Staphylococcus pyogenes citreus.

to contrast with
Micrococcus candicans.
Micrococcus agilis.

i. Prepare subcultivations from each:
Bouillon,

Agar streak,

.Jf , and incubate at 37° C.

Blood serum,

Litmus milk.
Agar streak,
Gelatine stab,
Potato.

and incubate at 20° C.

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' incu-
bation. Examine microscopically and compare. Note
the locomotive activity of M. agilis and the Brownian
movement of the remaining micrococci.

3. Prepare cover-slip films from the agar cultures,
after twenty-four hours' incubation. Stain for flagella
by the modified Pit field's method. Note M. agilis is
the only micrococcus showing flagella.

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, and Gram's method.
Examine the films microscopically and compare.
Note in the Gram preparation, the Gram negative
character of certain individual cocci in each film pre-
pared from the three days' growth — such cocci are dead.

5. Stain section of kidney tissue provided (showing

STUDY OF PATHOGENIC BACTERIA 411

abscess formation by Staphylococcus aureus) 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. Inoculate1 a white mouse subcutaneously with
three loopfuls of a forty-eight-hour agar cultivation of
the Staphylococcus aureus, emulsified with 0.2 c.c.
sterile broth.

Observe carefully during life, and when death occurs
make a careful post-mortem examination.
II. Pyo genie cocci.

Micrococcus gonorrhoea?.

Micrococcus intracellularis meningitidis

(meningococcus) .
Micrococcus catarrhalis.
Micrococcus tetragenus.
Micrococcus paratetragenus.

III. Pyo genie cocci.

Streptococcus pyogenes longus.
Streptococcus of bovine mastitis.
Streptococcus lanceolatus (Diplococcus

pneumonise or pneumococcus) .

to contrast with
Streptococcus brevis.
Streptococcus lebensis.

IV. Pyo genie bacilli.

Bacillus pneumonias (Friedlaender) .
Bacillus rhinoscleromatis.
Bacillus lactis aerogenes.

V. Pyogenic bacilli.

Bacillus pyocyaneus.

to contrast with

Bacillus fluorescens liquefaciens.
Bacillus fluorescens non -liquefaciens.

1 See note on Vivisection License, page 334.

412 STUDY OF PATHOGENIC BACTERIA

VI. Pneumonia group.

Streptococcus lanceolatus (pneumococcus) ,
Bacillus pneumoniae (Friedlaender) .
Streptococcus pyogenes longus.

•

VII. Diphtheroid group.

Bacillus diphtherias (Klebs-Lcefrler) .
Bacillus Hoffmanni.
Bacillus xerosis.
Bacillus septus.

VIII. Coli-typhoid group.

B. typhi abdominalis (B. typhosus).

B. coli communis.

B. enteritidis (Gaertner).

to contrast with
B. aquatilis sulcatus.

IX. Escherich group.

B. coli communis (Escherich).
B. coli communior.
B. lactis aerogenes.
B. cloacae.

X. Gaertner group.

Bacillus enteritidis (Gaertner).

B. paratyphosus A.

B. paratyphosus B.

Bacillus cholerae suum (Hog Cholera).

B. psittacosis.

XI. Eberth group.

B. typhosus (Eberth).
B. dysenteriae (Shiga).
B. dysenterias (Flexner).
B. fsecalis alcaligines.

STUDY OF PATHOGENIC BACTERIA 413

XII. Spirillum group.

Vibrio choleras.
Vibrio metschnikovi.

to contrast with

Vibrio proteus (Finkler and Prior).
Spirillum rubrum.
Spirillum rugula.

XIII. Anthrax group.

Bacillus anthracis.
to contrast with
Bacillus subtilis.
Bacillus mycoides.
Bacillus mesentericus fuscus.

XIV. Acid fast group.

Bacillus tuberculosis (human).

(bovine).

(avian).

(fish).

to contrast with

Bacillus phlei (Timothy grass bacillus) .
Butter bacillus of Rabino witch.

XV. Plague group.

Bacillus pestis.

B. septicaemias haemorrhagicse.

B. suipestifer.

XVI. Influenza group.
B. influenzas.

Bacillus asgypticus (Koch- Weeks).
Bacillus pertussis.

XVII. Miscellaneous.

Bacillus leprae.
Bacillus mallei.
Micrococcus melitensis.

414 STUDY OF PATHOGENIC BACTERIA

XVIII. Streptothrioc group.

Streptothrix actinomycotica.
Streptothrix madurae.

to contrast with
Cladothrix nivea.

XIX. Tetanus group.

Bacillus tetani.

Bacillus cedematis maligni.

Bacillus chauvei (symptomatic anthrax),

XX. Enteritidis sporogenes group.

Bacillus enteritidis sporogenes.
B. botulinus.
B. butyricus.
B. cadaveris.

XXI. BACTERIOLOGICAL ANALYSES.

EACH bacteriological or bacterioscopical analysis of
air, earth, sewage, various food-stuffs, etc., includes,
as a general rule, two distinct investigations yielding
results of very unequal value :

1. Quantitative.

2. Qualitative.

The first is purely quantitative and as such is of
minor importance as it 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 and more important is both qualitative
and quantitative in character since it seeks to accurately
identify such pathogenic bacteria as may be present
while, incidentally, the methods advocated are calcu-
lated to indicate, with a fair degree of accuracy, the
numerical frequency of such bacteria, 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; and the tech-
nique there set out will be found to be capable of expan-
sionand adaptation to any circumstance or set of cir-
cumstances which may confront the student.

Controls. — The necessity for the existence of ade-
quate controls in all experimental work cannot be
too urgently insisted upon. Every batch of plates that
is poured should include at least one of the presum-
es

41 6 BACTERIOLOGICAL ANALYSES

ably ' 'sterile" medium; plate or tube cultures should be
made from the various diluting fluids; every tube of
carbohydrate medium that is inoculated should go
into the incubator in company with a similar but un-
inoculated tube, and so on.

BACTERIOLOGICAL EXAMINATION OF WATER.

The bacteria present in the water may comprise
not only varieties which have their normal habitat in
the water and will consequently develop at 20° C., but
also if the water has been contaminated with excremen-
tal matter, varieties which have been derived from, 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 incubate the various cultivations at
each of these temperatures.

Further, the sample of water may contain moulds,
yeasts, or torulas, and the development of these will be
best secured by plating in wort gelatine and incubat-
ing at 20° C.

1. Quantitative. —

Collection of the Sample. — The most suitable vessels
for the reception of the water sample are small glass
bottles, 60 c.c. capacity, with narrow necks and over-
hanging glass stoppers (to prevent contamination of
the bottle necks by falling dust). These must be
carefully sterilised in the hot-air steriliser (vide page

(a) 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. Replace the stopper and
tie it down, but do not seal it.

(b) If the sample is obtained from a stream, tank,

WATER 417

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 downward, 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
centimetres of water from the bottle,
replace the stopper, and tie it down.

(c) If the sample is obtained from
a lake, river or the sea ; or when it is
desired to compare samples taken at
varying depths, the apparatus de-
signed by v. Esmarch (Fig. 203) is
employed. In this the sterilised
bottle is enclosed in a weighted metal
cage which can be lowered, by means
of a graduated line, until the required
depth is reached. At this point the
bottle is opened by a thin wire cord
attached to the stopper; when the
bottle is full (as judged by the air
bubbles ceasing to rise) the puU on Jr'cV.0£itoi£
the cord is released and the tension bottle for water
of the spiral spring above the stopper
again forces it into the neck of the bottle. When the
apparatus is taken out of the water, the small bottles
are filled from it, and packed in the ice-box mentioned
below.

An inexpensive substitute for Esmarch' s bottle can
be made in the laboratory thus :

Select a wide-mouthed glass stoppered bottle of about
500 c.c. capacity (about 20 cm. high and 8 cm. in
diameter) .

Remove the glass stopper and insert a rubber cork
with two perforations in its place.

Through one perforation pass a piece of glass tubing
about 5 cm. long and through the other a piece 22
27

4i8

BACTERIOLOGICAL ANALYSES

cm. long, reaching to near the bottom of the bottle,
each tube projecting about 2.5 cm. above the rubber
stopper. Plug the open ends of the tubes with cotton
wool. Secure the stopper in place with thin copper wire.
Sterilise the fitted bottle in the autoclave. Remove
the cotton wool plugs and connect the projecting
tubes by a piece of loosely fitting stout rubber pressure
tubing about 5 cm. long, previously
sterilised by boiling.

Take a piece of stout rubber cord
about 33 cm. long, and of 10 mm. diam-
eter (such as is used for door springs)
thread a steel split ring upon it and
secure the free ends tightly to the neck
of the bottle by cord or catgut.

Attach the cord used for lowering the
bottle into the water to the split ring
on the rubber suspender. The best ma-
terial for this purpose is cotton insulated
electric wire knotted at every metre.

Connect the split ring also with the
short piece of rubber tubing uniting
the two glass tubes by a piece of catgut
(or thin copper wire) of such length
that when the bottle is suspended there
Thresh's ' deep is no pull upon the rubber tube, but
boattie.Sampling which> however, will be easily jerked off
when a sharp pull is given to the sus-
pending cord.

Now wind heavy lead tubing about i cm. diameter
around the upper part of the bottle, starting at the
neck just above the shoulder. This ensures the sinking
of the bottle in the vertical position (Fig. 204).

The apparatus being arranged is lowered to the
required depth, a sharp jerk is then given to the sus-
pending cord, which detaches the rubber tube and so
opens the two glass tubes. Water enters through the

WATER

419

longer tube and the air is expelled through the shorter
tube. The bubbles of air can be seen or heard rising
through the water, until the bottle is nearly full, a
small volume of compressed air remaining in the neck
of the bottle.

As the apparatus is raised, the air thus imprisoned
expands, and prevents the entry of more water from
nearer the surface.

s/S '<• 2. -:' ' 2E-SII r' ^; ' " ''•' -'" -; '^LJi

&

I

I

FIG. 205. — Ice-box for transmission of water samples, etc.

Transport of Sample. — If the examination of the sam-
ple cannot be commenced immediately, steps must be
taken to prevent the multiplication of the bacteria con-
tained in the water during the interval occupied in transit
from the place of collection to the laboratory. To this
end an ice-box such as that shown (in Fig. 205) is essen-
tial. It consists of a double- walled metal cylinder into
which slides a cylindrical chamber of sufficient capacity
to accommodate four of the 60 c.c. bottles; this in turn
is covered by a metal disc — the three portions being

420 BACTERIOLOGICAL ANALYSES

bolted together by thumb screws through the over-
hanging flanges. When in use, place the bottles, rolled
in cotton-wool, in the central chamber, pack the space
between the walls with pounded ice, securely close the
metal box by screwing down the fly nuts, and place it
in a felt-lined wooden case. (It has been shown that
whilst bacteria will survive exposure to the temperature
of melting ice, practically none will multiply at this
temperature.)

On reaching the laboratory, the method of examina-
tion consists in adding measured quantities of the
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.

Apparatus Required:

Plate-levelling stand.

Case of sterile plates.

Case of sterile pipettes, i c.c. (in tenths of a cubic centimetre).

Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).

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, consecutively, i to 6;
the gelatine tubes, consecutively, i to 6, and the wort
tubes, i, 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

WATER 421

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
evenly throughout the water by shaking, cut the string
securing the stopper, and loosen the stopper, but do
not take it out.

FIG. 206. — Withdrawing water from water sample bottle.

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.
Tilt the bottle containing the water sample on the
bench holding the neck between the middle and ring
fingers of the left hand ; .grasp the head of the stopper
between the forefinger and thumb, and remove it from
the bottle.

6. Pass the pipette into the mouth of the bottle, hold-
ing its point well below the surface of the water (Fig. 206) .

422 BACTERIOLOGICAL ANALYSES

Suck up rather more than i c.c. into the pipette and
allow the pipette to empty ; this moistens the interior of
the pipette and renders accurate measurement possible.
Now draw up exactly i c.c. into the pipette. Withdraw
the pipette from the bottle, replace the stopper, and
stand the bottle upright.

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 replace it in the water-bath. 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. In a similar manner add i c.c. of the sample to the
contents of the fourth tube.

9. Similarly, add 0.5 c.c. and o.i c.c. respectively to
the contents of the fifth and sixth tubes.

10. Drop the pipette into the cylinder containing
lysol solution.

11. 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 number of the sample, (b) the quan-
tity of water sample it contains, and (c) the date.

1 2 . Pour the contents of a tube of liquefied agar —
not inoculated — into a Petri dish to act as a control to
demonstrate the sterility of the batch of agar employed.

13. Allow the plates to set, and incubate at 37° C.

14. Empty the water chamber of the levelling appa-
ratus and refill it with ice-water.

15. By means of the sterile 10 c.c. pipette deliver
9.9 c.c. sterile distilled water into a sterile glass
capsule.

1 6. Add o.i c.c. of the water sample to the 9.9 c.c.
sterile water in the capsule. This will give a dilution
of i in 100.

17. Plant the six tubes of nutrient gelatine in the
following manner: To the first tube add 0.5 c.c. of the

WATER 423

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.

18. 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.

19. Pour a control (uninoculated) gelatine plate.

20. Allow the plates to set, and incubate at 20° C.

21. 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, 0.2 c.c.

22. Label the plates, allow them to set, and incubate
at 20° C.

23. Count and record the number of colonies that
have developed upon the agar at 37° C. after forty-
eight hours* incubation.

24. Note the number of colonies present on each of
the gelatine and wort gelatine plates after forty-eight
hours' incubation.

25. Replace the gelatine and wort plates in the
incubator; observe again at three days, four days, and
five days.

26. Calculate and record the number of organisms
present per cubic centimetre of the original water from
the average of the six gelatine plates at the latest date
possible up to seven days — the presence of liquefying
bacteria may render the calculation necessary at an
earlier date, hence the importance of daily observations.

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

424

BACTERIOLOGICAL ANALYSES

(Fig. 207), each subdivision has an area of i square
centimetre; in Fakes' counter (Fig. 208), radii divide

FIG. 207. — Jeffer's disc, reduced.

FIG. 208. — Fakes' disc, reduced.

the circle into sixteen equal sectors, and counting is
facilitated by concentric circles equidistant from the
centre.

WATER 425

(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. In practice it will be found
that Pakes* disc is more suitable for the former class of
plate ; Jeffer's disc for the latter. It should be recol-
lected however that unless the plates have been care-
fully levelled and the medium is of equal thickness all
over it is useless to try and average from small areas —
since where the medium is thick all the bacteria will
develop, where the layer is a thin one, only a few bacteria
will find sufficient pabulum for the production of visible
colonies.

It will be noted that the quantities of water selected
for addition to each set of tubes of nutrient media
have been carefully chosen in order to yield workable
results even when dealing with widely differing samples.
Plates prepared in agar with o.i c.c. and in gelatin
with 0.02 c.c. can be counted even when large numbers
of bacteria are present in the sample ; whereas if micro-
organisms are relatively few, agar plate 4 and gelatine
plate i will give the most reliable counts. Again the
counts of the plates in a measure control each other ; for
example, the second and third plates of each gelatine
series should together contain as many colonies as the
first, and the second should contain about half as
many more than the third and so on.

426 BACTERIOLOGICAL ANALYSES

2. Qualitative Examination.—

Collection of Sample. — The water sample required for
the routine examination, which it will be convenient to
consider first, amounts to about no c.c. It is col-
lected in the manner previously described (vide page
416) ; similar bottles are used, and if four are filled the
combined contents, amounting to about 240 c.c., will
provide ample material for both the qualitative and
quantitative examinations. Unless the examination is
to be commenced at once, the ice-box must be employed,
otherwise water bacteria and other saprophytes will
probably multiply at the expense of the microbes in-
dicative of pollution, and so increase the difficulties
of the investigation.

In the routine examination of water supplies it is
customary to limit the qualitative examination to a
search for

A. B. coli and its near allies.

B. Streptococci,

organisms which are frequently spoken of as microbes
of indication, as their presence is held to be evidence of
pollution of the water by material derived from the
mammalian alimentary canal, and so to constitute a
danger signal.

C. Some observers still attach importance to the
presence of B. enteritidis sporogenes, but as the search
for this bacterium, (relatively scarce in water) necessi-
tates the collection of a fairly large quantity of water it
is not usually included in the routine examination.

In the case of water samples examined during the
progress of an epidemic, of new supplies and of unknown
waters the search is extended to embrace other mem-
bers of the eoli-typhoid group; and on occasion the
question of the presence or absence of Vibriocholerse or
(more rarely) such bacteria as B. anthracis or B.
tetani, may need investigation.

When pathogenic or excremental bacteria are pre-

WATER 427

sent in water, their numbers are relatively few, owing
to the dilution they have undergone, and it is usual in
commencing the examination, to adopt one or other of
the following methods :

A. Enrichment, in which the harmless non-pathogen-
ic bacteria may be destroyed or their growth inhibited,
whilst the growth of the parasitic bacteria is encouraged.

This is attained by so arranging the environment,
(i. e., Media, incubation temperature, and atmosphere)
as to favor the growth of the pathogenic organisms at
the expense of the harmless saprophytes.

B. Concentration, whereby all the bacteria present in
the sample of water, pathogenic or otherwise, are con-
centrated in a small bulk of fluid.

This is usually effected by filtration of the water
sample through a porcelain filter candle, and the
subsequent emulsion of the bacterial residue remaining
on the walls of the candle with a small measured quan-
tity of sterile bouillon.

A. Enrichment Method.

(Dealing with the demonstration of bacteria of in-
testinal origin.)

Apparatus Required (Preliminary Stage):

Incubator running at 42° C.

Case of sterile pipettes, i c.c. graduated in tenths.
Case of sterile pipettes, 10 c.c. graduated in c.c.
Case of sterile pipettes, graduated to deliver 25 c.c.
Tubes of bile salt broth ( vide page 180).
Flask of double strength bile salt broth (vide page 199).
Tubes of litmus silk.
Sterile flasks, 250 c.c. capacity.
Buchner's tubes.
Tabloids pyrogallic acid.
Tabloids sodium hydrate.
Bunsen burner.
Grease pencil.
(Later stage):

Incubator running at 37° C.

Surface plates of nutrose agar (see page 232).

428 BACTERIOLOGICAL ANALYSES

Aluminum spreader.

Tubes of various media, including carbohydrate media.

Agglutinating sera, etc.

METHOD.—

1. Number a set of bile salt broth tubes 1-5, and
a duplicate set ia-5a.

2. Number one flask 7 and another 8.

3. To Tubes No. i and la add o.i c.c. water sample.
To Tubes No. 2 and 2 a add 'i c.c. water sample.
To Tubes No. 3 and 3 a add 2 c.c. water sample.
To Tubes No. 4 and 4a add 5 c.c. water sample.
To Tubes No. 5 and 5a add 10 c.c. water sample.

4. Put up all the tubes in Buchner's tubes and incu-
bate anaerobically at 42° C.

NOTE. — The bile salt medium is particularly suitable for the
cultivation of bacteria of intestinal origin, and at the same time
inhibits the growth of bacteria derived from other sources.

The anaerobic conditions likewise favor the multipli-
cation of intestinal bacteria, and also their fermenta-
tive activity. The temperature 42° C. destroys or-
dinary water bacteria and inhibits the growth of many
ordinary mesophilic bacteria.

5. Pipette 25 c.c. of double strength bile salt broth
into flask 6, and 50 c.c. double strength bile salt broth
into flask 7.

6. Pipette 25 c.c. water sample into flask 6, and 50
c.c. water sample into flask 7.

7. Incubate the two flasks aerobically at 42° C.

8. After twenty-four hours incubation note in each
culture :

a. The presence or absence of visible growth.

b. The reaction of the medium as indicated by the
colour change, if any, the litmus has undergone.

c. The presence or absence of gas formation, as
indicated by a froth on the surface of the medium, and
the collection of gas in the inner "gas" tube.

WATER 429

9. Replace those tubes which show no signs of
growth in the incubator. Examine after another
period of twenty-four hours (total forty-eight hours
incubation) with reference to the same points.

10. Remove culture tubes which show visible growth
from the Buchner's tubes, whether acid production
and gas formation are present or not.

11. Examine all tubes which show growth by hang-
ing-drop preparations. Note such as show the presence
of chains of cocci.

12. Prepare surface plate cultivations upon nutrose
agar from each tube that shows growth either macro-
scopically or microscopically, and incubate for twenty-
four hours aerobically at 37° C.

13 . Examine the growth on the plates either with the
naked eye or with the help of a small hand lens. Prac-
tice will facilitate the recognition of colonies of the coli
group, the typhoid group and the paratyphoid group;
also those due to the growth of streptococci. The
investigation from this stage proceeds along two
divergent lines of enquiry — the first being concerned
with the identity of the bacilli — typhoid bacilli, the
second with that of the cocci.

