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Title: The Elements of Bacteriological Technique

Author: J. W. H. Eyre

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                Most recently updated: January 4, 2021

Language: English

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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. (EDIN.)

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
1913

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

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 forgotten 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--rendered necessary by
the introduction of new methods during recent years.

I take this opportunity of expressing my deep sense of obligation to my
confrère in the Physiological 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 experience 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.
    _July, 1913._




PREFACE TO THE FIRST EDITION


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

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

I make no apology for the space devoted to illustrations, 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 equipment.

The excellent and appropriate terminology introduced by Chester in his
recent work on "Determinative 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.

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 excellent manuals of bacteriology at present in use, but
aims only at supplementing the usually scanty details of technique, and
at instructing the student how to fit up and adapt apparatus for his
daily work, and how to carry out thoroughly and systematically the
various bacterioscopical analyses that are daily demanded of the
bacteriologist by the hygienist.

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

    JOHN W. H. EYRE.

    GUY'S HOSPITAL, S. E.




CONTENTS


                                                                 PAGE

I. LABORATORY REGULATIONS                                           1


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 Micrometry, 61.


V. MICROSCOPICAL EXAMINATION OF BACTERIA AND OTHER
MICRO-FUNGI                                                        69

    Apparatus and Reagents used in Ordinary
    Microscopical Examination, 69--Methods of
    Examination, 74.


VI. STAINING METHODS                                               90

    Bacteria Stains, 90--Contrast Stains,
    93--Tissue Stains, 95--Blood Stains,
    97--Methods of Demonstrating 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 Hyphomycetes,
    126--Morphology of the Blastomycetes,
    129.


IX. SCHIZOMYCETES                                                 131

    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


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
    Hæmolytic Serum, 327--The Titration of
    Hæmolytic Serum, 328--Storage of
    Hæmolysin, 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--Examination of Milk, 441--Ice Cream,
    457--Examination 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

[Illustration]




BACTERIOLOGICAL TECHNIQUE.




I. LABORATORY REGULATIONS.


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

     _Guy's Hospital._


     ~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.

     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.
     solution, carbolic acid 5 per cent. solution, or corrosive
     sublimate 1 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.

     5. All instruments must be flamed, boiled, or otherwise
     disinfected 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 after 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
     destructor, 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 bacteriologist 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 cleanliness 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. ~18 × 1.5~ cm., to contain media for ordinary tube cultivations.

2. ~18 × 1.3~ cm., to contain media used for pouring plate cultivations,
and also for holding sterile "swabs."

3. ~18 × 2~ cm., to contain wedges of potato, beetroot, or other vegetable
media.

4. ~13 × 1.5~ cm., to contain inspissated blood-serum.

The tubes should be made from the best German potash glass,
"blue-lined," stout and heavy, with the edge of the mouth of the tube
_slightly_ turned over, but not to such an extent as to form a definite
rim. (Cost about $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 calibre, and a batch of 18 by 1.5 cm. tubes
usually contains 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. ~5 × 0.7~ 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.

[Illustration: FIG. 1.--Bohemian flask.]

[Illustration: FIG. 2.--Pear-shaped flask.]

[Illustration: FIG. 3.--Erlenmeyer flask (narrow neck).]

~Bohemian Flasks~ (Fig. 1).--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, 100, 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 on account of the large nutrient surface available
for growth. A surface cultivation in one of these will yield as much
growth as ten or twelve "oblique" tube cultures. The wide mouth,
however, is a disadvantage, and for many purposes thin, flat culture
bottles known as ~Roux's bottles~ (Fig. 5) are to be preferred.

[Illustration: FIG. 4.--Kolle's culture flask.]

[Illustration: FIG. 5.--Roux's culture bottle.]

[Illustration: FIG. 6.--Guy's culture bottle.]

[Illustration: FIG. 7.--Filter flask.]

An even more convenient pattern is that used in the author's laboratory
(Fig. 6), as owing to the greater 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 contamination to a
minimum and the general shape permits the flasks to be stacked one upon
the other.

~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 cultivation. 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
diameter 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.

[Illustration: FIG. 8.--Petri dish (a), and capsules (b, c).]

[Illustration: FIG. 9.--Plate box with stirrup.]