A. B. Coli and its allies.

14. Pick off coliform or typhiform colonies; make
streak or smear subcultivations upon nutrient agar;
incubate aerobically for twenty-four hours at 37° C.

15. Examine the growth in each tube carefully both
macroscopically and microscopically. If the growth is
impure, replate on nutrose agar, pick off colonies and
subcultivate again. When the growth in a tube is pure,
add 5 c.c. sterile normal saline solution or sterile broth,
and emulsify the entire surface growth with it.

1 6. Utilise the emulsion for the preparation of a
series of subcultivations upon the media enumerated
below, using the ordinary loop to make the subcultures
upon solid media, but adding one-tenth of a cubic

430 BACTERIOLOGICAL ANALYSES

centimetre of the emulsion to each of the fluid media
by means of a sterile pipette.

Gelatine streak.
Agar streak.
Potato.

Nutrient broth. '
Litmus milk.

Dextrose peptone solution.
Lsevulose peptone solution.
Galactose peptone solution.
Maltose peptone solution. '
Lactose peptone solution.
Saccharose peptone solution.
Raffinose peptone solution.
Dulcite peptone solution.
Mannite peptone solution.
Glycerin peptone solution.
Inulin peptone solution.
Dextrin peptone solution.

17. Differentiate the bacilli after isolation by means
of their cultural reactions and biological characters into
members of:

I. The Escherich Group.

B. coli communis.

B . coli communior.

B. lactis aerogenes.

B. cloacae.

II. The Gaertner Group.

Bacillus enteritidis (of Gaertner).
B. paratyphosus A.
B. paratyphosus B.
Bacillus choleras suum.

WATER 431

III. The Eberth Group.

B. typhosus.
B. dysenteriae (Shiga).
B. dysenteriae (Flexner).
B. faecalis alcaligines.

18. Confirm these results by testing the organisms
isolated against specific agglutinating sera obtained
from experimentally inoculated animals.

If a positive result is obtained when using this
method, it only needs a simple calculation to determine
the smallest quantity (down to o.i c.c.) of the sample
that contains at least one of the microbes of indication.
For instance, if growth occurs in all the tubes from 4 to
10, and that growth is subsequently proved to be due
to the multiplication of 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 5 c.c. If, on the other
hand, the presence of the B. coli can only be proved
in flask No. 7, then the average number of colon bacilli
present in the sample is at least one in every 50 c. c.
(i. e., twenty per litre), but not one in every 25 c. c. and
so on.

The general outline of the method of identifying
the members of the coli-typhoid group is given in the
form of an analytical schema — whilst the full differen-
tial details are set out in tabular form.

432

BACTERIOLOGICAL ANALYSES

ANALYTICAL SCHEME FOR ISOLATION OF MEMBERS OF THE COLI AND
TYPHOID GROUPS.

Nutrose agar.

Red colonies.
Escherich group.

Blue colonies.
Gaertner and Eberth groups.

Lactose peptone solution.

Gas.

!

B. coli communis and its allies.

I

Acid and gas in glucrse peptone solution.

Acid and coagulation in1 milk.

General turbidity and indol in bouillon.

I
No gas.

Gaertner and Eberth groups.

Glucose peptone solution.

1

Gas. No gas.

1

Gaertner group. Eberth group.

- 1

1

1

Litmus milk.

Peptone solution. Litmus milk. Peptone solution.

1

1

Acid at first.

General turbidity. Acid. General turbidity.

Alkaline later.

No indol. No coagulation. No indol.

No coagulation.

Serum reaction. Serum reaction.

B. Streptococci.

19. Pick off streptococcus colonies and subcultivate
upon nutrient agar exactly as directed in steps 14, 15
and 1 6.

20. Differentiate the streptococci isolated into mem-
bers of the saprophytic group of short-chained cocci,
or members of the parasitic (pathogenic) group of
long-chained cocci, by means of their cultural charac-
ters, and record their numerical frequency in the
manner indicated for the members of the coli-typhoid
group.

WATER

433

• . 1

O O O O O O
+ + + + + + 1 O | | |

+ 111

4 =acid production.
— = alkali production.
O = no change in reaction.
C =clot.

rt

H

+ + + + + + +! + + + +

+ + + 1

«

•pauiJOj pput ou= —
•uorpnpoad jopui }u3iis = ^ 'uorpnpojd jopui = +

+ +I + I+ Ill+ll 11+11

9WOS 0

+ O O O

4 =acid or gas production.
± = slight acid production.
O = no change.

a,muBH 0

+ o + o

a^iornQ 0

oo o oooo

uuaoATO 0

t o o ]£ ooooo

oooo

UlOItBg °

oooooo o 4i oo o

oooo

O
'uTInuI <j

oooooo ooooo

oooo

uu^xaa

+ + 4 ° 4 4 ooooo

+ o o o

asourjB-g

+ + ° ° + + ooo

O O 41 O

asoa^ooBS §

o o o ooooo

oooo

1 O
asocpe'i ^

444444 ooooo

oooo

aso^^Bj^

++++++ f++++

+ 040

O

++++++ +++++ +++O

O
asojnAse'j

<^

+ + + + + + + + + + + + + + O

3S0^a »

4 4 4 O

+ + + + + + + + + + +

AimoW

++III+ +++++

4114

*i^ique ies gelatine.
+ = m )tile.
— = non-motile.

A = acid reaction
G = gas formation

SS * b » * * ' . • << PQ ' *

f -i I i • • • *:••!'

^§§S-SW' I ' 1 1 1 •

^HBg-^-s ^.a^^Sfe

iiijiis mm

4r.8lJlAi xsjalll

' Eberth Group.
typhosus
dysenteriae (Shiga)
dysenteriae (Flexner)
fjecalis alkaligines .

Kl ^

g pq pq pq n

28

434 BACTERIOLOGICAL ANALYSES

21. Determine the pathogenicity for mice (subcuta-
neous inoculation) and rabbits ( intravenous inocula-
tion) of the streptococci isolated.

On the facing insert page is reproduced a blank from
the author's Laboratory Water Analysis Book, by
means of which an exact record can be kept, with a
minimum of labour, of every sample examined.

B. Concentration Method.

The remaining organisms referred to on page 426
are more conveniently sought for by the concentration
method.

Collection of the Sample. — The quantity of water
required for this method of examination is about 2000
c.c., and the vessel usually chosen for its Vecep "^n is
an ordinary blue glass Winchester quart bottle, ster-
ilised in the hot-air oven, and over this a paper or parch-
ment 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
Examination; there is, however, not the same impera-
tive necessity to pack the sample in ice for transmission
to the laboratory.

Apparatus required:

Sterile Chamberland or Doulton " white " porcelain open mouth
filter candle, fitted with rubber washer.

Rubber cork to fit mouth of the filter candle, perforated with
one hole.

Kitasato serum flask, 2500 c.c. capacity.

Geryk air pump or water force pump.

Wulff's bottle, fitted as wash-bottle, and containing sulphuric
acid (to act as a safety valve between filter and pump).

Pressure tubing, clamps, pinch-cock.

Retort stand, with ring and clamp.

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 3 2 cm. in length,
the other 52 cm., the si orter arm being plugged with cotton- wool.
The rubber stopper must be sterilised by boiling and the glass
tubing by hot air, before use.

OIJX

WATER

435

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.

FIG. 209. — 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 Sprengel's pump.

Case of sterile pipettes, i c.c. in hundredths.

Tubes of various nutrient media (according to requirements).

Twelve Buchner's tubes with rubber stoppers.

Pyrogallic acid tablets.

Caustic soda tablets.

METHOD.—

. i . Fit up the filtering apparatus as in the accom-
panying diagram (Fig. 209), interposing the wash-

436

BACTERIOLOGICAL ANALYSES

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 Wulff's 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 pass through the
acid, and enter filter flask, and so 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 brush (Fig. 210)

Sterile test-tube emulsify the slimy residue which lines the
brush- candle, with the broth.

Practically all the bacteria contained in 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. (Some bacteria will of
course be left behind on the walls of the filter and in
its pores.)

Up to this point the method is identical, irrespective
of the particular organism whose presence it is desired
to demonstrate ; but from this point onward the methods
must be specially adapted to the isolation of definite
groups of organisms or of individual bacteria.

WATER 437

The Coli-Typhoid Group.—

i. Number nine tubes of bile salt broth (vide page
1 80), consecutively from i to 9.

2. To No i add i c.c.

2 add 2 c.c.

3 add 5 c.c.

of the original water sample
before the filtration is com-
menced.

3. To the remaining tubes of bile salt broth add
varying quantities of the suspension by means of
suitably graduated sterile pipettes, as follows :

No. 4 . . . 0.05 c.c. (equivalent to 10 c.c. of the original water sample).

No. 5 . . . 0.125 c.c. (equivalent to 25 c.c. of the original water sample).

No. 6 . . .0.25 c.c. (equivalent to 50 c.c. of the original water sample).

No. 7 . . .0.5 c.c. (equivalent to 100 c.c. of the original water sample).

No. 8 . . . i .o c.c. (equivalent to 200 c.c. of the original water sample).

No. 9 ... 2.5 c.c. (equivalent to 500 c.c. of the original water sample).

4. Put up each tube anaerobically in a Buchner's
tube and incubate at 42° C.

5. The subsequent steps are identical with those
described under the Enrichment method (see page 428
to 431; Steps 8 to 1 8) .

Alternative Methods. —

A few of the older methods for the isolation of the members of
the coli-typhoid groups are refered to but they are distinctly
inferior to those already described.

(A) The Carbolic Method :

1. Take ten tubes of carbolised bouillon (vide page 202) and
number them consecutively from i to 10.

2. Inoculate each tube with a different amount of the water
sample or 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 shows signs of growth, pour plates
in the usual manner, using carbolised gelatine (vide page 202) in
place of the ordinary gelatine, and incubate at 20° C. for three,
four, or five days as may be necessary.

6. Subucltivate from any colonies that make their appearance,
and determine their identity on the lines laid down in the previous
method.

(B) Parietti's Method:

i. Take nine tubes of Parietti's bouillon (vide page 202) —
i. e., three each of those containing o. i c.c., 0.2 c.c., and 0.5 c.c.

438 BACTERIOLOGICAL ANALYSES

of Parietti's solution respectively. 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 nutrient carbolised or potato
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 sub-
stituting Eisner's potato gelatine (vide page 204) for ordinary
nutrient gelatine in any of the previously mentioned methods.

(D) Cambier's Candle Method:

Treat a large volume of the water sample by the concentration
method (vide page 434).

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; replug 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.

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 associated 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 257), 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.

WATER 439

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 437).

6. Incubate each tube anaerobically at 37° C.
Anaerobic conditions can be obtained by putting the
cultures up in Buchner's tubes or in Bulloch's appa-
ratus. If, however, whole milk has been used in making
the litmus milk the layer of cream that rises to the sur-
face will be sufficient to ensure anaerobiosis ; whilst
if separated milk has been employed it will be sufficient
to pour a layer of sterile vaseline or liquid paraffin on the
surface of the fluid.

7. Examine after twenty-four hours' incubation.
Note (if 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 bub-
bles 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 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 each subcutaneously
with 0.5 c.c. of the whey and observe the result.

Vibrio Choleras.—

1. Number ten tubes of peptone water consecutively
from i to 10.

2. To each of the tubes of peptone water add a meas-
ured quantity of the suspension, corresponding to
those amounts employed in isolating the members of
the coli group (vide page 43 7) .

440 BACTERIOLOGICAL ANALYSES

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, both
by stained preparations or by fresh specimens with dark
ground illumination.

4. Inoculate fresh tubes of peptone water from such of
the tubes as exhibit pellicle formation — from the pel-
licle itself — and incubate at 3 7° C. for twenty-four hours.

5. Test the peptone water itself for the presence of
indol and nitrite by the addition of pure concentrated
H2S04.

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 ani-
mal immunised to the Vibrio choleras for agglutination.

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 257), and expose to a temperature
of 80° C. for twenty minutes.

3. Inoculate a young white rat subcutaneously (on
the inner aspect of one of the hind legs) with i c.c. of the
emulsion. Observe during life, and, if the animal
succumbs, make a complete post-mortem examination.

4. Melt three tubes of nutrient agar in boiling water
and cool to 42° C.

5. 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.

6. Incubate at 37° C. for twenty-four or forty -eight
hours.

MILK 441

7. Pick off any colonies resembling those of anthrax
and subcultivate on all the ordinary laboratory media.

8. Inoculate another young white rat as in 3, using
two loopfuls of the agar subcultivation emulsified with
i c.c. sterile bouillon. Observe during life, and if the
animal succumbs, make a complete post-mortem
examination.

B. TetanL—

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 3 7° C.

3. From such of the tubes as show visible growth
(with or without the production of gas) after twenty-
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, and, 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 and carry out further inoculation
experiments with the resulting growths.

EXAMINATION OF MILK.

"One-cow" or "whole" 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. In dealing with one-cow
milk, from a suspected, or an obviously diseased animal,
a complete analysis should include the examination
(both qualitative and quantitative) of samples of

442 BACTERIOLOGICAL ANALYSES

(a) fore-milk, (b) mid-milk, (c) strappings, and, if possi-
ble, from each quarter of the udder. " 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 indeed during the ex-
amination it is treated as such.

It is possible however to collect and store mixed
milk in so cleanly a manner that its germ content does
not exceed 5000 microorganisms per cubic centimetre.
Such comparative freedom from extraneous bacteria is
usually secured by the purveyor only when he resorts
to the process of pasteurisation (heating the milk to
65° C. for twenty minutes or to 77° C. for one minute)
or the simpler plan of adding preservatives to the
milk. Information regarding the employment of these
methods for the destruction of bacteria should always
be sought in the case of mixed milk samples, and in
this connection the following tests will be found useful :

1. Raw Milk (Saul).

To 10 c.c. milk in a test tube, add i c.c. of a i per cent, aqueous
solution of ortol (ortho-methyl-amino-phenol sulphate), recently
prepaied and mix. Next add 0.2 c.c. of a 3 per cent, peroxide of
hydrogen solution. The appearance of a brick red color within
30 seconds indicates raw milk. Milk heated to 74° C. for thirty
minutes undergoes no alteration in color; if heated to 75° C. for ten
minutes only, the brick red color appears after standing for about
two minutes.

2. Boric Acid.

Evaporate to dryness, 50 c.c. of the milk which has been
rendered slightly alkaline to litmus, then incinerate.

Dissolve in distilled water, add slight excess of dilute hydrochloric
acid and again evaporate to dryness.

Dissolve the residue in a small quantity of hot water and moisten
a piece of turmeric paper with the solution. Dry the turmeric
paper. Rose or cherry-red color = borax or boric acid.

3. Formaldehyde (Hehner).

To 10 c.c. milk in a test tube add 5 c.c. concentrated commercial
sulphuric acid slowly, so that the two fluids do not mix. Hold the
tube vertically and agitate very gently. Violet zone at the junc-
tion of the two liquids = formaldehyde.

4. Hydrogen Peroxide.

To 10 c.c. milk (diluted with equal quantities of water) in a test

MILK

443

tube add 0.4 c.c. of a 4 per cent, alcoholic solution of benzidine and
0.2 c.c. acetic acid. Blue coloration of the mixture = hydrogen
peroxide.

5. Salicylic Acid.

Precipitate the caseinogen by the addition of acetic acid and
filter. To the filtrate add a few drops of i per cent, aqueous solu-
tion of ferric chloride. Purple coloration = salicylic acid.

6. Sodium Carbonate or Bicarbonate.

To 10 c.c. of the milk in a test tube add 10 c.c. of alcohol and
0.3 c.c. of a i per cent, alcoholic solution of rosolic acid. Brownish
color = pure milk; rose color = preserved milk.

Quantitative.—

Collection of Sample. —

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

FIG. 211. — Milk-collecting bottle and dipper in case.

"pull-off" lid, containing a milk dipper, also made of
copper; and inside this, again, a wide-mouthed, stop-
pered glass bottle of about 250 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

/[/[/[ BACTERIOLOGICAL ANALYSES

contents, secured from shaking by packing with cotton-
wool, are sterilised in the hot-air oven (Fig. 26).
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. Pack the apparatus in the ice-box for transmission
to the laboratory in precisely the same manner as an
ordinary water sample.

"Whole" milk may with advantage be collected in
the sterile bottle directly since the mouth is sufficiently
wide for the milker to direct the stream of milk into it.

Condensed milk must be diluted with sterile distilled
water in accordance with the directions printed upon
the label, then treated as ordinary milk.

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.
Case of sterile plates.
Tubes nutrient gelatine or gelatine agar.
Tubes of wort gelatine.
Tubes of nutrient agar.
Water-bath regulated at 42° C.
Bunsen burner.
Grease pencil.

METHOD.—

i. Arrange four sterile capsules in a row; number
them I, II, III, and IV.

MILK 445

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 add it to the bouillon in
capsule I; mix thoroughly by repeatedly filling and
emptying the pipette.

4. Remove o.i c.c. of the milky bouillon from cap-
sule I, add it to the contents of capsule II, and mix as
before.

5. 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 con-
tents of capsule III to capsule IV.

Then i c.c. of dilution I contains o.i c.c. milk sample,

i c.c. of dilution II contains o.ooi c.c. milk sample,

i c.c. of dilution III contains o.ooooi c.c. milk sample,
i c.c. of dilution IV contains o.ooooooi c.c. milk sample.

6. Melt the gelatine and the agar tubes in boiling
water; then transfer to the water-bath and cool them
down to 42° C.

7. Number the gelatine tubes consecutively i to 12.

8 . Inoculate the tubes with varying quantities of the
material as follows :

To tube No. i add i.o c.c. of the milk sample.

2 add o. i c.c. of the milk sample.

3 add i.o c.c. from capsule I.

4 add o. i c.c. from capsule I.

5 add i.o c.c. from capsule II

6 add o. i c.c. from capsule II.

7 add 0.5 c.c. from capsule III.

8 add 0.3 c.c. from capsule III.

9 add 0.2 c.c. from capsule III.

10 add o. 5 c.c. from capsule IV.

11 add 0.3 c.c. from capsule IV.

12 add 0.2 c.c. from capsule IV.

9. Pour plates from the gelatine tubes; label, and
incubate at 20° C.

10. Liquefy five wort gelatine tubes and to them
add i.o c.c. of the milk sample and a similar quantity
of the diluted milk from capsules I, II, and III and IV
respectively.

446 BACTERIOLOGICAL ANALYSES

11. Pour plates from the wort gelatine; label, and
incubate at 20° C.

12. Inoculate the liquefied agar tubes as follows:

To tube No. i add o . i c.c. of the milk sample.

2 add o. i c.c. from capsule I.

3 add o. i c.c. from capsule II.

4 add o.i c.c. from capsule III.

5 add i.o c.c. from capsule IV. ^

6 add o. i c.c. from capsule IV. J

13. Pour plates from the agar tubes; label, and in-
cubate at 37° C.

14. After twenty-four hours' incubation " inspect,"
and after forty-eight hours' incubation, " 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 plates and estimate therefrom
the number of "organisms growing at 20° C." 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.

NOTE. — Many observers prefer to employ gelatine agar (see page
193) for the quantitative examination. In this case gelatine-
agar plates should be poured from tubes containing the quantities
of material indicated in step 8, incubated at 28° C. to 30° C.
and after five days the "total number of organisms developing at
28° C." recorded.

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 and when present to the estimation of their
numerical f re quency .

Members of the Coli-typhoid group.

Vibrio cholerae.

Streptococcus pyogenes longus.

Micrococcus melitensis.

MILK 447

Staphylococcus pyogenes aureus.

Bacillus enteritidis sporogenes.

Bacillus diphtherias.

Bacillus tuberculosis.

Some of these occur as accidental contaminations,
either from the water supply to the cow farm, or from
the farm employees, whilst others are derived directly
from the cow.

In milk, as in water examinations, two methods are
available, viz.: Enrichment and Concentration — the
former is used for the demonstration of bacteria of in-
testinal origin, the latter for the isolation of the micro-
organisms of diphtheria and tubercle. The first essen-
tial in the latter process is the concentration of the
bacterial contents of a large volume of the sample into
a small compass; but in the case of milk, thorough
centrifugalisation is substituted for filtration.

Apparatus Required:

A large centrifugal machine. This machine, to be of real
service 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 200 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 con-
siderable period of time. In other words, the machine
should run down very gradually and slowly after the
motive power is removed, thus obviating any dis-
turbance of the relative positions of particulate
matter in the solution that is being centrifugalised.

4. The machine should preferably be driven by electricity,

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 exertion.

448

BACTERIOLOGICAL ANALYSES

(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 212.

FIG. 212. — Electrically driven centrifugal machine, with flexible
(broken) spindle encircled by the field magnets of the motor.

Sterile centrifugal tubes, of some 60-70 c.c. capacity, tapering
to a point at the closed end, plugged with cotton-wool.

Small centrifugal machine to run two tubes of 10 c.c. capacity
at 2500 to 3000 revolutions per minute preferably driven by
electricity, of the type figured on page 327 (Fig. 162).

Sterile centrifugal tubes of 10 c.c. capacity with the distal
extremity contracted to a narrow tube and graduated in hundredths
of a cubic centimetre (Fig. 213).

Sterilised cork borer.

Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).

MILK

449

Case of sterile pipettes, i c.c. (in tenths of a cubic centimetre).

Sterile teat pipettes.

Flask of sterile normal saline solution.

METHOD.—

i. Fill 50 c.c. of the milk sample into each of four
tubes, and replace the cotton-wool plugs by solid rubber
stoppers (sterilised by boiling) , and fit the tubes in the
centrifugal machine.

NOTE. — One or two cubic centimetres of paraffinum liquidum in-
troduced into the buckets of the centrifuge before the glass tubes
are inserted will obviate any risk of breakage to the latter.

=r-==i2=_F-r

-===-—

FIG. 213. — Milk sediment-
ing tubes.

>— '

FIG. 214. — Milk in centrifuge
tube.

2. Centrifugalise the milk sample for thirty minutes
at a speed of 2500 revolutions per minute.

3. Remove the motive power and allow the machine
to slow down gradually.

4. Remove the tubes of milk from the centrifuge.
Each tube will now show (Fig. 214):

(a) A superficial layer of cream (varying in thickness
with different samples) condensed into a semi-solid
29

450 BACTERIOLOGICAL ANALYSES

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.

(c) A sediment or deposit consisting of the great
majority of the contained bacteria and leucocytes,
together with adventitious matter, such as dirt, hair,
epithelial cells, faecal debris, etc.

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 two
sterile capsules. Label C1 and C2.

6. Remove all but the last one or two 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 pair of tubes into
one sterile 10 c.c. tube (graduated) by means of sterile
teat pipettes, then fill to the 10 c.c. mark with sterile
normal saline solution and mix together. Label D1 and
D2.

8. Place the two tubes of mixed deposit in the
centrifuge, adjust by the addition or subtraction of
saline solution so that they counterpoise exactly, and
centrifugalise for ten minutes.

NOTE. — Each tube now contains the deposit from 100 c.c. of
the milk sample and the amount can be read off in hundredths
of a centimetre. The multiplication of this figure by 100 will
give the amount of "Apparent Filth," in "parts per million" — the
usual method of recording this quality of milk.

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 (C1) and deposit (D1)
microscopically —

(a) In hanging-drop preparations.

(b) In film preparations stained carbolic methylene-

MILS 451

blue, by Gram 's method, by Neisser 's method, and by
Ziehl-Neelsen 's method.

Note the presence or absence of altered and unaltered
vegetable fibres ; pus cells, blood discs ; cocci in groups
or chains, diphtheroid bacilli, Gram negative bacilli or
cocci, spores and acid fast bacteria.

ii. Adapt the final stages of the investigation to
the special requirements of each individual sample,
thus:

1 . Members of the Coli=typhoid Group.—

1. Emulsify the deposit from the second centrifu-
gal tube (D2) with 10 c.c. sterile bouillon and inocu-
late three tubes of bile salt broth as follows :

To Tube No. i add 2.5 c.c. milk deposit emulsion ( = 25 c.c. orig-
inal milk.)

To Tube No. 2 add i.o c.c. milk deposit emulsion ( = 10 c.c. orig-
inal milk.)

To Tube No. 3 add 0.5 c.c. milk deposit emulsion ( = 5 c.c. orig-
inal milk.)

2. Inoculate tube of bile salt broth No. 4 with i c.c.
of the original milk.

3. Inoculate further tubes of bile salt broth with
previously prepared dilutions (see page 445) as follows :

To tube No. 5 add i.o c.c. from capsule I.
To tube No. 6 add o. i c.c. from capsule I.
To tube No. 7 add i . o c.c. from capsule II.
To tube No. 8 add o. i c.c. from capsule II.
To tube No. 9 add i.o c.c. from capsule III.
To tube No. 10 add o. i c.c. from capsule III.
' To tube No. n add i.o c.c. from capsule IV.
To tube No. 12 add o. i c.c. from capsule IV.

and incubate anaerobically (in Buchner's tubes) at
42° C. for a maximum period of forty-eight hours.

4. If growth occurs complete the investigation as
detailed under the corresponding section of water ex-
amination (seepages 428 to 431).

NOTE. — The B. coli communis, derived from the alvine dis-
charges of the cow, is almost universally present in large or small

452 BACTERIOLOGICAL ANALYSES

numbers, in retail milk. Its detection, therefore, unless in enor-
mous numbers, (when it indicates want of cleanliness), is of little
value.

2. Vibrio Choleras. — Inoculate tubes of peptone
water by using the same amounts as in the search
for members of the Coli-typhoid groups (vide ante 1-3) ;
incubate aerobically at 37° C. and complete the exam-
ination as detailed under the corresponding section of
water examination (see page 43 9) .

3. B. Enteritidis Sporogenes. — Inoculate tubes of
litmus milk with similar amounts to those used in the
previous searches, omitting tube No. i (vide ante 1-3)
place in the differential steriliser at 80° C. for ten
minutes and then incubate anaerobically at 3 7° C. for
a maximum period of forty-eight hours. Complete
the investigation as detailed under the corresponding
section of water examination (see page 438).

4. B. Diphtherias.—

(A) i. Plant three sets of serial cultivations, twelve
tubes in each set, from (a) cream C2, (b) deposit D1
upon oblique inspissated blood-serum, and incubate
at37°C.

2. Pick off any suspicious colonies which may have
made their appearance twelve hours after incubation,
examine microscopically and subcultivate upon blood-
serum and place in the incubator; 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. Subcultivate such as
appear to be composed of diphtheria bacilli in glucose
peptone solution. Note those in which acid produc-
tion takes place.

5. Inoculate guinea-pigs subcutaneously with one
or two cubic centimetres forty-eight-hour-old glucose

MILK 453

bouillon cultivation derived from the first subcultiva-
tion of each glucose fermenter, and observe the result.

6. If death, apparently from diphtheritic toxaemia,
ensues, inoculate two more guinea pigs with a similar
quantity of the lethal culture. Reserve one animal
as a control and into the other inject 1000 units of
antidiphtheritic serum. If the control dies and the
treated animal survives, the proof of the identity of
the organism isolated with the Klebs-LoefHer bacillus
becomes absolute.

7. Inoculate guinea-pigs subcutaneously with filtered
glucose bouillon cultivations (toxins ?) and observe
the result.

(B) i. Emulsify the remainder of the deposit with

5 c.c. sterile bouillon and inoculate two guinea-pigs,
thus: guinea-pig a, subcutaneously with i c.c. emul-
sion; guinea-pig b, 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. Confirm their identity as in A 5 and

6 (vide supra).

5. Bacillus Tuberculosis. —

(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 deposit emulsion remaining in one or other
tube D'or D2).

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 ani-
mals. 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

454

BACTERIOLOGICAL ANALYSES

week and make a complete post-mortem examination.
5. If the result of the examination is negative or

FIG. 215. — Cadaver of guinea-pig experimentally infected with
B. tuberculosis.

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 care-

MILK 455

ful post-mortem examination. Examine material from
any caseous glands microscopically and inoculate
freely on to Dorset's egg medium.

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 (Fig. 215).

(B) i. Intimately mix all the available cream and
deposit from the milk sample, and transfer to a sterile
Erlenmeyer flask.

2. Treat the mixture by the antiformin method
(vide Appendix, page 502).

3. Inoculate each of two guinea-pigs, intraperi-
toneally, with half of the emulsion thus obtained.

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.

6. Utilise the remainder of the deposit for micro-
scopical examination and cultivations upon Dorsett's
egg medium.

NOTE. — No value whatever attaches to the result of a micro-
scopical examination for the presence of the B. tuberculosis
unless confirmed by the result of inoculation experiments.

6. Streptococcus Pyogenes Longus.—

(A) i . Spread serial surface plates upon nutrose agar.
Also plant serial cultivations upon sloped nutrient agar
(six tubes in series) .

2 . If the resulting growth shows colonies which resem-
ble those of the streptococcus, make subcultivations
upon agar and in bouillon, in the first instance, and
study carefully.

(B) i. Plant a large loopful of the deposit D2 into
each of three tubes of glucose formate bouillon, and
incubate anaerobically (in Buchner's tubes) for twenty-
four hours at 3 7° C.

456 BACTERIOLOGICAL ANALYSES

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.

7. Staphylococcus Pyogenes Aureus. —

1. Examine carefully the growth upon the serial
blood serum cultivations prepared to isolate B. diph-
therias and the serial agar cultivations to isolate strep-
tococci after forty-eight hours' incubation.

2. Pick off any suspicious orange coloured colonies,
plant on sloped agar, and incubate at 20° C. Observe
pigment formation.

3 . Prepare subcultivations from any suspicious
growths upon all the ordinary media, study carefully
and investigate their pathogenicity.

8. Micrococcus Melitensis. — The milk from an animal
infected with M. melitensis usually contains the organ-
isms in large numbers and but few other bacteria.

1. Spread several sets of surface plates upon nutrose
agar, each from one loopful of the deposit in tube D1
orD2.

2. Spread several sets of surface plates upon nutrose
agar, each from one drop of the original milk sample.

3. Incubate aerobically at 37° C. and examine daily
up to the end of ten days.

4. Pick off suspicious colonies, examine them micro-
scopically and subcultivate upon nutrose agar in tubes ;
upon glucose agar and in litmus milk.

5. Test the subsequent growth against the serum of
an experimental animal inoculated against M. melitensis
to determine its agglutinability.

ICE CREAM: BUTTER 457

6. If apparently M. melitensis, inoculate growth from
a nutrose agar culture after three days incubation in-
tracranially into the guinea-pig.

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 (vide page 471).

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 443).

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. (in hundredths).

Tubes of gelatine-agar (+ 10 reaction).

Plate-levelling stand, with its water chamber filled with water
at 42° C.

METHOD.—

i . Transfer a few grammes of the sample to a sterile
test-tube by means of the sterilised spatula.

458 BACTERIOLOGICAL ANALYSES

2. Place 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.,

To tube No. i add i c.c. liquefied butter.

2 add o . 5 c.c. liquefied butter.

3 add o . 3 c.c. liquefied butter.

4 add 0.2 c.c. liquefied butter.

5 add o . i c.c. liquefied butter.

6 add 0.05 c.c. liquefied butter.

7 add 0.03 c.c. liquefied butter.

8 add 0.02 c.c. liquefied butter.

9 add o.oi c.c. liquefied butter.

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.

Counterpoise for beaker.

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 Erlenmeyer flask which serves
to support the funnel. This piece of apparatus is sterilised
en masse in the hot-air oven.

Large centrifugal machine.

Sterile tubes (for the centrifuge) closed with solid rubber
Stoppers.

BUTTER 459

Case of sterile pipettes, 10 c.c.

Case of sterile graduated pipettes, i c.c. (in tenths of a cubic
centimetre) .

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.

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 warned 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 vigourously for a few minutes.

7. Centrifugalise again.

8. Pipette off the supernatant fluid; filling the tubes
with warm sterile bouillon, shake well, and again centri-
fugalise, 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 TV inch oil-immersion lens.

11. Proceed with the examination of the deposit
as in the case of milk deposit (see pages 450 et seq.) .

460 BACTERIOLOGICAL ANALYSES

EXAMINATION OF UNSOUND MEATS.

(INCLUDING TINNED OR POTTED MEATS, FISH, ETC.)

The bacterioscopic examination of unsound food is
chiefly directed to the detection of those members of
the Coli-typhoid group — B. enteriditis of Gaertnerand
its allies — which are usually associated with epidemic
outbreaks of food poisoning, and such anaerobic bac-
teria as initiate putrefactive changes in the food which
result in the formation of poisonous ptomaines, conse-
quently the quantitative examination pure and simple
is frequently omitted.

A. Cultural Examination.
Quantitative.—

Apparatus Required:

Sterilised tin opener, if (necessary.)

Erlenmeyer flask (500 c.c. capacity) containing 200 c.c. sterile
bouillon and fitted with solid rubber stopper.

Counterpoise.

Scissors and forceps.

Scales and weights.

Water steriliser.

Hypodermic syringe.

Syringe with intragastric tube.

Rat forceps.

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. —

i. Place the flask containing 200 c.c. sterile broth
on one pan of the scales and counterpoise accurately.

FOOD 461

2 . 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 to the extent of 20 grammes.

3. Make an extract by standing the flask in the
incubator running at 42° C. (or in a water-bath regu-
lated to that temperature) for half an hour, shaking its
contents from time to time. Better results are ob-
tained if an electrical shaker is fitted inside the incu-
bator and the flask kept in motion throughout the
entire thirty minutes.

Now every centimetre contains the bacteria washed
out from o.i gramme of the original food.

4. Inoculate tubes of liquefied gelatine as follows:

To tube No. i add i .o c.c. of the extract.

2 add 0.5 c.c. of the extract.

3 add 0.3 c.c. of the extract.

4 add o. 2 c.c. of the extract.

5 add o.i c.c. of the extract.

Pour plates from these tubes and incubate at 20° C.

5. Prepare a precisely similar set of agar plates and
incubate at 3 7° C. .

6. Pipette 5 c.c. of the extract into a sterile tube,
heat in the differential steriliser at 80° C. for ten
minutes.

7. From the heated extract prepare duplicate sets of
agar and gelatine plates and incubate anaerobically
in Bulloch's apparatusat 37° C. and 20° C. respectively.

8. After three days' incubation examine the agar
plates both aerobic and anaerobic and enumerate
the colonies developed from spores (7), and from vege-
tative forms and spores (5), and calculate and record
the numbers of each group per gramme of the original
food.

9. After seven days' incubation (or earlier if com-
pelled by the growth of liquefying colonies) enumerate
the gelatine plates in the same way.

462 BACTERIOLOGICAL ANALYSES

10. Subcultivate from the colonies that make their
appearance and identify the various organisms.

1 1 . Continue the investigations with reference to the
detection of pathogenic organisms as described under
water (page 429 et seq.).

Qualitative.—

I. Cultural.

The micro-organisms sought for during the examina-
tion of unsound foods comprise the following :

Members of the Coli-typhoid groups (chiefly those of
the Gaertner class).

B. anthracis.

Streptococci

Anaerobic Bacteria :

B. enteritidis sporogenes.
B. botulinus.
B. cadayeris.

The methods by which these organisms if present
may be identified and isolated have already been
discribed under the corresponding section of water
examination with the exception of those applicable to
B. botulinus, and B. cadaveris. These can only be
isolated satisfactorily from the bodies of experimen-
tally inoculated animals.

II Experimental
Tissue. —

1. 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.

OYSTERS 463

Extract.-

1. Introduce various quantities of the bouillon ex-
tract into the stomachs of several rats, mice and guinea-
pigs repeatedly over a period of two or three days by
the intragastric method of inoculation (see page 367)
and observe the result. Guinea-pigs and mice are very
susceptible to infection by B. botulinus by this method;
rabbits less so.

2. Inoculate rats, mice, and guinea-pigs subcuta-
neously into deep pockets, and intraperitoneally with
various quantities of the bouillon extract, and observe
the result.

3. Filter some of the extract through a Chamberland
candle and incubate the filtrate to determine the
presence of soluble toxins.

4. If any of the animals succumb to either of these
methods of inoculation, make careful post-mortem
examinations and endeavour to isolate the pathogenic
organisms.

THE EXAMINATION OF OYSTERS AND OTHER SHELLFISH.

On opening the shell of an oyster a certain amount
of fluid termed "liquor" is found to be present. This
varies in amount from a drop to many cubic centi-
metres (o.i c.c. to 10 c.c.) — in the latter case the bulk
of the fluid is probably the last quantum of water in-
gested by the bivalve before closing its shell. In
order to obtain a working average of the bacterio-
logical flora of a sample, ten oysters should be taken
and the body, gastric juice and liquor should be
thoroughly mixed before examination. The examina-
tion, as in dealing with other food stuffs, is directed to
the search for members of the Coli-typhoid group,
sewage streptococci and perhaps also B. enteritidis
sporogenes.

464 BACTERIOLOGICAL ANALYSES

Apparatus Required:

Two hard nail brushes.

Liquid soap.

Sterile water in aspirator jar with delivery nozzle controlled
by a spring clip.

Sterile oyster knives.

Sterile glass dish, with cover, sufficiently large to accommodate
ten oysters.

Sterile forceps.

Sterile scissors.

Sterile towels or large gauze pads.

Sterile graduated cylinders 1000 c.c. capacity, with either the
lid or the bottom of a sterile Petri dish inverted over the
open mouth as a cover.

Glass rods.

Corrosive sublimate solution, i per mille.

Bile salt broth tubes.

Litmus milk tubes.

Surface plates of nutrose agar.

Case of sterile pipettes, i c.c. (in tenths of a c.c.)

Case of sterile pipettes, 10 c.c. (in tenths of a c.c.)

Case of sterile glass capsules.

Erlenmeyer flasks, 250 c.c. capacity.

Double strength bile salt broth.

METHOD. —

1. Thoroughly clean the outside of the oyster shells
by scrubbing each in turn with liquid soap and nail
brush under a tap of running water. Then, holding
an oyster shell in a pair of sterile forceps wash every
part of the outside of the shell with a stream of sterile
water running from an aspirator jar; deposit the oyster
inside the sterile glass dish. Repeat the process with
each of the remaining oysters.

2. Before proceeding further, cleanse the hands
thoroughly with clean nail brush, soap and water,
then plunge them in lysol 2 per cent, solution, and
finally in sterile water.

3. Spread a sterile towel on the bench.

4. Remove one of the oysters from the sterile glass
dish and place it, resting on its convex shell, on the

OYSTERS 465

towel. Turn a corner of the sterile towel over the
upper flat shell to give a firmer grip to the left hand,
which holds the shell in position.

5. With the sterile oyster knife (in the right hand)
open the shell and separate the body of the oyster
from the inner surface of the upper flat shell. Bend
back and separate the flat shell, leaving the body of the
oyster in and attached to the concave shell. Avoid
spilling any of the liquor.

(Some dexterity in opening oysters should be
acquired before undertaking these experiments).

6. Cut up the body of the oyster with sterile scissors
into small pieces and allow the liquor freed from the
body during the process to mix with the liquor previ-
ously in the shell.

7 . Transfer the comminuted oyster and the liquor to
the cylinder.

8. Treat each of the remaining oysters in similar
fashion.

9. Mix the contents of the cylinder thoroughly by
stirring with a sterile glass rod. The total volume will
amount to about 100 c.c.

10. Use o.i c.c. of the mixed liquor to inseminate
each of a series of three nutrose surface plates.

11. Inoculate o.i c.c. of the mixed liquor into each
of three tubes of litmus milk.

1 2 . Add sterile distilled water to the contents of the
cylinder up to 1000 c.c. and stir thoroughly with a
sterile glass rod and allow to settle. The bacterial
content of each oyster may be regarded, for all practi-
cal purposes, as comprised in 100 c.c. of fluid.

13. Arrange four glass capsules in a row and number
I, II, III, IV. Pipette 9 c.c. sterile distilled water into
each.

14. To capsule No. I add i c.c. of the diluted liquor,
etc. from the cylinder, and mix thoroughly. To cap-
sule II add i c.c. of dilution in capsule I and mix thor-

3°

466 BACTERIOLOGICAL ANALYSES

oughly. Carry over i c.c. of fluid from capsule II to
capsule III, afterwards adding i c.c. of fluid from capsule
III to capsule IV.

15. Label tubes of bile salt broth and inoculate with
the following amounts of diluted oysters :

No. 6 with 10 c.c. cylinder fluid =0.1 oyster.

No. 5 with i c.c. cylinder fluid = 0.01 oyster.

No. 4 with i c.c. capsule I fluid = 0.001 oyster.

No. 3 with i c.c. capsule II fluid = 0.0001 oyster.

No. 2 with i c.c. capsule III fluid = 0.00001 oyster.

No. i with i c.c. capsule IV fluid = 0.000001 oyster.