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

1. Pipettes of 1 c.c. capacity graduated in 0.1 c.c.

2. Pipettes of 1 c.c. capacity graduated in 0.01 c.c. (Fig. 10, a).

3. Pipettes of 10 c.c. capacity graduated in 0.1 c.c. (Fig. 10, b).

These should be about 30 cm. in length (1 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.

[Illustration: FIG. 10.--Measuring pipettes, a and b.]

The laboratory should also be provided 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. 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., 1 c.c., 0.5 c.c., 0.25 c.c., 0.2 c.c.,
0.1 c.c., 0.05 c.c., and 0.01 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-worker'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.

[Illustration: FIG. 11.--Glass-cutting knife. a. handle. b. double
edged blade. c. shaft. d. locking nut. e. spanner for nut.]

1. The glass-cutting knife. This article is sold in two forms, a bench
knife (Fig. 11) and a pocket knife. The former is provided with a blade
some 8 cm. in length 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 stroppings on a sandstone hone. The pocket form, about 6-cm.
long 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 concertina 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 preparation 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 associated
result--cracking.

[Illustration: FIG. 12.--Glass blower's table with Enfer's foot
bellows.]

6. ~Sedimentation tubes 5×0.5~ cm., for sedimentation reactions, etc., and
for containing small quantities of fluid to be centrifugalised in the
hæmatocrit. These are made by taking 14-cm. lengths of stout glass
tubing of the requisite diameter and heating the centre in the Bunsen or
blowpipe 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 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 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 blowpipe
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 capillary 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 capillary pipette. After a number have been
prepared, they are sterilised and stored in batches, either in metal
cases similar to those used for the graduated pipettes or in large-sized
test-tubes--sealed ends downward and plugged ends toward the mouth of
the case.

[Illustration: FIG. 13.--Capillary pipettes. a, b, c.]

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 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
relaxation 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. 14).

[Illustration: FIG. 14.--Filling the capillary teat-pipette.]

A modification of this pipette, in which a constriction 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 1 cm. bore, in a
similar manner to the Pasteur pipettes, except that the point of the
blowpipe flame must be used in order to obtain the sharp shoulder at
either end of the central bulb. The terminal tubes must retain a
diameter of at least 1 mm., in order to avoid capillary action during
the collection of the fluid.

[Illustration: FIG. 15.--Blood pipettes and hair-lip pin in a
test-tube.]

[Illustration: FIG. 16.--Blood-pipette in metal thermometer case.]

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, 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
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.

[Illustration: FIG. 17.--Capillary graduated pipettes.]

~Automatic ("Throttle") Pipettes.~--These ingenious pipettes, introduced
by Wright, can easily be calibrated in the laboratory and are
exceedingly useful for graduating small pipettes, for measuring small
quantities of fluids, in preparing 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
passage 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.

4. Adjust a rubber teat to the pipette and by pressure 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 projecting about 0.5 cm.
beyond the tapering end of the barrel.

[Illustration: FIG. 18.--Throttle pipette--small capacity.]

8. Drop a small pellet of sealing wax into the barrel by the side of the
capillary tube and then warm the tube at the gas flame until the wax
becomes softened and makes an air-tight joint 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.:

1. 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 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. 19, A).

[Illustration: 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).

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 "b," 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 one extremity, blowing a
small bulb at the centre, and plugging the open end with cotton-wool;
after sterilisation the open end is provided with a short piece of
rubber tubing and a glass mouthpiece. When it is necessary to observe
sedimentation reactions in very small quantities of fluid, these tubes
will be found much more convenient than the 5 by 0.5 cm. test-tubes
previously mentioned.

[Illustration: FIG. 20.--Sedimentation pipette.]

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

[Illustration: FIG. 21.--Fermentation tubes.]

~Fermentation Tubes~ (Fig. 21).--These are used for the collection and
analysis of the gases liberated from the media during the growth of some
varieties of bacteria 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 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 completely.

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

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

~To Clean New Graduated Pipettes.~--

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

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

3. Rinse the pipettes in running water and drain.

4. Run distilled water through the pipettes and drain.

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

6. Place the pipettes in the hot-air oven (_vide_ page 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 autoclave (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.

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 completely.

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

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

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


~Infected Pipettes.~--

1. Plunge infected pipettes immediately after use into tall glass
cylinders containing a 2 per cent. solution 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.

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 employed for blood or
serum work (whether new or infected), much time is consumed in the
various steps from 5 onward, and the cleansing process can be materially
hastened if the following device is adopted.