1 6. Transfer 100 c.c. cylinder fluid (= i oyster) to an
Erlenmeyer flask and add 50 c.c. double strength bile
salt broth, and label 7.

17. Duplicate all the above indicated cultures.

18. Put up the tube cultures in Buchner's tubes and
incubate anaerobically at 42° C.

If growth occurs in tube I the organism finally iso-
lated, e.g., B. coli, must have been present to the extent
of one million per oyster.

19. Complete the examination for members of the
Coli-typhoid group and sewage streptococci, as directed
under Water Examination, page 429 (steps 11-21).

20. Inoculate a series of 6 tubes of litmus milk with
quantities of the material similar to those indicated in
step 1 5 ; heat to 80° C. for ten minutes, and incu-
bate under anaerobic conditions at 37° C. Examine
for the presence of B. enteritidis sporogenes as directed
under Water Examination, page 438 (steps 7-10).

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

SEWAGE 467

quantitative examination and transmitted in the ice
apparatus used in packing those samples.

Apparatus Required. — As for water (vide page 420) .

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
thoroughly.

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.

Now i c.c. of dilution No. I contains o.i c.c. of the original sewage,

i c.c. of dilution No. II contains o.ooi c.c. of the original sewage,

i c.c. of dilution No. Ill contains o.ooooi c.c. of the original sewage,
i c.c. of dilution No. IV contains o.ooooooi c.c. of the original sewage.

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 concerned with the identification and enumeration
of the same bacteria dealt with under the corresponding
section of water examination ; it is consequently con-
ducted on precisely similar lines to those already in-
dicated (vide pages 426 to 441).

468 BACTERIOLOGICAL ANALYSES

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.

Erlenmeyer flask, 250 c.c. capacity (having a wide mouth
properly plugged with wool), containing 50 c.c. sterile water.

Rubber stopper to fit the mouth of the flask, perforated with
two holes, and fitted as follows:

Take a 9 cm. length of glass tubing and bend up 3 cm. at
one end at right angles to the main length of tubing. Pass
the long arm of the angle through one of the perforations in the
stopper; plug the open end of the short arm with cotton- wool.

Take a glass funnel 5 or 6 cm. in 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.

A battery jar or a small water-bath to hold the Erlenmeyer
flask when packed round with ice.

Supply of broken ice.

Rubber tubing.

Screw clamps and spring clips, for tubing.

Water steriliser.

Retort stand and clamps.

Apparatus for plating (as for enumeration of water organisms,
vide page 420).

METHOD.—

1. Fill 10 litres of water into the aspirating bottle
and attach a piece of rubber tubing with a screw clamp
to the delivery tube. Open the taps fully and regulate
the screw clamp, by actual experiment, so that the tube
delivers i c.c. of water every second. The screw clamp
is not touched again during the experiment.

At this rate the aspirator bottle will empty itself
in just under three hours. Shut off the tap and make
up the contents of the aspirator bottle to 10 litres again.

2. Sterilise the fitted rubber cork, with its funnel
and tubing, by boiling in the water steriliser for ten
minutes.

AIR

469

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.

FIG. 216. — Arrangement of apparatus for air analysis.

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 (Fig. 216).

6. Open the tap fully, 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 water in the Erlen-
meyer flask.)

7. When the aspiration is completed, disconnect the
flask and remove it from its ice packing.

470 BACTERIOLOGICAL ANALYSES

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 water 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.

1 2 . 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. water only represent those con-
tained in 10 litres of air, the resulting figure must be
multiplied by 100.

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 water, and incubate at 37° C.

3. Pour plates of nutrient agar with similar quan-
tities of the water 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.

The bacteriological examination of soil yields in-
formation of value to the sanitarian during the pro-
gress of the process of homogenisation of "made soil"
(e. g., a dumping area for the refuse of town) and

SOIL 471

determines the period at which such an area may with
propriety and safety be utilised for building purposes ;
or to the agriculturalist in informing him of the suita-
bility of any given area for the growth of crops.

The surface of the ground, exposed as it is to the
bactericidal influence of sunlight and to rapid alterna-
tions of heat and cold, rain and wind, contains but
few micro-organisms. Again, owing to the density
of the molecules of deep soil and lack of aeration on the
one hand, and the filtering action of the upper layers
of soil and bacterial antagonism on the other, bacterial
life practically ceases at a depth of about 2 metres.
The intermediate stratum of soil, situated from 25 to
50 cm. below the surface, invariably yields the most
numerous and the most varied bacterial flora.

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. 217. — Soil scoop.

whether we require surface samples or soil from vary-
ing depths.

(a) For surface samples, use an iron scoop, shaped
like a shoe horn, but provided with a sharp spine
(Fig. 217). This is wrapped in asbestos cloth and
sterilised in the hot-air oven. When removed from the
oven, wrap a piece of oiled paper, silk, or gutta-percha
tissue over the asbestos cloth, and secure it with
string, as a further protection against contamination.

472

BACTERIOLOGICAL ANALYSES

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 capsule for transmission.

FIG. 218. — Fraenkel's borer.

(b) For deep samples collected at various distances
from the surface, an experimental trench may be cut
to the required depth and samples collected at the
required points on the face of the section. It is, how-
ever, preferable to utilise some form of borer, such
as that designed by Fraenkel (Fig. 218).

Fraenkel's Earth Borer. — This instrument consists
of a stout hard-steel rod, 150 cm. long, marked in centi-

SOIL 473

metres from the drill-pointed extremity. It is pro-
vided with a cross handle (adjustable at any point
along the length of the rod by means of a screw nut) .
The terminal centimeters are thicker than the remainder
of the rod, and on one side a vertical cavity about
0.5 cm. deep is cut. This is covered by a flanged sleeve
so long as the borer is driven into the soil clockwise,
and is opened for the reception of the sample of soil,
when the required depth is reached, by reversing the
screwing motion, and again closed before withdrawal
of the borer from the earth by resuming the original
direction of twist. It can be sterilised in a manner
similar to that adopted for the scoop, or by repeatedly
filling the cavity with ether and burning it off.

Quantitative .-«r-Four distinct investigations are in-
cluded in the complete quantitative bacteriological
examination of the soil :

i. 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 .

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.

474 BACTERIOLOGICAL ANALYSES

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. 107).

Sterile metal (nickel) spoon or spatula.

Fractional steriliser (Fig. 140).

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 Erlen-
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,
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 thirty
minutes. One cubic centimetre of the turbid" broth
contains the washings from o.i gramme of soil.

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.

SOIL 475

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.
Then i c.c. fluid from capsule II contains soil water from .0001

gm. earth.
Then i c.c. fluid from capsule III contains soil water from

.000001 gm. earth.
Then i c.c. fluid from capsule IV contains soil water from

.0000000 1 gm. earth.

(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 0.4 c.c. of the diluted soil
water. Label and incubate.

12. Pour similar sets of wort gelatine plates from
the contents of capsules 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. Weigh out a second sample of soil — 10 grammes
—dry over a water-bath until of constant weight and
calculate the ratio

wet soil weight
dry soil weight

15. " Count" the plates after incubation for three,
four, or five days, and correcting the figures thus ob-
tained by means of the "wet" to "dry" soil ratio
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.

(c) The number of aerobic organisms "growing at
37° C." present per gramme of the soil.

476 BACTERIOLOGICAL ANALYSES

(B) Anaerobes (Vegetative Forms and Spores). —

16. Pour similar sets of plates in glucose formate
gelatine and agar and incubate in Bulloch's anaerobic
apparatus.

(C) Aerobes and Anaerobes (Spores Only). —

17. Pipette 5 c.c. soil water into a sterile tube.

1 8. Place in the differential steriliser at 80° C. for
ten minutes.

19. 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.

20. "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.

21. Calculate the ratio existing between spores and
spores + vegetative forms under each of the two groups,
aerobic and anaerobic micro-organisms.

Qualitative Examination. — The qualitative examina-
tion of soil is usually directed to the detection of one
or more of the following :

Members of the Coli-typhoid group.

Streptococci.

Bacillus anthracis.

Bacillus tetani.

Bacillus cedematis maligni.

The nitrous organisms.

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

V

SOIL 477

bouillon (technique similar to that described for the
water sample, vide pages 434-436).

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 onward the process is
varied slightly for each particular bacterium.

I. The Coli=typhoid Group.—

II. Streptococci.—

III. Bacillus Anthracis. —

IV. Bacillus TetanL—

The methods adopted for the isolation of these
organisms are identical with those already described
under water (page 43 7 et seq.) .

V. Bacillus (Edematis Maligni. — Method precisely
similar to that employed for the B. tetani.

VI. The Nitrous Organisms.—

1. Take ten tubes of Winogradsky's solution No I
(vide page 198) and number them consecutively from
i to 10.

2. Inoculate each tube with varying quantities of
the material as follows :

To tube No. i add i . o c.c. of the soil water.

To tube No. 2 add o . i c.c. of the soil water.

To tube No. 3 add i.o c.c. from Capsule I.

To tube No. 4 add o. i c.c. from Capsule I.

To tube No. 5 add i . o c.c. from Capsule II.

To tube No. 6 add o. i c.c. from Capsule II.

To tube No. 7 add i.o c.c. from Capsule III.

To tube No. 8 add o.i c.c. from Capsule III.

To tube No. 9 add i .o c.c. from Capsule IV.

To tube No. 10 add o. i c.c. from Capsule IV.

Label and incubate at 30° C.

478 BACTERIOLOGICAL ANALYSES

VII. The Nitric Organisms.—

3. Take ten tubes of Winogradsky's solution No II,
number them consecutively from i to 10 and inoculate
with quantities of soil water similar to those enum-
erated in section VI step 2. Label and incubate at
30° C.

4. 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.

5. Make further subcultivations from such of those
tubes as show growth, and again incubate.

6. If growth occurs in these subcultures, make
surface smears on plates of Winogradsky's silicate
jelly (vide page 198).

7. Pick off such colonies as make their appearance
and subcultivate in each of these two media.

TESTING 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 only the passage of germ-free
filtrates. In order to determine the freedom of the
filter from flaws and cracks which would permit the
passage of bacteria no matter how perfect the general
structure of the candle might be, the candle must first
be attached by means of a long piece of pressure tubing,
to a powerful pump, such as a foot bicycle pump, fitted
with a manometer. The candle is then immersed in
a jar of water and held completely submerged whilst
the internal pressure is gradually raised to two atmos-
pheres by the action of the pump. Any crack or flaw
will at once become obvious by reason of the stream
of air bubbles issuing from it.

The examination for permeability is conducted as
follows :

FILTERS 479

Apparatus Required:

Filtering apparatus: The actual filter candle that is used must
be the one it is intended to test and must be previously care-
fully sterilised; the arrangement of the apparatus will natur-
ally vary with each different form of filter, one or other of those
already described (vide pages 42-48).

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 mycoides, 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
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 agar
plates each containing i c.c. of the filtrate.

6. Incubate at 37° C. until, if necessary, the comple-
tion of seven days.

7. If the filtrate is not sterile, subcultivate the organ-
ism passed and determine its identity with the test
bacterium before rejecting the filter — since the filtrate
may have been accidentally contaminated.

8. If the filtrate is sterile, resterilise the candle and
repeat the test now substituting a cultivation of B.
prodigiosus — a bacillus of smaller size.

9. If the second test is satisfactory, test the candle
against a cultivation of a very small coccus, e. g.f
Micrococcus melitensis, in a similar manner; in this
instance continuing the incubation of cultivations from
the filtrate for fourteen days.

480 BACTERIOLOGICAL ANALYSES

TESTING OF DISINFECTANTS.

Methods have already been detailed (page 310) for
the purpose of studying the vital resistance offered by
micro-organisms to the lethal effect of germicides. But
it frequently happens that the bacteriologist has to
determine the relative efficiency of " disinfectants"
from the standpoints of the sanitarian and commercial
man rather than from the research worker's point of
view. In pursuing this line of investigation, it is con-
venient to compare the efficiency, under laboratory
conditions, of the proposed disinfectant with that of
some standard germicide, such as pure phenol. In so
doing, and in order that the work of different observers
may be compared, conditions as nearly uniform as
possible should be aimed at. The method described is
one that has been in use by the writer for many years
past, modified recently by the adoption of some of
the recommendations of the Lancet Commission on the
Standardisation of Disinfectants— particularly of the
calculation for determining the phenol coefficient.

This method has many points in common with that
modification of the "drop" method known as the
Rideal- Walker test.

General Considerations. —

These may be grouped under three headings : Test
Germ, Germicide, and Environment.

i. Test Germ. — B. coli.

As disinfectants are tested for sanitary purposes, it is
obvious that a member of the coli-typhoid group should
be selected as the test germ. B. coli is selected on ac-
count of its relative nonpathogenicity, the ease with
which it can be isolated and identified by different ob-
servers in various parts of the world, the stability of its
fundamental characters, and evenness of its resistance
when utilised for these tests; finally since the colon

DISINFECTANTS 481

bacillus is an organism which is slightly more resistant
to the lethal action of germicides than the more patho-
genic members of this group, a margin of safety is in-
troduced into the test which certainly enhances its value.
B. coli should be recently isolated from a normal
stool, and plated at least twice to ensure the purity of
the strain; and a stock agar culture prepared which
should be used throughout any particular test. For any
particular experiment prepare a smear culture on agar
and incubate at 3 7° C. for 2 4 hours anaerobically. Then
emulsify the whole of the surface growth in 10 c.c. of
sterile water. Transfer the emulsion to a sterile test-
tube with some sterile glass beads and shake thoroughly
to ensure homogenous emulsion. Transfer to a centri-
fuge tube and centrifugalise the emulsion to throw
down any masses of bacteria which may have escaped
the disintegrating action of the beads. Pipette off the
supernatant emulsion for use in the test.

2. Germicide. —

a. Disinfectant to be tested. —

The first essential point is to test the unknown disin-
fectant, which may be referred to as germicide-x, on
the lines set out on page 3 1 1 to determine its inhibition
coefficient.

This constant having been fixed, prepare various solu-
tions of germicide-x with sterilised distilled water by ac-
curate volumetric methods, commencing with a solution
somewhat stronger than that representing the inhibition
coefficient. The solutions must be prepared in fairly
large bulk, not less than 5 c.c. of the disinfectant being
utilised for the preparation of any given percentage
solution.

b. Standard Control. — Phenol.

The standard germicide used for comparison should
be one which is not subject to variation in its chemical
composition, and the one which has obtained almost
universal use is Phenol. r

482

BACTERIOLOGICAL ANALYSES

The following table shows the effect of different per-
centages of carbolic acid upon B. coli for varying con-
tact times, compiled from an experiment conducted
under the standard conditions referred to under Envi-
ronment. The results closely correspond to those
recorded by the Lancet Commission on Disinfectants,
1909.

Percentage of phenol

Contact time in minutes.

2$

5

10

15

20

25

3°

35

i 20

+
+
+
+
+
+
+

+
+
+
+
+

+
+
+
+

+
+
+

+
+
+

+
+

+

-

i.io

i.o

0.9
o 8t

o 80 ...

O 7 C

0-7

O 6=r .

— =No growth, i.e., bacteria killed.
+ = Growth, i.e., bacteria still living.

From this it will be seen that the following per-
centage solutions will need to be prepared, namely:
i.i per cent., i.o per cent., 0.9 per cent., 0.75 per cent.,
0.7 per cent., as controls for each experiment.

Prepare solutions of varying percentages by weighing
out the quantity of carbolic acid required for each and
dissolving in 100 c.c. of pure distilled water in an ac-
curately standardised measuring flask. The solutions
must be prepared freshly as required each day.

Environment.—

a. General. —

Close the windows and doors of the laboratory in
which the investigation is carried out, to avoid
draughts. Flush over the work bench and adjacent
floor with i : 1000 solution of corrosive sublimate.

DISINFECTANTS 483

Caution the assistant, if one is employed, to avoid
unnecessary movement or speech.

b. Contact Temperature, 15-18° C. —

This is the temperature at which contact between the
germicide and the test germ takes place, and is of
importance, since some germicides (e. g., Phenol) appear
to be more powerful at high temperatures. 18° C. —
practically the ordinary room temperature — is a tem-
perature at which the multiplication of B. coli is a com-
paratively slow process, but variation of a degree above
this temperature or of two or three degrees below is of
no moment. If the room temperature is below 15° C.
when the experiments are in progress, arrange a water-
bath regulated at 18° C. for the reception of the tubes
containing the mixture of germ and germicide ; if above
19° C. immerse the tubes in cold water, to which small
pieces of ice are added from time to time to prevent the
temperature rising above 18° C.

•c. Relative Proportional Bulk of Test Germ and
Germicide, 50 : 1 . —

Five cubic centimetres is a convenient amount of
germicidal solution to employ, and to this o.i c.c. of
the emulsion of test germ should be added.

d. Bulk of Sample Removed from Germ -{-Germicide
Mixture at Each of the Time Periods, 0. 1 c.c. —

This is sufficient to afford a fair sample of the germ
content of the mixture, and at the same time is insuffi-
cient to exert any inhibitory action when transferred to
the subculture medium.

e. Subculture Medium. Bile Salt Broth. —

A fluid medium is essential in order to obtain imme-
diate dilution of the germicide carried over ; at the same
time it is advantageous to employ a selective medium
which favours the growth of the test germ to the

484 BACTERIOLOGICAL ANALYSES

exclusion of organisms likely to contaminate the prep-
aration, and if possible one which affords character-
istic cultural appearances.

Bile Salt Broth (page 180) combines these desiderata;
it permits only the growth of intestinal bacteria, whilst
the formation of an acid reaction and the production
of gas in subcultures prepared from the germ-germicide
mixture is fairly complete evidence of the presence of
living B. coli.

The amount of medium present in each test-tube is a
matter of importance, since the medium not only pro-
vides pabulum for the test germ, but also acts as a
diluent to the germicide, to reduce its strength below
its inhibition coefficient. For routine work each sub-
culture tube contains 10 c.c. of medium, but it is
obvious that if germicide-x possesses an inhibition
coefficient of o.i per cent, the addition of o.i c.c. of
a 10 per cent, solution to 10 c.c. of medium would effectu-
ally prevent the subsequent growth of the test germ
after a contact period insufficient to destroy its vitality.
Hence the preliminary tests may in some instances
indicate the necessity for the presence of 12 c.c., 15 c.c.
or more of the fluid medium in the culture tubes.

/. Incubation Temperature, 37° C. —
g. Observation Period of the Subcultivations, Seven
Days.—

In order to determine whether or no the test germs
have been destroyed, observations must always be con-
tinued— when growth appears to be absent — up to the
end of seven days before recording ' ' no growth. ' '

h. Identification of the Organisms Developing in the
Subcultivations after Contact in the Germ + Germicide
Solution. —

This is based on the naked eye characters of the
growth in the bile salt broth, supplemented where

DISINFECTANTS 485

necessary by plating methods, further subcultivations
upon carbohydrate media and agglutination experi-
ments. The sign ( + ) is used to indicate that growth
of the test organism occurred in the subcultivations,
and the sign ( — ) to indicate that the test germs have
been destroyed and no subsequent growth has taken
place.

METHOD. —

Apparatus Required:

Sterile test-tubes (narrow, not exceeding 1.3 cm. diameter).

Test-tube rack (Fig. 219).

Sterile graduated pipettes in case, i c.c. (in tenths).

Sterile graduated pipettes in case, 5 c. c.(m c.c.).

Circular rubber washers, 2.5 cm. diameter with central hole,
sterilised by boiling immediately before use, then transferred to
sterilised glass double dish.

Electric signal clock or stop watch.

Sterile forceps.

Sterilised glass beads. .

Shaking machine.

Grease pencil.
Material Required:

Percentage solutions of germicide-x (vide page 481).

Percentage solutions of pure phenol (vide page 482).

Aqueous emulsion of B. coli (vide page 481).

Tubes of bile salt broth.

Preliminary Tests.—

a. Inhibition Coefficient. —

Determine the lowest percentage of germicide-x
which inhibits growth of B. coli in the bile salt broth,
and the highest percentage which fails to inhibit (page
311). On the result of this experiment determine the
bulk of medium required in the subculture tubes and
the percentage solutions to be employed in the trial
trip. Assuming the inhibition coefficient to be i : 1000,
it will be quite safe to employ the ordinary culture
tubes containing 10 c.c. medium in the subsequent
experiments.

486 BACTERIOLOGICAL ANALYSES

b. Trial Trip. —

Determine the lethal effect of a series of five solutions
of germicide-x (say 1:100, 1:250, 1:300, 1:500,
1:600) at contact times of 2j, 5, 25 and 30 minutes
in the following manner:

1. Arrange five test-tubes marked A to E in the lower
tier of the test-tube rack.

2. Into tube A pipette 5 c.c. germicide-x 1:100
solution.

Into tube B pipette 5 c.c. germicide-x 1:200 solution.
Into tube C pipette 5 c.c. germicide-x i :3oo solution.
Into tube D pipette 5 c.c. germicide-x i :5oo solution.
Into tube E pipette 5 c.c. germicide-x i :6oo solution.

3 . Arrange 20 tubes of bile salt broth in the upper tier
of the test-tube rack in two rows, those in the front
row numbered consecutively from left to right i-io,
those in the back row 11-20.

4. Place a square wire basket of about 50 tubes
capacity close to the left of the test-tube rack, for the
reception of the inoculated tubes.

5. Take a sterile i c.c. pipette from the case, pick up
a sterile rubber washer with forceps and push the point
of the pipette into the central hole.

6. Put down the forceps on the bench with the sterile
points projecting over the edge. Without taking. the
tube from the rack remove the cotton- wool plug from
tube A, and lower the pipette, with the rubber washer
affixed, on to the open mouth of the tube; with the
help of the forceps to steady the washer, push the
pipette on through the hole until the point of the
pipette has reached to within a few millimetres of the
bottom of the tube (see fig. 219).

7. Adjust in the same way a pipette and a washer in
the mouth of each of the other tubes, B, C, D and E.

8. Set the electric signal clock to ring for the com-
mencement of the experiment and at subsequent inter-
vals of 2j, 5, 25 and 30 minutes.

DISINFECTANTS

487

9. Take up 0.5 c.c. of B. coli emulsion in sterile
pipette graduated in tenths of a cubic centimetre and
stand by.

10. As soon as the bell rings lift the pipette from
tube A with the left hand and from the charged pipette
held in the right hand deliver o.i c.c. of B. coli emulsion
into the i : 100 solution. Then replace the pipette and
washer.

FIG. 219. — Test-tube rack.

IT. Raise the tube with the left hand and shake it to
mix germ and germicide, whilst returning the delivery
pipette in the right hand.

12. Repeat the process with tubes B, C, D and E;
then drop the infected delivery pipette in the lysol jar.
The inoculation of the five tubes can be carried out
very expeditiously, but a period of 10 seconds must
be allowed for each tube.

13. When the bell rings at 2j minutes blow through
the pipette in tube A (this agitates the germ + germicide
mixture and ensures the collection of a fair sample) ;
allow the mixture to enter the pipette, and as the
column of fluid extends well above the terminal
graduation, the right forefinger adjusted over the
butt-end of the pipette before it is lifted will retain

488 BACTERIOLOGICAL ANALYSES

more than o. i c.c. of the mixture within the bore when
the point of the pipette is clear of the fluid in the tube.
Touch the point of the pipette on the inner wall of the
tube, and allow any excess of fluid to escape, only
retaining o.i c.c. in the pipette.

14. At the same time, with the left hand remove Bile
Salt Tube No. i from the upper tier of the rack, take
out the cotton-wool plug with the hand already holding
the pipette (the relative positions of pipette, plug and
culture tubes being practically the same as those of
platinum loop, plug and culture tube shown in Fig. 68,
page 74).

15. Insert the point of the pipette into the subculture
tube, and blow out the mixture into the medium — re-
plug the tube and drop it into the wire basket. Re-
place the washer-pipette in tube A.

As soon as the point of the pipette has entered the
mouth of tube A it may be released, since it has already
been so adjusted that it just clears the bottom of the
test-tube, and the elastic washer will prevent any
damage to the tube.

Steps 13, 14 and 15 occupy on an average 10 seconds.

1 6. Repeat steps 13, 14 and 15 with each of the other
tubes B, C, D and E.

17. Repeat these various steps 13—16 when the bell
rings at 5, 25 and 30 minutes.

18. Place all the inoculated tubes in the incubator
at 37° C.

19. Examine the tubes at intervals of 24 hours, and
record the results in tabular form as shown in Table
page 491 (the figures in the squares indicate the number
of hours at which the changes in the medium due to the
growth of B. coli first appeared).

20. If a consideration of the tabulated results indi-
cates strengths of Germicide-x lethal at 2j and 30
minutes the final test can be arranged, but if this
result has not been attained, sufficient evidence will

DISINFECTANTS 489

probably be available to enable a second trial test to
be planned which will give the required information.

Final Test. —

c. Determination of Phenol Coefficient. —

X-Disinfectant. — This comprises two distinct tests,
one of the Germicide-x, the other of the standard
phenol.

1 . Arrange five test-tubes clearly marked in the lower
tier of the rack.

2. Pipette into each 5 c.c. respectively of the five
percentage solutions of x-disinfectant which the trial
run has already shown will include those affording
lethal values at 2^ and 30 minutes.

3. Arrange 20 tubes of bile salt broth in the upper tier
of the test-tube rack in two rows, those in the front
row numbered consecutively from left to right i-io,
those in the back row 11-20.

4. Arrange further 20 tubes of bile salt broth num-
bered 2 1-40 in two rows in a second smaller rack which
can be stood on the upper tier of the rack as soon as
the first 20 tubes have been inoculated.

5. Place a square wire basket of about 50 tube
capacity close to the left of the test-tube rack, for the
reception of the inoculated tubes.

6. Adjust a sterile i c.c. pipette in the mouth of each
of the tubes, A, B, C, D and E, by means of a washer, as
previously described.

7. Set the electric signal clock to ring for the com-
mencement of the experiment and subsequently at
2j, 5, 10, 15, 20, 25, 30 and 35 minutes.

8. Complete precisely as indicated in Trial Runs,
steps 9-19.

Control Phenol. —

Immediately the subculture tube from the 3o-minute
contact period have been inoculated, carry out a pre-

490 BACTERIOLOGICAL ANALYSES

cisely similar experiment, in which five percentage
strengths of Phenol, (e. g., i.i, i.o, 0.9, 0.75, 0.7) are
arranged in the lower tier of the test-tube rack in
place of the five strengths of Germicide-x.

Calculate the phenol coefficient by the following
method :

(a) Divide the figure representing the percentage
strength of the weakest lethal dilution of the carbolic
acid control at the 2j-minute contact period by the
figure representing the percentage strength of the
weakest lethal dilution of the x-disinfectant at the
same period. The quotient = phenol coefficient at 2^
minutes.

(b) Similarly obtain the phenol coefficient at 30
minutes contact period.

(c) Record the mean of the two coefficients obtained
in (a) and (b) as the mean phenol coefficient, or simply
as the Phenol Coefficient.

The details of the Final Test of an actual determina-
tion are set out in the accompanying table.

DISINFECTANTS

491

TABLE 27

Organism employed, B. Coli Communis.

Culture Medium, Nutrient Agar (+ 10). Age, 24 hrs. Temp, of Incubation, 37° C.

. / Culture 1 ,-, . .

Quantities used < ^ . . > ri/mulsion o.i c.c. +5 c.c. Germicide.
\ Emulsion j

Room Temperature during Experiments, 17° C.

Germicide

Strength

Time of exposure

Incubation

2*

5

10

15

20

25

30

35

Time

Temp.

z

Germicide-x.

4%

—

—

—

—

—

' —

_

7 days.

37° C.

a

Germicide-x.

3%

48

—

—

—

7 days.

37° C.

3
4

Germicide-x.

2%

24

24

24

24

48

72

7 days.

37° C.

Germicide-x.

1% 24

24

24

24

72

24

72

7 days.

37° C.

S

a
3

Germicide-x.

0.5%

24

24 24

24

24

24

24

24

24hours.

37° C.

—

—

—

Phenol

I. 10%

—

—

7 days.

37° C.

Phenol

I .00%

24

7 days.

37° C.

Phenol

o.7S%

24

24

24

24

48

7 days.

37° C.

4
5

Phenol

o. 70%

24

24

24

24 \ 24

72

...

7 days.

37° C.

Phenol

0.65%

24

24

24

24

24

48

24

24

2 days.

37° C.

Phenol Coefficient :

I^O + O^X

4.00 2.0/

0.27+ 0.35 _ 62 _

2 2 ~°'31

APPENDIX.

METRIC AND IMPERIAL SYSTEMS OF WEIGHTS AND
MEASURES.

The initial unit of the metric system is the Metre (m.)
or unit of length, representing one-fourth-millionth
part of the circumference of the earth round the poles.

The unit of mass is the Gramme (g.), and repre-
sents the weight of one cubic centimetre of water at
its maximum density (viz. 4° C. and 760 mm. mercury
pressure) .

The unit of the measure of capacity is the Litre (/.) ,
and represents the volume of a kilogramme of distilled
water at its maximum density.

The decimal subdivisions of each of the units are
designated by the Latin prefixes niilli = ^-^\ centi=
•J-O-Q-; deci — -^\ the multiples of each unit by the
Greek prefixes deka= 10; hecto= 100; kilo= 1000; myria
= 10,000.

For a comparison of the values of some of the more
frequently employed expressions of the Metric System
and the Imperial System, the following may be found
convenient for reference :

Length :

i millimetre ( = i mm.) = -fa of an inch.

*• centimetre (= i cm.) = f of an inch.

L inch (i") =25 millimetres or 2j centimetres.

Mass:

i milligramme ( = i mg.) = 0.01543 grain (or approximately
& grain) .

i gramme (= i g.) = 15.4323 grains.

i "kilo" or kilogramme (=i kgm.) = 2 pounds, 3^ ounces
avoirdupois.

i pound avoirdupois ( = i ^0=453.592 grammes.

i ounce avoirdupois ( = i oz.) =28.35 grammes.

i grain = 0.0648 gramme or 64.8 milligrammes.

4Q2

APPENDIX 493

Capacity :

i cubic centimetre (=i c.c.) = i6.9 rninirns imperial measure.

i litre (=i /.) =35.196 fluid ounces imperial measure.

i fluid ounec imperial measure (= ig) = 28.42 cubic centimetres.

i pint imperial measure(— xO.) =568.34 cubic centimetres.

i gallon imperial measure ( = iC.)=4-546 litres, or 10 pounds
avoirdupois, of pure water at 62° F. and under an atmospheric
pressure of 30 inches of mercury.

FACTORS FOR CONVERTING FROM ONE SYSTEM TO THE OTHER.

To convert grammes into grains X 15. 43 2.

To convert grammes into ounces avoirdupois X 0.03527.

To convert kilogrammes into pounds ... X 2.2046.
To convert cubic centimetres into fluid

ounces imperial X 0.0352.

To convert litres into fluid ounces imperial . X 3 5 . 2 .

To convert metres into inches X39-37-

To convert grains into grammes X 0.0648.

To convert avoirdupois ounces into grammes . X 28 . 3 5.
To convert troy ounces into grammes . . . X 31 • 104.
To convert fluid ounces into cubic centi-
metres X28.42.

To convert pints into litres X 0.568.

To convert inches into metres X 0.0254.

494

APPENDIX

TABLE FOR THE CONVERSION OF DEGREES CENTL
GRADE INTO DEGREES FAHRENHEIT.

F.

Cent.

Faht.

1

Cent.

Faht.

Cent.

Faht.

0

32 . o

34

93-2

68

154-4

i

33-8

35

95-°

69

156 . 2

2

35-6

36

96.8

70

158.0

3

37-4

37

98.6

7i

159.8

4

39-2

38

100.4

72

161.6

5

41.0

39

102 . 2

73

163.4

6

42.8

40

104 . o

74

165.2

7

44-6

4i

105.8

75

167 . o

8

46.4

42

107 . 6

76

168.8

9

48.2

43

109.4

77

170. 6

10

5° • °

44

III. 2

78

172.4

1 1

5i-8

45

II3.0

79

174.2

12

53-6

46

II4.8

80

176.0

13

55-4

47

116.6

81

177.8

14

57-2

48

118.4

82

179.6

15

59-°

49

120 . 2

83

181.4

16

60.8

50

122 .O

84

183.2

i7

62.6

51

123.8

85

185.0

18

64.4

52

125 . 6

86

186.8

19

66.2

53

127.4

87

188.6

20

68.0

54

129. 2

O O
00

190.4

21

69.8

55

I3I.O

89

192 .2

22

71.6

56

132.8

90

194.0

23

73-4

57

134.6

91

195.8

24

75-2

58

136.4

92

197.6

25

77.0

59

138.2

93

199.4

26

78.8

60

140. o

94

201 . 2

27

80.6

61

I4I.8

95

203.0

28

82.4

62

143.6

96

204.8

29

84.2

63

145 -4

97

206 . 6

30

86.0

64

147.2

98

208.4

31

87.8

65

149.0

99

210.2

32

89.6

66

150.8

TOO

212 . O

33

91.4

67

152.6

APPENDIX

495

TABLE FOR THE CONVERSION OF DEGREES FAHREN-
HEIT INTO DEGREES CENTIGRADE.

1

a

0>

O

1

1

+3

aS
PH

1

fe

6

a3 i <D
PH O

32

o .

69

20. 6

I05

40. 6

141

60.6

177

80.6

33

0.6

70

21 . I

106

41. i

142

61. i

I78

81.1

34

i . i

7i

21.7

107

41.7

M3

61. 7

179

8i.7

35

i .7

72

22 . 2

108

42.2

144

62.2

180

82.2

36

2 . 2

73

22.8

109

42 .8

145

62.8

181

82.8

37

2.8

74

23-3

I 10

43-3

146

63-3

182

83-3

38

3-3

75

23-9

iii

43-9

147

63 .9

183

83-9

39

3-9

76

24.4

112

44-4

148

64.4

184

84.4

40

4.4

77

25.0

H3

45-°

149

65.0

185

85.0

4i

5-°

78

25.6

114

45-6

150

65.6

186

85.6

42

5-6

79

26.1

115

46.1

151

66.1

187

86.1

43

6.1

80

26.7

116

46.7

152

66.7

188

86.7

44

6.7

8 1

27.2

117

47-2

153

67 . 2

189

87.2

45

7.2

82

27.8

118

47-8

154

67.8

190

87.8

46

7-8

83

28.3

119

48.3

155

68.3

191

88.3

47

8-3

84

28.9

120 48.9

156

68.9

192

88.9

48

8.9

85

29.4

121 49.4

157

69.4

193

89.4

49

9-4

86

30.0

122 l 50.0

158

70. o

194

90.0

5°

10 . 0

87

30. 6

I23

50. 6

159

70 . 6

195

90. 6

5i

10. 6

88

31-1

124

51-1

160

71.1

196

91.1

52

1 1 . i

89

31 .7

125

5r-7

161

71.7

197

91.7

53

11.7

90

32.2

126

52.2

162

72 . 2

198

92.2

54

12 . 2

91

32-8

I27

52.8

163

72.8

199

92.8

55

12.8

92

33-3

128

53-3

164

73-3

200

93-3

56

13-3

93

33-9

I29

. 53-9

165

73-9

201

93-9

57

13 .9

94

34-4

I30

54-4

166

74-4

2O2

94-4

58

14.4

95

35-°

J3!

55-°

167

75-°

203

95-°

59

15-0

96

35-6

I32

55-6

168

75-6

2O4

95-6

6o

15-6

97

36.1

133

56. i

169

76.1

205

96. i

61

16.1

98

36.7

134

56.7

170

76.7

2O6

96.7

62

16.7

99

37-2

135

57-2

171

77.2

207

97.2

63

17.2

IOO

37-8

136

57-8

172

77-8

208

97.8

64

17.8

101

38.3

*37

58-3

173

78.3

2O9

98-3

65

18.3

IO2

38-9

138

58.9

174

78.9

210

98.9

66

18.9

I03

39-4

J39

59-4

175

79-4

211

99-4

67

19.4

I04

40.0

140

60 .0

176

80.0

212

IOO.O

68

20. o

496

APPENDIX

Percentage Formula for addition of salts, etc., to completed
media.

Formula for preparing any desired percentage of a given salt,
etc., in tubed media; e. g., to make 4 per cent, solution of KNO3
in a series of tubes of broth each containing 10 c.c. of medium,
when there is already available a 25 per cent, stock aqueous
solution of potassium nitrate.

(AT+X) Y^A (X)

IOO IOO

N = number of cubic centimetres contained in each tube.
X = amount of stock solution to be added to each tube.
Y = percentage required in the medium.
A = percentage of stock solution.
Then

(ip + X) 4^5X

IOO IOO

Therefore,

Therefore,

X= i . 9 c.c.
This allows for solution added to the original bulk of medium.

Therefore, 10 c.c. broth +1.9 c.c. of a 25 per cent, aqueous
solution KNO3 makes 11.9 c.c. medium containing 4 per cent.
KN03.

TABLES FOR PREPARING DILUTIONS

(of Serum, Disinfectants or other substances.)
In estimating the agglutinin content or litre of a serum,
testing disinfectants and for many other purposes, it becomes
necessary to prepare a series of dilutions of the material under
examination, and in order to avoid unnecessary expenditure of
labour it is convenient to adhere to some definite scale of incre-
ment, such for example as the following :

10 to

From dilutions of i :

of 5-

From dilutions of i :
of 10.

From dilutions of i .
of 25.

From dilutions of i :
of 50.

From dilutions of i :
of TOO.

From dilutions of i :
of 250.

From dilutions of i :
of 1000.

80 to i

200 to i :

400 to

500 to i

1000 to i :

5000 to i

80 rise by increments
200 rise by increments
400 rise by increments
500 rise by increments
1000 rise by increments
5000 rise by increments
10,000 rise by increments

APPENDIX 497

From dilutions of i : 10,000 to i : 100,000 rise by increments
of 5000.

From dilutions of i : 100,000 to i : 1,000,000 rise by increments
of 100,000.

When dealing with a substance of unknown powers — and this
is especially true with regard to agglutinating sera — it is customary
to run a preliminary test, using a few widely separated dilutions
such as may be obtained in the following manner:

FIRST DILUTION — I.

i c.c. serum + 9 c.c. normal saline solution = 10 per cent,
solution or i: 10 dilution (of which i c.c. contains o.i c.c. of the
original serum).

When dealing with fluids other than serum the diluent is
usually distilled water; whilst if the original substance is a solid
the instructions would read :

i gram o.s. + 10 c.c. distilled water = 10 per cent, solution, etc.

SECOND DILUTION — II.

i c.c. first dilution + 9 c.c. normal saline solution = i per
cent, solution or 1:100 dilution.

THIRD DILUTION — III.

i c.c. second dilution + 9 c.c. normal saline solution = i per
mille solution or i: 1000 dilution.

FOURTH DILUTION — IV.

i c.c. second dilution + 9 c.c. normal saline solution = o. i per
mille solution or i : 10,000 dilution.

The following tables showing the secondary dilutions that can
readily be prepared from each of these four primary dilutions for
use in the subsequent determination of the exact titre will probably
be found of service by those who are not ready mathematicians.

498

APPENDIX

TABLES FOR PRE-

TABLE I

Using 10% stock solution
First
dilution

+ Diluent

TABLE II
Using i% stock solution

Second )

... . > + Diluent

dilution f

I

10 = I C.C.

+ 0 C.C.

I

: ioo = i c.c.

+ 0 C.C.

I

15 = i c.c.

+ 0.5 c.c.

I

: no = i c.c.

+ O.I C.C.

I

20 = I C.C.

+ 1.0 C.C.

I

: 120 = i c.c.

+ 0.2 C.C.

I

25 = i c.c.

+ 1.5 c.c.

[I

: 125 = i. c.c.

+ 0.25 c.c.]

I

30 = i c.c.

+ 2.0 C.C.

I

: 130 = i c.c.

+ 0.3 c.c.

I

35 = i c.c.

+ 2.5 c.c.

I

: 140 = i c.c.

+ 0.4 c.c.

I

40 = i c.c.

+ 3-0 c.c.

I

: 150 = i c.c.

+ 0.5 c.c.

I

45 = i c.c.

+ 3-5 C.C.

I

: 160 = i c.c.

+ 0.6 c.c.

I

50 = i c.c.

+ 4.0 c.c.

I

: 170 = i c.c.

+ 0.7 c.c.

I

55 = i c.c.

+ 4-5 c.c.

[t

: 175 = i c.c.

+ 0.75 c.c.]

I

60 = i c.c.

+ 5.0 c.c.

I

: 180 = i c.c.

+ 0.8 c.c.

I

65 = i c.c.

+ 5-5 c.c.

I

: 190 = i c.c.

+ 0.9 c.c.

I

70 = i c.c.

+ 6.0 c.c.

I

: 200 = i c.c.

+ 1.0 C.C.

J

1 r = T P ^

+ 6.5 c.c.

I

/ D 1 U. ^.

80 = i c.c.