Fit up a large-sized Kitasato's filter flask to a Sprengel'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, place the pipette point downward
in the beaker of water and start the pump (Fig. 22).

[Illustration: FIG. 22.--Cleaning blood pipettes.]

When all the water has been aspirated through the pipette into the
filter flask, fill the beaker with rectified 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, 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 1 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.

[Illustration: 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 00). A piece of
hard wood 76×26×26 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 sections; both varieties must be of "No. 1" thickness,
which varies between 0.15 and 0.22 mm., that they may be available for
use with the high-power immersion lenses.

Cover-slips should be cleaned in the following manner:

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

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

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

3. Turn the cover-slips out into a flat glass dish and wash in running
water under the tap until all trace of yellow colour has disappeared.
During the washing 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.


_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 adherent 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 1 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 interleaved
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-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 projecting.

[Illustration: FIG 24..--Plugging test-tubes: a, cylinder of wool
being rolled; b, cylinder of wool being doubled; c, cylinder of wool
being inserted in tube.]

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

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, pending 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, 0.1 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 penetrating 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 experiments conducted by the aid of
apparatus sterilised in this manner.

     NOTE.--Tubes, flasks, filter flasks, pipettes, glass tubing,
     etc., may be rapidly sterilised, in case of emergency, by
     washing, in turn, with distilled water, perchloride of
     mercury solution, alcohol, 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, pipettes, etc., a very short exposure to
this heat being sufficient.

_Ether Flame._--In an emergency 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 process twice. It may then be safely assumed
that the apparatus so treated is sterile.

[Illustration: FIG. 25.--Muffle furnace.]

_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
blocking the interstices of the filter.

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

1. Roll each bougie up in a piece of asbestos cloth, secure the ends of
the cloth with a few turns of copper wire, and place inside the muffle
(a small muffle 76×88×163 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 applicable 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
sterilised 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. 18). 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 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 mercurial thermo-regulator may be used but it is preferable 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., 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.

[Illustration: FIG. 26.--Hot-air oven.]


TO USE THE HOT-AIR OVEN.--

1. Place the crates of test-tubes, metal cases containing 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,
otherwise 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 160° 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 100°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 175°C.

5. Extinguish the gas at once, and allow the apparatus 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.

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

~Moist Heat.~--_Water at 56° C._--This temperature, if maintained for
thirty minutes, is sufficient to destroy the vegetative forms of
bacteria, but has practically no effect on spores. Its use is limited to
the sterilisation of such albuminous "fluid" media as would coagulate 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 albuminous 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, extinguish 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.

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

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

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

_Water at 100°C._ destroys the vegetative forms of bacteria almost
instantaneously, and spores in from five to fifteen minutes. This method
of sterilisation is applicable to the metal instruments, such as knives,
forceps, etc., used in animal experiments; syringes, rubber corks,
rubber and glass tubing, and other small apparatus, and is effected in
what is usually spoken of as the "water steriliser" (Fig. 27).

[Illustration: FIG. 27.--Water sterilizer.]

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

METHOD.--

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

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

[Illustration: FIG. 28.--Koch's steriliser.]

[Illustration: 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 boiling for fifteen minutes.

5. After disinfection, and whilst still hot, take out 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 modifications, 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 tubulure; it is mounted on a tripod stand and heated from
below by a Bunsen burner. The more elaborate steriliser is cased with
felt or asbestos board, and provided 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 compartment until it rests on the diaphragm,
and replace the lid.

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

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

     NOTE.--After removing tubes, flasks, etc., from the steam
     steriliser, they should be at once separated freely in order
     to prevent moisture condensing upon 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 destroyed by repeating the process twenty-four hours
later; a third sterilisation after a similar interval makes assurance
doubly sure.

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

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 attained in ten
minutes. This method was formerly employed 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 temperature
undergo hydrolytic changes which render them unsuitable for the
cultivation of the more delicate micro-organisms. The use of superheated
steam should be restricted almost entirely to the disinfection of such
contaminated articles, old cultivations, etc., 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 cylinder, provided with a
copper or gun-metal lid, which is secured in place by means of bolts and
thumbscrews, the joint between the cylinder and its lid being
hermetically sealed by the 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 circular rows of Bunsen
burners, each circle having an independent gas-supply, occupying the
lower half. In the interior of the boiler is a large 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 bottom of the basket;
also fill the contained flasks and tubes with water.