+ 7.0 c.c.

I

: 200 = i c.c.

+ 1.0 C.C.

I

80 = i c.c.

+ 7.0 c.c.

I

I

'. 225 == i c.c.
: 250 = i c.c.

~f~ 1.25 c.c.
+ 1.5 c.c.

I

90 = i c.c.

+ 8.0 c.c.

I

: 275 = i c.c.

+ 1.75 c.c.

I

100 = I C.C.

+ 9.00 c.c.

I

: 300 = i c.c.

+ 2.0 C.C.

I

no = i c.c.

+ 10.0 c. :.

I

: 325 = i c.c.

+ 2.25 c.c.

I

120 = I C.C.

+ II .0 C.C.

I

: 350 = i c.c.

+ 2.5 c.c.

[I

125 = i c.c.

+ ii. 5 c.c.]

I

: 375 = i c.c.

+ 2.75 c.c.

I

130 = i c.c.
140 = i c.c.

+ 12.0 C.C.

I

: 400 = i c.c.

+ 3.0 c.c.

I

150 = i c.c.

+ 14.0 c.c.

I

: 400 = i c.c.

-f- 3.0 c.c.

I

160 = i c.c.

+ 15.0 c.c.

I

: 450 = i c.c.

+ 3- 5 c.c.

*

170 = i c.c.
175 == icc

+ 16.0 c.c.

I

: 500 = i c.c.

+ 4.0 c.c.

I

180 = i c.c.

i 10.5 c.c.j
+ 17.0 c.c.

I

: 500 = i c.c.

+ 4-0 c.c.

I

190 = i c.c.

+ 18.0 c.c.

I

: 600 = i c.c.

+ 5-0 c.c.

I

200 = I C.C.

+ 19.0 c.c.

I

'. 700 = i c.c.

+ 6.0 c.c.

1 /c - •» ~ 1

I

200 = I C.C.

4- 19.0 c.c.

I
I

: 750 = i c.c.
: 800 = i c.c.

T 0.5 C.C.J

+ 7.0 c.c.

I

225 = i c.c.

+ 21.5 c.c.

I

• 900 = i c.c.

+ 8.0 c.c.

I

250 = i c.c.

+ 24.0 c.c.

I

: 1000 = i c.c.

+ 9.0 c.c.

I

275 = icc

1 z-

I

300 = i c.c.

+ 29.0 c.c.

I

: 1000 = i c.c.

-f- 9.0 c.c.

I

325 = i c.c.

+ 31-5 C.C.

I

: 2000 = i c.c.

4- 19.0 c.c.

I

350 = i c.c.

+ 34.0 c.c.

I

: 3000 = i c.c.

+ 29.0 c.c.

I

375 = i c.c.

+ 36. 5 C.C.

I

: 4000 = i c.c.

+ 39.0 c.c.

I

400 = i c.c.

+ 39.0 c.c.

I

: 5000 = i c.c.

+ 49.0 c.c.

I

400 = i c.c.

+ 39-0 C.C.

I

450 = I C.C.

+ 44.5 c;c.

I

500 = i c.c.

+ 49.0 c.c.

APPENDIX

499

PARING DILUTIONS.

TABLE III

Using 0.1% stock solution
Third
dilution

Diluent

TABLE IV

Using 0.01% stock solution
Fourth
dilution

Diluent

x

1000

I C.C.

+ 0 C.C.

x

10,000

I

C.C. + 0 C

.c.

I

1250

=

I C.C.

4- 0.25 c.c.

I

15,000

=

I

c.c. + 0.5

c.c.

I

1500

—

I C.C.

+ 0.5 c.c.

I

20,000

—

I

C.C. + 1.0

c.c.

I

1750

—

I C.C.

+ 0.75 c.c.

I

25,000

«•

I

c.c. +

i-5

c.c.

I

2000

=

I C.C.

+ 1.0 C.C.

I

30,000

=

I

c.c. +

2.0

c.c.

I

2250

=

I C.C.

+ 1.25 c.c.

I

35,ooo

=

I

c.c. -f- 2.5

c.c.

I

2500

=

I C.C.

+ 1.5 c.c.

I

40,000

=

I

c.c. +

3-o

c.c.

I

2750

=

I C.C.

+ 1.75 C.C

I

45,000

=

I

c.c. -f- 3.5

c.c.

I

3000

=

I C.C.

+ 2.0 C.C.

I

50,000

=

I

c.c. +

4-o

c.c.

I

3250

=

I C.C.

+ 2.25 c.c.

I

55,ooo

—

I

c.c. +

4.5

c.c.

I

3500

=

I C.C.

+ 2.5 c.c.

I

60,000

=

I

c.c. +

5-0

c.c.

I

3750

=

I C.C.

+ 2.75 c.c.

I

65,000

=

I

c.c. +

5-5

c.c.

I

4OOO

=

I C.C.

+ 3.0 c.c.

I

70,000

=

I

c.c. +

6.0 c.c.

I

4250

=

I C.C.

+ 3.25 c.c.

I

75,000

=

I

c.c. 4-

6.5

c.c

I

4500

=

I C.C.

+ 3-5 C.C.

I

80,000

BB

I

c.c. +

7.0 c.c.

I

4750

=

I C.C.

+ 3.45 c.c.

I

85.000

=

I

c.c. +

7-5

c.c.

I

5000

=

I C.C.

+ 4.0 c.c.

I

90,000

=

I

c.c. -f

8.0

0 -

c.c.

I

5000
6OOO

-

I C.C.

+ 4.0 c.c.

I

I

95,000

100,000

-

I
I

c.c. ~f~
c.c. +

0-5

9-0

c.c.
c.c.

I

I

7000

=

I C.C.
I C.C.

~r~ 5-O C.C.

+ 6.0 c.c.

i : 100,000

a.

0.

I C.C.

+ 0

9 c.c.

[I

7500

=

I C.C.

+ 6-Sc.c.]

i : 200,000

—

o .

I C.C.

+ i

.9 c.c.

I

8OOO

.

I C.C.

+ 7.0 c.c.

[i : 250,000

—

o .

I C.C.

+ 2

4 c.c.

I

9000

=

I C.C.

+ 8.0 c.c.

i : 300,000

m*

0.

I C.C.

+ 2

9 c.c.

I

10,000

=

I C.C.

+ 9.0 c.c.

i : 400,000

—.

0.

I C.C.

+ 3

+ A

9 c.c.

i . 500,000

0.

I C.C.

4

9 c.c.

I

15,000

=

I C.C.
I C.C.

+ 14.0 c.c.

i : 500,000

=

0.

I C.C.

+ 4

9 c.c.

I

20,000

=

I C.C.

+ 19.0 c.c.

i : 600,000

-

0.

I C.C.

+ 5

9 c.c.

I

25,000

=

I C.C.

+ 24.0 c.c.

i : 700,000

=

0.

I C.C.

+ 6.9 c.c.

i : 30,000

=

I C.C.

+ 29.0 c.c.

[i : 750,000

=

o .

I C.C.

+ 7

4 c.c.

i i 800,000
i : 900,000

=

o .

0.

I C.C.
I C.C.

+ 7 • y ^•<^-
+ 8.9 c.c.

i : 1,000,000

=

0.

I C.C.

+ 9

9 c.c.

500

APPENDIX

TEMPERATURE PRESSURE TABLE.

Temperature
Centigrade

Mm. of Hg.

Pounds per sq. in.
absolute pressure

Atmospheres

98°

707. i

13-7

°-93

99°

733-i

14.2

0.96

100°

760 .0

14.7

I . OO

101°

787.8

15.2

1-03

102°

816.0

15-8

1.07

103°

845-2

16.3

i . ii

104°

875-4

16 . 9

i . 15

105°

906 .4

17-5

1.19

106°

938.3

18.1

1.23

107°

971 .1

18.8

1.27

108°

1004 . 9

19.4

1.32

109°

1039.6

20 . I

1.36

110°

I075-3

20.8

1.41

in0

I I 12 . 0

21.5

i .46

112°

1 149 . 8

22.2

i.5i

H3°

1188.6

22.9

i.56

114°

1228 .4

23 • 7

1.61

"5°

1269 .4

24.5

1.67

116°

I311 -4

25-3

1.72

117°

1354.6

26 . 2

1.78

118°

1399.0

27.0

1.84

119°

1444.5

27.9

i . 90

120°

I491-2

28.8

i .96

121°

1539-2

29-7

2.02

122°

1588.4

3°-7

2 .09

I23°

1638.9

3r-7

2.15

124°

1690 . 7

32.7

2 . 22

I25°

1743-8

33-7

2 . 29

APPENDIX

501

TABLE FOR DESICCATION AT LOW TEMPERA
TURES IN VACUO.

Temperature
Centigrade

Mm. of Hg.

21°

18.4

22°

19.6

23°

20.8

24°

22 . I

25°

23-5

26°

24.9

27°

26.4

28°

28.0

29°

29.7

30°

31-5

3i°

33-3

32°

35-3

33°

37-3

34°

39-5

35°

41.7

36°

44-1

37°

46.6

38°

49-2

39°

51.9

40°

54-8

41°

57-8

42°

61.0

43°

64.3

44°

67.7

45°

71-3

46°

75.i

47°

79.0

48°

83.1

49°

87-4

50°

91.9

502 APPENDIX

ANTIFORMIN METHOD

For the detection of B. Tuberculosis.

Antiformin was introduced into bacteriological
technique by Uhlenhuth in 1908 for the purpose of
demonstrating tubercle bacilli when present in small
numbers, in sputum or other material. It is a
powerful oxidising agent and rapidly destroys most
bacteria, but tubercle and other acid-fast organisms
resist its lethal action for considerable periods, and
upon this fact the method is based.

To prepare Antiformin measure out and mix : —

Eau de Javelle (Liquor sodas chlorinatae — B.P.) 50 c.c.
Sodic hydrate 15 per cent, aqueous solution .... 50 c.c.

METHOD.

1. Introduce the sputum or other material (e.g.
milk deposit and cream; pus; minced gland or other
organ ; caseous material ; broken down foci, etc.) into
a sterile tube and then add an equal volume of anti-
formin.

2. Close the tube with a rubber cork and shake
vigorously (a sample of antiformin that does not
"foam" at this stage is of little use). Disintegration
of the material at once starts, associated bacteria are
destroyed and the mixture rapidly becomes a homo-
genous but turbid fluid — a process which may be
hastened by : —

3. Placing the tube in the incubator at 37° C. for 30
minutes — shaking from time to time.

4. Centrifugalise the fluid thoroughly, at high
speed.

5. Pipette off the supernatant fluid, fill up with
sterile distilled water, cork the tube and shake to dis-
tribute the deposit throughout the water. Again
centrifugalise.

6. Repeat steps 4 and 5 twice more.

APPENDIX 503

7. Employ one portion of the final deposit to inoc-
ulate guinea pigs.

8. Plant the remainder of the deposit freely on
Dorset's Egg medium; cap and incubate at 37° C.

NOTE.. — If only microscopical films are needed, fill up the centri-
fuge tube with Ligroin (a petroleum ether) in place of sterile dis-
tilled water in step 5 and prepare the films from the surface of the
fluid to stain by the Ziehl-Neelsen process.

INDEX

ABBA'S condenser, 7
Abbott's stain for spores, 107
Aberration, chromatic, 56

spherical, 55
Absolute alcohol as a fixative, 82

as an antiseptic, 27
Absorbent paper for drying cover-
slips, 69

A. C. E. mixture, 345
Acetic acid for clearing films, 82
Achromatic condenser, 54
Acid haematin, 96

production, analysis table, 283
by bacteria, 145
investigation of, 280
qualitative examination, 283,
~ 284

quantitative examination, 280
Acid-fast bacilli in tissues, to

stain, 124

Action of various gases on bac-
teria, 295

Active immunisation, illustra-
tive example, 322
Adjustable water bath, 299
Aerobic cultures, 221
Aerogenic bacteria, 131
^Esculin agar, 204
Agar gelatine (guarniari), 194
methods of preparation, 167
surface plates, 232
Agar-agar, preparation of, 167
Agglutination reaction, macro-

scopical, 386
microscopical, 385
Agglutinin, 381
Air, analysis of, 468
filter, 40

Eump, Geryk, 43
umin solution, Mayer's, 120

Alcohol production, test for, 285

Alkaline pyro, 239

Alum carmine, 96

Ammonia production test for, 285

Amphitrichous bacteria, 136

Anaerobic cultures, 236
Botkin's method, 243
Buchner's method, 238
Bulloch's method, 245
Hesse's method, 237
McLeod's method, 240
media, 180
Novy's method, 244

Anaerobic cultures, Roux's bio-
logical method, 237
physical method, 237
vacuum method, 238
Wright's method, 239
Anaesthetics, 345
Analysis of air, apparatus for, 469
method of, 468
qualitative bacteriological,

470

quantitative bacteriologi-
cal, 468

of butter, qualitative bacterio-
logical, 458
quantitative bacteriological,

457

of cream, qualitative bacterio-
logical, 458
quantitative bacteriological,

457

of fish, 460

of ice cream, qualitative bac-
teriological, 457
of meat, apparatus for, 460
method of, 460
qualitative bacteriological,

462

of milk, apparatus for, 444
collection of samples, 441
method of, 441
qualitative bacteriological,

446
quantitative bacteriological,

444

of oysters, 463

of sewage, qualitative bacter-
iological, 467
quantitative bacteriological,

466

of shellfish, 463
of soil, apparatus for, 473
collection of samples, 471
method of, 470
qualitative bacteriological,

476
quantitative bacteriological,

473
of water, apparatus for, 420,

427 ^

collection of samples, 416
method of, 416
qualitative bacteriological,

426

505

INDEX

Analysis of water, quantitative

bacteriological, 420
Aniline dyes, 83

Gentian violet, 95
water, to prepare, 108
Animal tissue media (Frugoni),

210

Animals, natural infections of,

337

Antiformin method for B. tuber-
culosis, 502

Antigen, definition of, 324

Antiseptics, 27
action of, 310

Apparent filth in milk, 450

Arnold's steam steriliser, 34

Arthrogenous spores, 138

Ascitic bouillon, 210

fluid agar (Wassermann), 213

Ascomycetas, 128

Ascopores, 129

Asparagin Media (Frankel and

Voges), 183
(Uschinsky), 183

Aspergillus, 127

Atmospheric conditions, 295

Attenuating the virulence of
organisms, 321

Autoclave, 37
to use, 37

Automatic pipettes, 13

Autopsies, 396

Autopsy, card index for, 402

BACILLI, morphology of, 132
Bacillus anthracis in soil, 477

in water, 440

coli in water, detection of, 429
diphtherias in milk, 452
enteritidis in water, 437
sporogenes in milk, 452

in water, 438

cedematis maligni in soil, 477
tetani in soil, 477

in water, 441
tuberculosis in milk, 453

antiformin method, 502
typhosus in water, 441
Bacteria, anatomy of, 134
classification of, 131
grouping of, for study, 410
in tissues, demonstration of , 114
influence of environment on,

142

metabolic products of, 143
methods of identification, 259
microscopical examination of,
stained, 81

unstained, 74
physiology of, 136

Bacteria, simple stains for, 90
Bacterial emulsion, preparation

of, 389

enzymes, 144, 277,
ferments, 144
food stuffs, 142
toxins, 144

Bacteriological analyses, gen-
eral considerations, 415
examination of blood, 377
Base of microscope, 50
Basidium, 128

Beer wort, preparation of, 175
Beetrpot media, 200
Beggiotoa, morphology of, 133
Benzole bath, 256
Berkefeld filter, 42
Beyrinck's solution I, 197

II, 198

Bile salt agar (MacConkey), 205
broth, double strength, 199

(MacConkey), 180
Biochemical examination of cul-
tures, 276

Biochemistry of bacteria, 276
Biological differentiation of bac-
teria, 249

Bipolar germination, 140
Bismarck brown, 94
Blastomycetes, morphology of,

129

Blood agar, 171, 214
plates, animal, 251

human, 250
(Washbourn), 214
bacteriological examination of,

377

cells, washing of, 388
collection of, for serological

examination, 379
films, preparations of, 376

staining of, 97
histological examination of,

373

pipettes, ii

serological examination of, 378

stains, 97

Blood-serum (Councilman and
Mallory), 208

inspissated, 168

(Loeffler), 208

(Lorrain Smith), 208
Blowpipe table, 9
Body tube of microscope, 50
Bohemian flask, 4
Boiling water, 33
Bone marrow, films, preparation

of, 400

Bordet-Gengou reaction, 393
Boric acid in milk, test for, 442

INDEX

507

Botkin's anaerobic method, 243

Bouillon, preparation of, 163

Brain extract, 149

Bread paste, 193

Brilliant green agar (Conradi),

206
bile salt agar (Fawcus), 206

Brownian movement, 79

Buchner's anaerobic method, 238

Bulloch's anaerobic method, 245
tubes for permanent prepara-
tions, 407

Bunge's mordant, 104

Burri's Chinese ink stain, 77

Butter, analysis of, 457
qualitative analysis of, 458
quantitative analysis of, 457

CADAVER, preparation of, for

autopsy, 397
Cages for guinea-pigs, 343

for laboratory animals, 341

for mice, 342

for rabbits, 343

for rats, 342
Calculated figure for weight of

medium mass, 166, 167
Cambier's candle method of iso-
lating colityphoid groups, 438
Camera lucida, 62
Capaldi-Proskauer medium, No

I, 186
No II, 187
Capillary pipettes, 10

graduated, 13
Capitate bacilli, 139
Capsule formation, 134

of bacteria, 134

thermo-regulator, 218
Capsules, collodion, inoculation

of, 357

preparation of, 357
glass, 6
to clean infected, 20

new, 1 8
to stain, 99
to sterilise, 31

Carbohydrate media, prepara-
tion of, 177
Carbolic acid as a germicide, 27,

481

method of isolating coli- ty-
phoid group, 437
Carbolised agar, 202
bouillon, 202
gelatine, 202
Carbon dioxide in cultures, test

for, 289

Card index, 336, 402
Carrot media, 200

Cedarwood oil for immersion

lens, 88

Cell wall of bacteria, 134
Celloidin sacs, manufacture of,

358

Cellular incubator, 216
Centrifugal machine for blood

and serum work, 327
for milk work, 447
Centrifugalised milk, 449
Centrigade degrees, conversion

of, 494
Chemical products of bacteria,

145
China green agar (Werbitski),

207

Chloroform as an antiseptic, 27
Chromatic aberration, 56
Chromogenic bacteria, 131
Chromoparous bacteria, 144
Chromophorus bacteria, 144
Citrated blood agar, 191
Cladothrix, morphology, 193
Classification of bacteria, 131

of fungi, 126
Clavate bacilli, 139
Clearing films with acetic acid, 82
Clostridium, 139
Coarse adjustment, 51
Cobweb micrometer, 66
Cocaine, 345

Cocci, morphology of, 131
Coccidium infection, 339
Coefficient, inferior lethal, 312

of inhibition, 311

phenol, 489

superior lethal, 313
Cohn's solution, 191
Cold incubator, 217
Coli-typhoid group, differential

table, 433
in milk, 451
in soil, 477
isolation of, 432
members of, 430

Collection of blood for bacterio-
logical examination, 378
for media making, 168

of milk samples, 443

of pathological material during
life, 373

of pus, 373

of soil sample, 471

of water samples, 416
Collodion capsules, 357

sacs, manufacture of, 357
Colonies of bacteria, edges, 267
Coloured light, action of, 309
Columella, 127
Comparative haemocytology, 374

5o8

INDEX

Complement, definition of, 325

fixation test, 393
Concentration method in water,

analysis, .434
Condenser achromatic, 54

dark ground, 60

paraboloid, .60

substage, 54
Condidium, 128
Continuous .sterilisation, 36
Contrast stains, 93
Corrosive sublimate (Lang), 82
Cotton-wool filter, 40
Counterstaining films, 84
Counting plate colonies, 423
Cover-slip films, 81

to clean new, 22

used, 24-

Crates for test-tubes, 3 1
Creanv analysis of, 457

qualitative analysis of, 458

quantitative analysis of, 457
Crenothrix morphology, 133
Criteria of infection, 370
Criterion of immunity, 324
• Cultural characters, macroscop-

ical examination, 261
Culture flask, Guy's, 5
Kolle, 4 '
Rpux, 5

Cuneate bacillif 139
Cutaneous inoculation, 352

DARK ground condenser, 60

illumination, 87
Daughter cells, 129
Daylight, diffuse, action of, 308
Decimal scales, 340
Decolourising agents, 84
Definition of objective, 56
Depilatory powder, 346
Description of plate culture, 261
Descriptive terms, 261
Desiccation, effects of, 306

table, 501

Desiccator, Mueller's, 307
Dextrose solution, preparation of,

.J78
Diaphragm, iris, 53

Diastatic enzymes, tests for, 278
Differential atmosphere cultiva-
tion, 257

incubation, 255

media, 255

staining, 108

sterilisation, 256
Diluting chamber, 248
Dilution by teat pipette, 383

of serum, 382

tables, 498

Dilutions, preparations of, 496
Diphtheria, bacillus of, in milk,

452

Diplobacilli, morphology of, 133

Diplococci, morphology of, 133

Diplococcus pneumonias, immuni-
sation against, 322

Discontinuous sterilisation; 36

Discs of plaster-of- Paris, 192

Disinfectants, action of, 310
chemical, 27
testing of, 480

Dissociating fluid, Price Jones,
400

Dosage of inoculum, 316

Double nosepiece, 58
stains for spores, 106
sugar agar (Russell), 207

Drop-bottle, 73

Dry heat, 28

Dunham's solution, 177

Dyes, aniline, 83 .