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

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

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

6. Wait until the index of the manometer records a temperature of 120°
C., then regulate the gas and the spring safety valve in such a manner
that this temperature 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 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.

[Illustration: 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 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 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.

[Illustration: FIG. 31.--Electric signal timing clock.]

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

Perhaps the best example of such a filter is the cotton-wool plug which
closes the mouth of a culture tube. Not only does ordinary diffusion
take place through it, but if a tube plugged in the usual manner with
cotton-wool is removed from the hot incubator, the temperature 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 becomes moist, either by
absorption from the atmosphere, or from liquids coming into contact with
it, micro-organisms (especially the mould fungi) commence to multiply,
and the long thread forms rapidly penetrate the substance of the plug,
and gain access to and contaminate the interior of the tube.

[Illustration: FIG. 32.--Cotton-wool air filter.]


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:

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

[Illustration: FIG. 33.--Ruffer's flask.]

3. Sterilise a short length of rubber tubing by boiling. 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.

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 cylindrical vessel, closed at one end
like a test-tube, and made either of porous "biscuit" porcelain,
hard-burnt and unglazed (Chamberland system), or of Kieselguhr, a fine
diatomaceous earth (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 unequal 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 overcome 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.

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 interposed between the filter
flask and the pump, in order to prevent moist air reaching the oil in
the pump.

5. Air filter (_vide_ page 40) sterilised.

6. Pressure tubing.

7. Screw clamps (Fig. 36).

METHOD.--

1. Couple the exhaust pipe of the suction pump with the lateral tube of
the filter flask (first removing the cotton-wool plug from this latter),
by means of pressure tubing, interposing, if necessary, the wash-bottle
of sulphuric acid.

[Illustration: FIG. 34.--Porcelain filter candle.]

[Illustration: FIG. 35.--Geryk air pump.]

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

[Illustration: 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).

[Illustration: 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 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 addition 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 useful, 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 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.~--

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

[Illustration: FIG. 38.--Pakes' 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.

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.

[Illustration: 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 dioxide, 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.

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 filtration 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:

[Illustration: FIG. 40.--Microscope stand.]

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

[Illustration: FIG. 41.--Foot, three types.]

~1. The Base or Foot~ (Fig. 40, a).--Two elementary forms--the tripod
(Fig. 41, a) and the vertical column set into a plate known as the
"horse-shoe" (Fig. 41, b)--serve as the patterns for countless
modifications in shape and size of this portion of the stand. The chief
desiderata--stability and ease of manipulation--are attained in the
first by means of the "spread" of the three feet, which are usually shod
with cork; in the second, by the dead weight of the foot-plate. The
tripod is mechanically the more correct form, and for practical use is
much to be 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 advantage 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
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.

[Illustration: FIG. 42.--Coarse adjustment.]

[Illustration: FIG. 43.--Fine adjustment.]


     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.

~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 _AA_, Fig. 42).

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

[Illustration: FIG. 44.--Spindle head to fine adjustment.]

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 12 cm.--flat and rigid, in order to afford a safe support
for the Petri dish used for plate cultivations; and should be supplied
with spring clips (removable at will) to secure the 3 by 1 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 mechanical 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
stationary milled heads (Fig. 45, _AA'_). The shape of the aperture is a
not unimportant point; it should be square to allow of free movement
over the substage 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, _BB'_), such as is usually
fitted to this class of stage, or a Maltwood finder, may be employed.

[Illustration: FIG. 45.--Mechanical stage.]

[Illustration: FIG. 46.--Iris diaphragm.]

~6. Diaphragm.~--Separate single diaphragms must be avoided; a revolving
plate pierced with different sized apertures and secured below the stage
is preferable, but undoubtedly the best form is the "iris" diaphragm
(Fig. 46) which enters into the construction 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 thickness of the slides.

The form in most general use is that known as the Abbé (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 f), and must be accurately
adjusted so that its optical axis coincides with that of the objective.
Vertical movement of the entire substage apparatus effected by means of
a rack and pinion is a decided advantage, and some means should be
provided for temporarily removing the condenser from the optical axis of
the microscope.

[Illustration: FIG. 47--Optical part of Abbé illuminator.]

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 Abbé condenser transmits a cone of about
.45 whilst the aperture of the 1/12 inch immersion lenses of different
makers varies from 1.0 to 1.4, hence, the efficiency of these lenses is
much curtailed if the condenser is merely the Abbé. 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 attached 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 expensive "compensation" oculars. Two or three, magnifying 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 square
aperture can be reduced to three-quarters, one-half or one-quarter of
the original size.

~10. Objectives.~--Three objectives are necessary: one for low-power
work--e. g., 1 inch, 2/3 inch, or 1/2 inch; one for high-power
work--e. g., 1/12 inch oil immersion lens; and an intermediate
"medium-power" lens--e. g., 1/6 inch or 1/8 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 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
refrangibilities 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 although 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 centering of the whole of the lenses in the objective.

[Illustration: FIG. 48.--Huyghenian eyepiece.]

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

(e) _Numerical Aperture_ (_N. A._).--The numerical aperture may be
defined, in general terms, as the ratio of the _effective_ diameter of
the back lens of the objective to its equivalent focal length. The
determination of this point is a process requiring considerable
technical skill and mathematical ability, and is completely beyond the
powers of the average microscopist.[1]

Although with the increase in power it is correspondingly difficult to
combine all these corrections in one objective, they are brought to a
high pitch of excellence 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    }     { 1", 2/3", 1/2":
"blood film," stained with Jenner's } for { 1/6", 1/8"
or Romanowsky's stain.              }     { 1/12" oil

2. A thin cover-slip preparation    }
of a young cultivation of           }     { 1/8" dry
_B. diphtheriæ_ (showing            } for {
segmentation) stained with          }     { 1/12" oil
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, preferably 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 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.

[Illustration: FIG. 49.--Eye shade.]

For critical work and particularly for photo-micrography, 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 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.

[Illustration: FIG. 50.--Double nosepiece.]

[Illustration: FIG. 51.--Triple nosepiece.]

_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 circulated. It is made to clamp on to the stage of the
microscope by the screws _A A'_, 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
temperature 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.

[Illustration: FIG. 52.--Warm stage.]

A better form is the electrical hot stage designed by Lorrain Smith;[2]
it requires the addition of a lamp resistance and sliding rheostat, also
a delicate ammeter reading to .01 of an ampère. 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 accommodates
the thermometer. Two coils of manganin wire run in the paraffin at
opposite sides of the bulb (outside 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 about 10 ohms. A
current of 2-1/2 ampères 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 1/12-inch oil immersion lens.

[Illustration: 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 particular make of condenser but generally 1 mm.) and specially
thin cover-glasses (not more than 0.17 mm.) The objective must not have
a higher NA than 1.0, consequently immersion lenses must be fitted with
an internal stop to cut down the aperture.

_Micrometer._--Some form of micrometer for the purpose of measuring
bacteria and other objects is also essential. Details of those in
general use will be found in the following pages.

[Illustration: FIG. 54--Diamond Object marker.]

_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 permanently record the position of an interesting
portion of the specimen. The diamond is mounted on a spring which
regulates the pressure, and the size of the circle can be adjusted 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 (0.001 mm.),
denominated a _micron_ (sometimes, and erroneously, referred to as a
micro-millimetre), and indicated in writing by the Greek letter µ. 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.

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

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

For each of these methods a ~stage micrometer~ is necessary. This is a 3
by 1 inch glass slip having engraved on it a scale divided to hundredths
of a millimetre (0.01 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.

[Illustration: FIG. 55.--Camera lucida, Abbé pattern.]

(a) By means of the Camera Lucida.

1. Attach a camera lucida (of the Wollaston, Beale, or Abbé pattern)
(Fig. 55) to the eyepiece of the microscope.

2. Adjust the micrometer on the stage of the microscope 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 10µ. Mark on the paper the optical combination (ocular
objective and tube length) employed to produce this particular
magnification.

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

To measure an object by this method simply project the image on to the
scale corresponding to the particular optical combination in use at the
moment. Read off the number of divisions it occupies and express them as
_micra_.

In place of preserving a scale for each optical combination, the object
to be measured and the micrometer 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.

[Illustration: FIG. 56.--Eyepiece micrometer, ordinary.]

[Illustration: 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 (0.1 mm.) (Fig. 56), or the
entire surface ruled in 0.1 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 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 diminishing the tube length; and note the number of
included divisions.

4. Calculate the value of each division of the eyepiece micrometer in
terms of µ, by means of the following 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 micrometer value.

Repeat this process for each of the other combinations. 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 _micra_ by
a reference to the standard value for the particular optical combination
employed.