EARTHENWARE box for dirty
slides, 70

Earthy salts agar (Lipman and
Brown), 197

Edge of individual colonies, char-
acters of, 267

Egg albumin agar, 213

broth, (Lipschuetz), 213
media (Dorset), preparation of,

. J74

inspissated, 212
(Lubenan), 209
(Tarchanoff and Kolesmi-

koff), 212
to clear nutrient media with,

166

Ehrlich's eyepiece, 55
Eikonometer, 65
Eisenberg's milk-rice medium,

189

Electric dental engine, 360
signal clock, 38
warm stage, 59
Elevation of colonies, 263
Eisner's gelatine, 204

method of isolating coli: ty-
phoid group, 438
Endogenous spores, 138

varieties of, 139
Endo-germination, 139
English proof agar, Blaxall, 193
Enumerating colonies on plates,

discs, Jeffer's, 424

Pakes', 424

Enrichment method in water
analysis, 427

INDEX

509

Enumeration of micro-organisms,

423

Environmental conditions, 142
Enzyme production, investiga-
tion of, 277
Eosin, 93

Equatorial germination, 140
Erlenmeyer flask, 4
Ernstschen Koerner, 136
Esmarch's roll culture, 226

water collecting bottle, 417
Estimation of reaction of media,

280
Ether flame, 28

soluble acids, 284
Eucaine, 345
Exalting virulence of organisms,

320

Examination of milk, 441
Experimental infections, study
of, during life, 370

inoculation of animals, 332
Extracellular toxins, 144
Eyepiece, Ehrlich, 55

micrometer, 63
Eyepieces, 55
Eye-shade, 57

FAHRENHEIT degrees, conversion

of, 495

Feeding experiments, 369 •
Fermentation reactions, 279

tubes, 17

Field of objective, 56
Filar micrometer, 66
Filling tubes, etc., with medium,

1 60
Film preparations, 81

fixing, 8 1

making, 81

mounting, 85

staining, 83
Filter candle, closed, 47

open, 43

testing efficiency ^of , 478

to disinfect, 28

to sterilise, 29
flask, 6

gapers, to fold, 156
;er.s, cotton-wool, 40

porcelain, 42

testing of, 478
Filtration, 40

by aspiration, 42

of media, 156

under pressure, 45
Fine adjustment, 51

spindle head, 52
Fish, analysis of, 460

bouillon, 190

Fish gelatine, 190

gelatine-agar, 190
Fishing colonies, 253
Fission, reproduction by, 136
Fixation, 81

by heat, 81

of tissues, 1 14
Fixing fluids, for films, 82
Flagella, classification of bacilli
•by, 136

to* stain, 101
Flask 'Bohemian, 4

Erlenmeyer, 4

filter, 6

Kitasato'a serum, 6

Kolle's culture, 4
Flasks and'test tubes, to plug, 24

to clean dirty, 20 •
new, 1 8

to sterilise, 31
Fleischwasser, 148
Fluid cultures, description of,
271

media, 146

Foot of microscope, 50
Formaldehyde in milk, Hehner's

test for, 442
Formalin method o£ preserving

cultures, 407
tissues, 404

Fractional sterilisation, 33 "
Fraenkel and Voge's solution, 183
Fraenkel's earth borer, 472
Freezing method for sections, 115
French Mannite Agar (Sabour-
aud), 193

proof agar (Sabouraud), 193
Fresh preparations of bacteria, 7 4
Friedlander's capsule stain for

sections, 123

Frost's mounting fluid, 406
Frozen sections, rapid method,

116
Fuchsin, 92

agar (Braun), 205

sulphite agar (Endo), 206
•

GAS analysis, qualitative, 290
quantitative, 290

collecting apparatus, 291

generators, 242

production by bacteria, 289

tubes for media, 161
Gasperini's solution, 193
Gelatin agar, 193

preparation of, 164

surface plates, 231
General anaesthetics, 345
Gentian violet, 9 1
German lined paper, 69

510

INDEX

Germicides, 27

testing power of, 480
Germination, 140
Geryk air-pump, 43
Glass apparatus in common use,

3

to clean, 18
Glass-cutting knife, 8
Glucose formate agar (Kitasato),

180

bouillon (Kitasato), 180
gelatine (Kitasato), 180
Glycerinated potato, 209
Glycerine agar, 209
blood-serum, 208
bouillon, 209
potato bouillon, 203

broth, 203

Goadby's gelatine, 214
Gonidium, 128
Goniodophore, 128
Graduated capillary pipettes, 13

pipettes, 6
Gram-Claudius* differential stain,

109

Gram's differential stain, 108
Gram-Wei-gert for sections, 121,

122

Gram-Weigert's differentiail stain,

109
modified, no

Grease pencils, 72

Grouping of bacteria for study,
410

Guarded trepine, 360

Guarniari's agar gelatine, 194

Guinea-pig cages, 343
holder, 350

Gulland's solution, 82

Gum solution, preparation of , 116

Guy's culture bottle, 5

Gypsum blocks (Engel and Han-
sen), 192

ILEMATIN, 95

Haematocytometer, 248
Haematoxilin, 95
Hasmolysin, definition of, 326

preparation of, 327

storage of, 331
Haemolytic serum, titration of,

328
Hanging-block culture (Hill) ,

235
Hanging-drop cultures, 233

examination of, 86, 79

preparation of, 78

permanent staining of, 80

slides, 70
Hardening tissues, 114

Haricot agar, 200

bouillon, 200
Hay infusion, 200
Hearson's water bath, 299
Heat effect of, 299
Hehner's test, 442
Heiman's serum agar, 210
Hesse's anaerobic culture method;

237
Histological examination of blood,

373

Holder for guinea-pigs, 350
Hot air, 29

steriliser, 30
to use, 31
incubator, 217
Hot-water funnel, 158
Human blood agar plates, 250
Huyghenian eyepiece, 55
Hydrogen, generating apparatus,

242

in culture, test for, 289
peroxide in milk, test for, 442
Hyphomycetes, morphology of,

126
reproduction of, 126

ICE-BOX, for water samples, 419
Ice cream, analysis of, 457
Illuminant for microscope, 67
Immune body, 393
Immunisation, methods of, 321
Imperial system, 492

factors for converting, 493
Impression films, 85
Incubators, 216
Index cards, 336, 403 -
Indol, test for, 286
Infection, definition of, 370

general observations during
life, 371

results of, 404
Influence of environment on

bacterial growth, 142
Inhalation, fluid inoculum, 365

powdered inoculum, 366
Inhibition coefficient, 310, 311
Inoculation card index, 336

cutaneous, 352

intracranial, 360

intramuscular, 355

intraocular, 362

intraperitoneal, 355

intrapulmonary, 363

intravenous, 363

of collodion capsules, 357

subcutaneous, 353

syringe, 344
Inoculum, character of, 346

preparation of, 346

INDEX

Inosite-free media — bouillon (Dur-
ham), 183

Inseparate toxins, 144
Intermittent sterilisation, 36
Intracellular toxins, 144
Intracerebral inoculation, 362
Intracranial inoculation, 360
'Intragastric inoculation, large

animals, 367
Marks method, 367
Intramuscular inoculation, 355
Intraocular inoculation, 362
Intraperitoneal inoculation, 355
Intrapulmonary inoculation, 363
Intravenous inoculation, 363
In vacuo anaerobia cultures, 289
Invertin enzymes, tests for, 279
Involution forms, 137
Iodine solution, 108
Iron bouillon, 185

peptone solution (Pakes), 185
Isolation by animal experiments,

258

by differential atmosphere ,257
incubation, 255
media, 255
sterilisation, 256
by dilution, 248
by plate cultures, 250
subcultures, preparatioin of,
254

JEFFER'S counting disc, 424

Jenner's stain, 97

Jores' mounting fluid, 405

KAISERLING fixing solution, 405
Kanthack's serum agar, 211
Killed cultivations, 318
Kipp's hydrogen apparatus, 242
Kitasato's serum flask, 6
Klebs-Loeffler bacillus in milk,

452

Koch's steam steriliser, 34
Kohle's culture flask, 4

LAB enzymes, test for, 279
Laboratory animals, 335

comparative haematocytol-

ogy of, 374

normal temperature, 372
regulations, I

Lactose litmus agar (Wurtz), 203
bouillon, 203
gelatine (Wurtz), 203
Lakmus Molke, 203
Lang's solution, 82
Lead bouillon, 185
peptone solution, 186

Leishman's stain, 98
for sections, 1 25
Lemco broth, 163
Leptothrix, morphology, 133
Lethal dose, minimal, 316
Leviditi's staining method, 124
Light, action of, 308
Liquefiable media, 147
Liquid soap, 346
Lithium carmine, 96
Litmus bouillon, 186
gelatine, 202
milk cultures, description of, 272

preparation of, 172
nutrose agar (Drigalski-Con-

radi), 205
whey, 195
agar, 196
gelatine, 196
(Petruschky), 195
Local anaesthetics, 345

reaction to infection, 372
Locomotive movement, 80
Lcefner's capsule stain, 103

serum, 208

Lophotrichous bacilli, 136
Lorrain Smith electric warm

stage, 59
serum, 208

Lugol's solution, to prepare, 108
Lysol, 27

MACCONKEY'S capsule stain, 99

media, 180, 199, 205
MacCrorrie's capsule stain, 103
Macroscopical examination of

cultures, 261
Malachite green agar (Lceffler),

207
Malt extract solution (Herschell),

196

Margin of individual colonies, 267
Martin's filtering apparatus, 320
Material for inoculation, 346
Mayer's albumin, 120
Mean phenol coefficient, 490
Measuring bacteria, 61
Meat, bacteriological analysis of,

460
extract preparation of, 148

reaction of, 149

Mechanical separation of bac-
teria, 249
stage, 52

Media, filtration of, 156
preparation of, 163
aerobic culture, 222
aesculin agar, 204
agar- agar, 167
agar gelatine (Guarniari), 194

INDEX

Media, preparation of anaerobic

culture, 1 80

animal tissue (Frugoni), 210
ascitic bouillon, 210

fluid agar (Wassermann),

213
asparagin (Fraenkel and

Voge's), 183
(Uschinsky), 183
beer wort, 175
beetroot, 200
Beyrinck's solution I, 197

II, 198
bile salt agar (MacConkey),

205

broth (MacConkey), 180
double strength, 199
blood agar (Washbourn), 214
blood-serum, 168

(Councilman and Mai-
lory), 208
(Loeffler), 208
(Lorrain Smith), 208
bouillon, 163
bread paste, 193
brilliant green agar (Con-

radi), 206
bile salt agar (Fawcus),

206
Capaldi-Proskauer, No. 1, 186

No. II, 187
carbohydrate, 177
carbolised agar, 202
bouillon, 202
gelatine, 202
carrot, 200
China green agar (Werbit-

ski), 207

citrated blood agar, 171
Cohn's solution, 191
dextrose solution, 178
double sugar agar (Russell),

207
earthy salt agar (Lipman and

Brown), 197
egg Dorset, 174
Lubenau, 209

egg-albumen, inspissated, 212
(Tarchanoff and Koles-

nikoff), 212
egg-albumin agar, 213

broth (Lipschuetz), 213
English proof agar (Blaxall),

193

fish bouillon, 190
gelatine, 190

agar, 190
fluid, 146

French mannite agar (Sab-
ouraud), 193

Media, preparation of French
proof agar(Sabouraud),i93

Fuchsin agar (Braun), 205
sulphite agar (Endo), 206

gelatine, 193
agar, 193

glucose formate agar (Kit-

asato), 1 80

bouillon (Kitasato), 180
gelatine (Kitasato), 180

glycerinated broth, 209
potato, 209

glycerine agar, 209
blood-serum, 208, 209
bouillon, 209
potato bouillon, 203

gypsum blocks (Engel and
Hansen), 192

haricot agar, 200
bouillon, 200

hay infusion, 200

inosite free-bouillon (Dur-
ham), 183

iron bouillon, 185

peptone solution (Pakes),

185

lactose litmus agar (Wurtz),
203

bouillon, 203
gelatine (Wurtz), 203
lakmus molke, 203
lead bouillon, 185

peptone solution, 186
lemco broth, 163
liquefiable, 147
litmus bouillon, 186
gelatine, 202
milk, 172
nutrose agar (Drigalski-

Conradi), 205
whey, 195
agar, 196
gelatine, 196
(Petruschky), 195
malachite green agar (Lceff-

ler), 207
malt extract solution (Hers-

chell), 196
milk, 172

rice (Eisenberg), 189

(Soyka), 189
Naegeli's solution, 191
Naehrstoff agar (Hesse and

Niedner), 199

neutral litmus solution, 179
nitrate bouillon, 185

peptone solution (Pakes),

186

nutrient, 146
agar-agar, 167

INDEX

513

Media, preparation of nutrient
bouillon, 163

gelatine, 164
nutrose agar (Eyre), 172
oleicacid agar (Fleming), 201
Omeliansky's nutrient fluid,

189

Parietti's bouillon, 202
parsnip, 200
Pasteur's solution, 191
peptone rosolic acid water,

1 86

water (Dunham), 177
plaster- of -Paris discs, 192
potato, 174

gelatine (Eisner), 204

(Goadby), 214
proteid free broth (Uschin-

sky), 183
rosolic acid peptone solutions

186
serum, bouillon, 210

dextrose water, (Hiss), 188

sugar, (Hiss), 188

water, 170
serum-agar (Heiman), 210

(Kanthack and Stevens),

211

(Lib man), 212

( Wertheimer) , 211
silicate jelly (Winogradsky),

198

solid, 147
special, 182
stock nutrient, 163
sugar, 177

agar, 185

(dextrose) bouillon, 184

gelatine, 184
sulphindigotate agar, 181

bouillon (Weyl), 181

gelatine (Weyl), 181
tissue (Noguchi), 214
turnip, 200
urine agar, 188

bouillon, 187

gelatine, 187

(Heller), 188
wheat bouillon (Gasperini),

193

whey agar, 195
gelatine, 195
wine must, 192
Winogradsky 's solution (for
nitric organisms), 198
(for nitrous organisms),

198

wood ash agar, 201
wort agar, 176
gelatine, 176
33

Media, preparation of yeast
water (Pasteur), 191

standardisation of, 154

storage of, in bulk, 159

storing tubes of, 161

sore boxes, 162

titration of, 150

tubing of nutrient, 160
Merismopedia, morphology of,

132
Mesophilic bacteria, 143

pathogenic effects, 315
Metabolic end-products, 145
Metrachromatic granules, 136
Metal instruments, to sterilise, 28
Metatrophic bacteria, 131
Methods of cultivation, 221

of identification of bacteria, 259

of inoculation, 352

of isolation, 248

of sterilisation, 26
Methylene-blue, 90
Metric system, 492

factors for converting, 493
Meyer's carmine, 96
Microbes of indication, 426
Micrococci, morphology, 132
Micrococcus, melitensis in milk,

456
Micrometer, filar, 66

net, 63

ocular, 63

stage, 62

Micrometry, methods of, 61
Micron, 61
Microscope, 49
Microscopical examination of

bacteria, 86
stained, 88
unstained, 86

observations of cultures, 272
Milk, analysis of, qualitative, 446
quantitative, 444

condensed, analysis of, 444

media, 193

preparation of, 172

rice (Eisenberg), 193
(Soyka), 189

samples, collection of, 443

sedimenting tubes, 449
Minimal lethal dose, 316
Mirror for microscope, 55
Moeller's stain for spores, 107
Moist heat, 32
Molecular movement, 79
Monotrichous bacilli, 136
Motility, examination for, 79

true, 80
Moulds, examination of, 126

for paraffin imbedding, 117, 119

INDEX

Mounting film preparations, 85

paraffin sections, 119
Mouse cages, 342

holder, 351

scales, 341
Mucor mucedo, 126
Muco rinse, 126
Mueller's desiccator, 307
Muffle furnace, 28 _
Muirs's capsule stain, 100

flagella stain, 101
Museum preparations of bacteria,

497

of tissues, 404

sealing of, 406
Mycelium, 126
Mycoprotein, 135

NAEGELI'S solution, 191
Naehrstoff agar (Hesse and Nied-

ner), 199
Naked flame, 28
Neisser's stain modified, 1 1 1
Net micrometer, 63
Neutral litmus solution, prepara-
tion of, 179

red, 94
Nitrate bouillon, 185

peptone solution (Pakes), 186
Nitric organisms in soil, 478
Nitroso-indol reaction, 287
Nitrous organisms in soil, 477
Normal averages (t.p.r.), 372

serum, 375
Nosepiece, 57

double, 58

triple, 58
Novy's anaerobic method, 244

jars, 245

Nuclei, to stain, 105
Nucleus of bacteria, 135
Numerical aperature, 56
Nutrient media, 146
Nutrose agar (Eyre), preparation

of, 172

OBJECT marker, 61

Objectives, 55

Oblique tube cultures, 223

Ocular micrometer, 63

Oculars, 55

Oese, platinum, 71

Oidium, 128

Oil of garlic, 27

of mustard, 27

Oleic acid agar (Fleming), 201
Omeliansky's nutrient fluid, 189
Operation tables (Eyre's), 352

(Tatin's), 351
Opsonic index, 393

Opsonic index, determination of,

390

Opspnin, 387
Optical characters of colonies,

267
Optimum reaction of medium,

determination of, 305
temperature, determination of,

298

Organisms of suppuration, 409
Orsat-Lunge gas apparatus, 292
Orth's carmine, 96
Oxford stain for Actinomyces, 112
Oysters, analysis of, 463

Pake's counting disc, 424

filter reservoir, 45
Papier char din, 158
Pappenheim's stain, in
Paraboloid condenser, 60
Parachromophorous bacteria, 144
Paraffin method for sections, 117

sections, mounting of, 119

to stain, 121

Paratrophic bacteria, 131
Parietti's bouillon, 202

method of isolating coli-ty-

phoid group, 437
Parsnip medium, 200
Passages of virus, 320
Pasteur-Chamberland filter, 42
Pasteur's pipettes, 10

solution, 191
Pathogenesis, investigation of,

315

Pathogenic bacteria, 131
study of, 408

Pediococci, morphology of, 132

Penicillium, 128

Peptone rosolic acid water, 1 86
water (Dunham), preparation
of, 177

Percentage formula, 496

Perchloride of mercury, 27

Perisporacae, 127

Peritrichous bacilli, 136

Permanent preparations of bac-
teria, 407

of tissues, 404

Petri's dishes, 6

Phagocytic index, 392

Phenol coefficient, 489
production, test for, 287

Photogenic bacteria, 131, 144

Physiological filter, 156

Picric acid solution, 121
(Spengler's), 112

Picrocarmine, 97

Pigment production, observations
on, 288

INDEX

515

Pipettes, automatic, 13

blood, II

capillary, 10

cases for, 7

graduated, 6
capillary, 13

Pasteur's, 10

sedimentation, 16

standard graduated, 7

teat, 10

throttle, 13

to clean infected, 20
new, 1 8

to sterilise, 31
Piridin method of staining spiro-

chaetes, 124

Pitfield's flagella stain, 103
Plasmolysis, 135
Plaster-of- Paris discs, 192
Plate box, 7

cultures, description of, 261
preparation of, 226

levelling stand, 228
Plates, Petri's, 6

to clean infected, 20
new, 1 8

to sterilise, 31
Platinum needles, 71

method of mounting, 71
Pleomorphism, 133
Polar germination, 140

granules, 136
Polkoerner, 136
Polychrome blood stains, 97
Pooled serum, 379
Porcelain filter, 42
Berkefeld, 42
Chamberland, 42
Doulton, 42

Post-mortem examination of ex-
perimental animals, 396
Potato gelatine (Eisner), 204
(Goadby), 214

medium, preparation of, 174
Potted meat, analysis of, 460
Pouring plates, 227
Preparation of experimental ani-
mals, 335

Preservatives in milk, 442
Pressure temperature table, 500
Primary colours, action of, 309
Proteid free broth (Uschinsky ) ,

183
Proteolytic enzymes, tests for,

277

Prototrophic bacteria, 131
Psychrophilic bacteria, 143
pathogenic effects, 315
Pus, collection of, 373
Pyrogallic acid solution, 293

QUALITATIVE analysis of air, 470

of milk, 446

of sewage, 467

of soil, 476

of unsound meat, 462

of water, 426
Quantitative analysis of air, 468

of milk, 444

of sewage, 466

of soil, 473

of unsound meat, 460

RABBIT cages, 343

scabies, treatment of, 338
scales, 340

Raising virulence of organisms,
320

Ramsden's micrometer, 66

Range of medium reaction, meas-
urement of, 305
of temperature, measurement
of, 298

Rat cages, 342

Raw milk, Saul's test for, 442

Reaction of medium, 305
optium, 305
range of, 305
scale, 153

Reduced pressure and tempera-
ture table, 501

Reducing agents, production, 389
tests for, 289

Reduction of nitrates, 389

Reichert's thermo-regulator, 218

Relation of bacteria to environ-
ment, 142

Removal of material from culture
tubes, 74

Rennin enzymes, tests for, 279

Reproduction of bacteria, 136

Resistance glass, 6
to lethal agents, 306

Resting stage of bacteria, 137

Restrictions upon experimental
inoculations, 334

Ribbert's capsule stain, 101

Roll cultures, 226

Rosplic acid peptone solution, 186

Rosindol reaction, 286

Roux's anaerobic culture method,

237
culture bottle, 5

SABOURAUD'S medium, 193
Saccharomyces, morphology of,

129

Safranine, 94

Salicylic acid in milk, test for, 443
Saprogenic bacteria, 131
Sarcinae, morphology of, 132

INDEX

Saul's test, 442
Scales, decimal, 340

trip, 164

Scalpels, to sterilise, 32, 33
Schallibaum's solution, 121
Scheme for study of bacteria, 259
Schizomycetes, classification of,

131

morphology of, 131
Scissors, to sterilise, 32
Sealing museum jars, 406
Searing iron, 397
Sections, special staining methods

for, 121
Sedimentation pipettes, 16

tubes, 9

Selecting objectives, 57
Sensitising red blood cells, 395
Serial cultivations, 251
Serological examination of blood,

378
Serum agar (Heiman), 210

(Kanthack and Stevens),

211

(Lib man), 212
plates, 250
(Wertheimer), 211
bouillon, 210
collection of, 379
dextrose water (Hiss), 1 88
inspissator, 169
sugar media (Hiss), 188
water, preparation of, 170
Sewage, analysis of, qualitative,

467

quantitative, 466
Shake cultivations, 225
description of, 271
Shape of colonies, 262
Shaving experimental animals,

349

Shellfish, analysis of, 463
Silicate jelly (Winogradsky), 198
Single stain for spores, 106
Size of colonies, 262
Slanted tube cultures, 223
Slides, to clean new, 22

used, 23
Smear culture, 224

description of, 268
Soap liquid, 346
Soda solution, storage of stock,

^ J54

Sodium bicarbonate in milk, test

for, 443

Soil, analysis of, qualitative, 476
quantitative, 473
collection of samples, 471
Solid media, 147
Soluble toxins, 144

Soyka's milk rice, 189
Spear-headed spatula, 402
Special media, 182
Specific serum, 379
dilution of, 382
Spherical aberration, 55
Spirillum, morphology of, 133
Spirochasta, morphology of, 133
Spirochsetes in tissues, to stain,

124

Spleen extract, 149
Sporangium, 127
Spore formation, arthrogenous,

138

endogenous, 138
method of, 138, 273

germination, method of, 140,

274

observation of, 140, 273
Spores, characters of, 139

classification of, 139

double stain for, 106

to stain, 106
Stab culture, 224

description of, 265
Stage micrometer, 62

of microscope, 52
Staining methods, 90

paraffin sections, 121

reactions of bacteria, 274
Stains intra-vitam, 77

negative (Burn), 77

rack for, 72
Standard graduated pipettes, 7

soda solution, 154
Standardisation of media, 154
Standardising bouillon, 155
Staphylococci, morphology, 132
Staphylococcus in milk, 456
Steam steriliser, Arnold, 35
Koch, 35
to use, 35

streaming, 35
Sterigma, 127
Sterilisation by chemicals, 27

by dry heat, 28

by filters, 40

by moist heat, 32

by streaming steam, 35

by superheated steam, 36

of albuminous liquids, 32

of gases, 40
Sterilising agents, 26
Stichcultur, 224
Stock dilutions, 497

nutrient media, 163

plate for isolation work, 253
Storage of media in bulk, 159

of tubed media, 161
Store boxes for media, 161

INDEX

517

Streak culture, 224

description of, 268
Streaming movement, 80

steam, 35 ^

Streptobacilli, morphology, 133
Streptococci in soil, 477

in water, detection of, 432

morphology of, 132
Streptococcus pyogenes longus in

milk, 455

Streptothrix, morphology of, 133
Strichcultur, 223
Structure, internal, of colonies,

265

Study of pathogenic bacteria, 408
Subcutaneous inoculation, 353
Subdural inoculation, 361
Substage condenser, 54
Sugar agar,. 185

dextrose bouillon, 184

gelatine, 184

media, preparation of, 177
Sulphindigotate agar, 181

bouillon (Weyl), 181

gelatine (Weyl), 181
Sulphuretted hydrogen in cul-
tures, test for, 290
Sun-light, action of, 309
Superheated steam, 36
Superior lethal coefficient, 310,

313

Suppuration, organisms of, 409
Surface characters of colonies, 264

plates, 230

Surgical motor, electric, 360
Swarm spores, 127
Syringe for subcutaneous inocu-
lation of solid material, 354

hypodermic, 344

TATIN'S operating table, 351
Taxonomy, 262
Teat-pipettes, 10
Temperature, action of, 299

optimum, 298

pressure table, 500

range, 298

taking, 340

Test objects for objectives, 57
Testing niters, 478
Test-tubes, 3

to clean infected, 19
new, 1 8

to plug, 24

to sterilise, 31

Tetracocci, morphology of, 132
Thermal death-point, 143
determination of, 298
of spores, 301, 304
of vegetative forms, 298, 303

Thermophilic bacteria, 143
Thermo-regulators, Hearson's

capsule, 218
Rei chert's, 218
Thionine blue, 92
Thiothrix, morphology of, 133
Thresh' s water collecting bottle,

418

Throttle pipettes, 13
Tinned meat, analysis of, 460
Tissue medium (Noguchi), 214

stains, 95
Tissues for sectioning, fixing. 114

freezing, 116

hardening, 114

imbedding, 118

preparation of, 114

washing, 115
Titration of media, 150
Tortulae, differentiation from sac-

charomyces, 130
Total acidity, 280
Toxins, testing of, 318
Trephines, 360
Triple nosepiece, 58
True motility, 80
Tube cultures, preparation of, 222

length, 50
Tubercle bacillus in milk, 453

to stain, no, 124
Tuberculous guinea-pig, cadaver

of, 454

Tubing nutrient media ,160
Turnip media, 200

UNNA-PAPPENHEIM'S stain for

sections, 123

Unsound meat, analysis of, 460
Urine agar, 188

gelatine, 187
(Heller), 188

media bouillon, 187
Uschinsky's solution, 183

VALENCY of specific sera, 386
Van Ermengem's flagella stain,

104

Vegetative stage of bacteria, 136
Vesuvin, 94

Vibrio cholerae in milk, 452
in water, 439

morphology of, 133
Virulence, attenuating, 321

of organisms, 320

raising, 320

Vivisection license, 334
Voges holder, 350
Volatile oils as disinfectants, 27

WARM stage, 58

INDEX

Washing red blood cells, 388

tissues, 115

Water, analysisbf,qualitative, 426
quantitative, 416

steriliser, 33
Weighing animals, 340
Welch's capsule stain, 101
Wertheimer's serum agar, 211
Wheat bouillon (Gasperini), 193
Whey agar, 195

gelatine, 195
Wine must, 192
Winogradsky's solution I, 198
II, 198

Wire crates for test-tubes, 3 1
Wood ash agar, 201
Working up plates, 252
Wort agar, 176
gelatine, 176
Wright's anaerobic method, 239

YEAST water (Pasteur), 191

•ZIEHL-NEELSEN'S stain, no
Zooglcea, 134
Zymogenic bacteria, 131

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Diseases of the Intestines and Peritoneum Second Edition

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Tuberculosis and Acute General Miliary Tuberculosis

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Malarial Diseases, Influenza, and Dengue

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Diseases of Kidneys and Spleen, and Hemorrhagic Diatheses

By DR. H. SENATOR, of Berlin, and DR. M. LITTEN, of Berlin. The entire
volume edited, with additions, by JAMES B. HERRICK, M. D., Professor of the
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Diseases of the Heart

By PROF. DR. TH. VON JURGENSEN, of Tubingen ; PROF. DR. L. KREHL,

of Greifswaid ; and PROF. DR. L. VON SCHROTTER, of Vienna. Edited by

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Goepp's State Board Questions

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State Board Questions and Answers. By R. MAX GOEPP, M.D.,
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Stevens' Therapeutics New (sth) Edition

A TEXT-BOOK OF MODERN MATERIA MEDICA AND THERAPEUTICS.
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THERAPEUTICS AND MA TERIA MEDICA

Hinsdale's Hydrotherapy

Hydrotherapy : A Treatise on Hydrotherapy in General ; Its
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and a Brief Chapter on the Use of Waters Internally. By GUY HINS-
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Octavo of 466 pages, illustrated. Cloth, $3.50 net.

INCLUDING CROUNOTHERAPY

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important place in medical practice that a good, practical work on the subject
is an essential in every practitioner's armamentarium. This new work supplies
all needs. It describes fully the various kinds of baths, douches, sprays ; the
application of heat and cold ; the internal use of mineral waters and all other
procedures included under hydrotherapeutic measures.

The Medical Record

" We cannot conceive of a work more useful to the general practitioner than this, nor one
to which he would resort more frequently for reference and guidance in his daily work."

Kelly's Cyclopedia of Ameri-
can Medical Biography

Cyclopedia of American Medical Biography. By HOWARD A.
KELLY, M. D., Johns Hopkins University. Two octavos, averaging 525
pages each, with portraits. Per set : Cloth, $10.00 net ; Half Morocco,

$13.00 net.

IN TWO VOLUMES

Dr. Kelly, in these two handsome volumes, presents concise, yet complete,
biographies of those men and women who have contributed noteworthily to the
advancement of medicine in America. Dr. Kelly's reputation for painstaking
care assures accuracy of statement. There are about one thousand biographies
included.

Swan* s Prescription- writing and Formulary

PRESCRIPTION-WRITING AND FORMULARY. By JOHN M. SWAN, M. D., formerly
Director Glen Springs Sanitarium, Watkins, N. Y. i6mo of 185 pages. Flexible
leather, $1.25 net.

Stewart's Pocket Therapeutics and Dose-book |jsj£

POCKET THERAPEUTICS AND DOSE-BOOK. By MORSE STEWART, JR., M.D. 32mo
of 263 pages. Cloth, $1.00 net.

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The American Illustrated Medical Dictionary — By W. A. NEW-
MAN BORLAND, M. D., Editor of "The American Pocket Medical Dic-
tionary." Large octavo of 1107 pages, bound m full flexible leather.
Price, $4.50 net; with thumb index, $5.00 net.

KEY TO CAPITALIZATION AND PRONUNCIATION— ALL THE NEW WORDS

Howard A. Kelly, ^/[^O»tProfessor of Gynecologic Surgery, Johns Hopkins University.

" Dr. Dorland's dictionary is admirable. It is so well gotten up and of such convenient
size. No errors have been found in my use of it."

Thornton's Dose-Book. New (4th) Edition

DOSE-BOOK AND MANUAL OF PRESCRIPTION-WRITING. By E. Q. THORNTON, M.D.,

Assistant Professor of Materia Medica, Jefferson Medical College, Philadelphia. Post-
octavo, 410 pages, illustrated. Flexible leather, $2.00 net.

" I will be able to make considerable use of that part of its contents relating to the correct
terminology as used in prescription-writing, and it will afford me much pleasure to recom-
mend the book to my classes, who often fail to find this information in their other text-
books."— C. H. MILLER, M. D.', Professor of 'Pharmacology, Northwestern University Medi-
cal School.

Lusk on Nutrition New (2d) Edition

ELEMENTS OF THE SCIENCE OF NUTRITION. By GRAHAM LUSK, PH. D., Professor
of Physiology in Cornell University Medical School. Octavo of 402 pages. Cloth,
$3.00 net.

" I shall recommend it highly. It is a comfort to have such a discussion of the subject."
— LEWELLYS F. BARKER, M. D., Johns Hopkins University.

Camac's "Epoch-making Contributions"

EPOCH-MAKING CONTRIBUTIONS IN MEDICINE AND SURGERY. Collected and
arranged by C. N. B. CAM AC, M. D., of New York City. Octavo of 450 pages, illus-
trated. Artistically bound, $4.00 net.

" Dr. Camac has provided us with a most interesting aggregation of classical essays^
We hope that members of the profession will show their appreciation of his endeavors."—
THERAPEUTIC GAZETTE.

PRACTICE, MATERIA MEDICA, Etc. 15

The American Pocket Medical Dictionary New (8th) Edition

THE AMERICAN POCKET MEDICAL DICTIONARY. . Edited by W. A. NEWMAN DOR-
LAND, M. D., Editor " American Illustrated Medical Dictionary." 677 pages. Flexible
leather, with gold edges, $1.00 net; with thumb index, #1.25 net.

Pusey and Caldwell on X-Rays Second Edition

THE PRACTICAL APPLICATION OF THE RONTGEN RAYS IN THERAPEUTICS AND
DIAGNOSIS. By WILLIAM ALLEN PUSEY, A. M., M. D., Professor of Dermatology in
the University of Illinois; and EUGENE W. CALDWELL, B. S., Director of the Edward
N. Gibbs X-Ray Memorial Laboratory of the University and Bellevue Hospital Medical
College, New York. Octavo of 625 pages, with 200 illustrations. Cloth, $5.00 net;
Half Morocco, $6.50 net.

Cohen and Eshner's Diagnosis. Second Revised Edition

ESSENTIALS OF DIAGNOSIS. By S. SOLIS-COHEN, M. D., Senior Assistant Professor
in Clinical Medicine, Jefferson Medical College, Phila. ; and A. A. ESHNER, M. D.,
Professor of Clinical Medicine, Philadelphia Polyclinic. Post-octavo, 382 pages ; 55
illustrations. Cloth, $l.oo net. In Saunders1 Question- Compend Series.

Morris' Materia Medica and Therapeutics. New (7th) Edition

ESSENTIALS OF MATERIA MEDICA, THERAPEUTICS, AND PRESCRIPTION-WRITING.
By HENRY MORRIS, M. D., late Demonstrator of Therapeutics, Jefferson Medical
College, Phila. Revised by W. A. BASTEDO, M. D., Instructor in Materia Medica and
Pharmacology at Columbia University. 1 2mo, 300 pages. Cloth, #1.00 net. In Saunders'
Question- Compend Series.

Williams' Practice of Medicine

ESSENTIALS OF THE PRACTICE OF MEDICINE. By W. R. WILLIAMS, M.D.,
formerly Instructor in Medicine and Lecturer on Hygiene, Cornell University ; and
Tutor in Therapeutics, Columbia University, N. Y. I2mo of 456 pages, illustrated.
In Saunders1 Question- Compend Series. Double number, $1.75 net.

Todd's Clinical Diagnosis

The New (2d) Edition

A MANUAL OF CLINICAL DIAGNOSIS. By JAMES CAMPBELL TODD, M. D., Professor
of Pathology, University of Colorado. I2mo of 469 pages, with 164 text-illustrations
and 10 colored plates. Cloth, $2.25 net.

Bridge on Tuberculosis

TUBERCULOSIS. By NORMAN BRIDGE, A. M., M. D., Emeritus Professor of Medicine
in Rush Medical College. I2mo of 302 pages, illustrated. Cloth, $1.50 net.

Oertel on Bright's Disease illustrated

THE ANATOMIC HISTOLOGICAL PROCESSES OF BRIGHT'S DISEASE. By HORST
OERTEL, M. D., Director of the Russell Sage Institute of Pathology, New York. Octavo
of 227 pages, with 44 text-cuts and 6 colored plates. Cloth, $5.00 net.

Arnold's Medical Diet Charts

MEDICAL DIET CHARTS. Prepared by H. D. ARNOLD, M. D., Dean of Harvard
Graduate Medical School. Boston. Single charts, 5 cents ; 50 charts, #2.00 et ; 500
charts, $18.00 net; 1000 charts, $30.00 net.

Eggleston's Prescription Writing J«*

F^KNTTATS OF PRESCRIPTION WRITING. By GARY EGGLESTON, M. D., Instructor
in PhTmlcology Cornelf UnUsity Medical School. i6mo of 1 25 pages. Cloth, *.oo

1 6 SAUN'DERS' BOOKS ON PRACTICE, Etc.

Jakob and Eshner's Internal Medicine and Diagnosis

ATLAS AND EPITOME OF INTERNAL MEDICINE AND CLINICAL DIAGNOSIS. By DR.
CHR. JAKOB, of Erlangen. Edited, with additions, by A. A. ESHNER, M. D., Pro-
fessor of Clinical Medicine, Philadelphia Polyclinic. With 182 colored figures on
68 plates, 64 text- illustrations, 259 pages of text. Cloth, $3.00 net. In Saunders*
Hand-Atlas Series.

Prartirp of Medicine Second Edition,

fractice 01 m< Revised and Enlarged

A MANUAL OF THE PRACTICE OF MEDICINE. By GEO. ROE LOCKWOOD, M. D.,
Attending Physician to the Bellevue Hospital, New York City. Octavo, 847 pages,
with 79 illustrations in the text and 22 full-page plates. Cloth, $4.00 net.

Barton and Wells' Medical Thesaurus

A THESAURUS OF MEDICAL WORDS AND PHRASES. By W. M. BARTON, M. D., and
W. A. WELLS, M. D., of Georgetown University, Washington, D. C. I2mo of 535
pages. Flexible leather, $2.50 net; thumb indexed, $3.00 net.

Jelliffe's Pharmacognosy

AN INTRODUCTION TO PHARMACOGNOSY. By SMITH ELY JELLIFFE, PH. D., M. D..,
of Columbia University. Octavo, illustrated. Cloth, $2.50 net.

Stevens' Practice of Medicine New (9th) Edition

A MANUAL OF THE PRACTICE OF MEDICINE. By A. A. STEVENS, A. M., M. D.,

Professor of Pathology, Woman's Medical College, Phila. Specially intended for

students preparing for graduation and hospital examinations. Post-octavo, 573 pages,
illustrated. Flexible leather, $2.50 net.

Saunders' Pocket Formulary New (oth) Edition

SAUNDERS' POCKET MEDICAL FORMULARY. By WILLIAM M. POWELL, M. D.
Containing 1831 formulas from the best-known authorities. With an Appendix con-
taining Posologic Table, Formulas and Doses for Hypodermic Medication, Poisons and
their Antidotes, Diameters of the Female Pelvis and Fetal Head, Obstetrical Table,
Diet-list, Materials and Drugs used in Antiseptic Surgery, Treatment of Asphyxia from
Drowning, Surgical Remembrancer, Tables of Incompatibles, Eruptive Fevers, etc.,
etc. In flexible leather, with side index, wallet, and flap, $1.75 net.

Tousey's Medical Electricity and X-Rays

MEDICAL ELECTRICITY AND THE X-RAYS. By SINCLAIR TOUSEY, M. D., Consulting
Surgeon to St. Bartholomew's Hospital, New York. Octavo of 1116 pages, with 750
practical illustrations, 16 in colors. Cloth, $7.00 net ; Half Morocco, $8.50 net.

Hatcher and Sollmann's Materia Medica

A TEXT-BOOK OF MATERIA MEDICA : including Laboratory Exercises in the Histo-
logic and Chemic Examination of Drugs. By ROBERT A. HATCHER, PH. G., M. D.,
and TORALD SOLLMANN, M. D. i2mo of 411 pages. Flexible leather, $2.00 net.

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