Zeiss prepares a compensating eyepiece micrometer for use with his
apochromatic objectives, the divisions of which are so computed that
(with a tube length of 160 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 microscopical 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 measurement, 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. Observation 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 accurately fill one of the divisions
of the eikonometer scale; this indicates a magnifying power of 500 as
the constant for that particular optical combination, and a record
should be made of the fact.

The magnification constants of the various other 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 eyepiece and the size of
the object read off on the eikonometer scale as millimetres, and the
actual size calculated by dividing the observed size by the
magnification constant for the particular optical combination employed
in the observation.

(c) By means of the filar micrometer.

[Illustration: FIG. 58.--Ramsden's Filar micrometer.]

[Illustration: FIG. 59.--Ramsden's micrometer field, a, fixed wire;
b, reference wire (fixed); c, travelling wire.]

The ~Filar~ or cobweb Micrometer (Ramsden's micrometer) eyepiece (Fig. 58)
consists of an ocular having a fine "fixed" wire stretching horizontally
across the field (Fig. 59), a vertical reference wire--fixed--adjusted
at right angles to the first; and a fine wire, parallel 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 intervals between its teeth
corresponding to one complete revolution of this screw-head.

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 micrometer 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 coincide
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 revolutions (by means of the recording comb) and the fractions
of a revolution (by means of scale on the head of the micrometer screw),
which are required to measure the 0.01 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 µ.

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 revolution of the screw-head required to
traverse such object from edge to edge, and express the result as
_micra_ by reference to the recorded values for that particular optical
combination.

_Microscope Illuminant._--In tropical and subtropical regions diffuse
daylight is the best illuminant. In temperate climes however daylight of
the desirable 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 curved sheet-iron reflector. An obscured metal filament 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.

[Illustration: FIG. 60.--Electric microscope lamp.]

FOOTNOTES:

[1] Its importance will be realised, however, when it is stated in the
words of the late Professor Abbé: "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."

[2] Made by Mr. Otto Baumbach, 10, Lime Grove, Manchester.




V. MICROSCOPICAL EXAMINATION OF BACTERIA 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 drying cover-slips and
slides.

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

[Illustration: FIG. 61.--Disinfectant Jar.]

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

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 lining 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).

[Illustration: 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 1 to 2 mm. in height, to the centre of a 3 by
1 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 1 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.

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 1 mm. in
its short diameter, and is termed a loop or an oese; the terminal 3 or 4
mm. of another wire is flattened 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 used for inoculating culture
tubes and preparing specimens for microscopical examination.

[Illustration: FIG. 63.--Ends of platinum rods. a, loop; b, spatula;
c, needle.]

The method of mounting these wires may be described as follows:

Take a piece of aluminium wire 25 cm. long and about 0.25 cm. in
diameter, and drill a fine hole completely 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 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.

[Illustration: 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 1 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.

    Neutral red, 1 per cent. aqueous solution.

    Leishman's modified Romanowsky stain.

    Carbolic acid, 5 per cent. aqueous solution.

    Acetic acid, 1 per cent. solution.

    Sulphuric acid, 25 per cent. solution.

    Xylol.

[Illustration: FIG. 65.--Staining rack, rubber change mat and lysol
pot.]

[Illustration: FIG. 66.--Drop bottle.]

[Illustration: 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 respectively 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 cultivation for examination by
these methods may be described as follows:

~1. Living, Unstained.~--(a) _"Fresh" Preparation._--

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

[Illustration: FIG. 68.--Holding tubes for removing bacterial growth, as
seen from the front.]

     TECHNIQUE OF OPENING AND CLOSING A CULTURE TUBE.

     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 intended to destroy any micro-organisms that
     may have become entangled in the loose fibres of the
     cotton-wool, and which, if not thus destroyed, might fall
     into the tube when the plug is removed and so accidentally
     contaminate the cultivation.)

     3. Hold the tube at or near its centre between the ends of
     the thumb and first two fingers of the left hand, and allow
     the sealed end to rest upon the back of the hand between the
     thumb and forefinger, the plug pointing to the right. Keep
     the tube as nearly in the horizontal position 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 "screwing" motion of the right
     hand.

     6. Introduce the platinum loop into the tube and hold it in
     this position until satisfied that it is quite cool. (The
     cooling may be hastened by touching the loop on one of the
     drops of moisture which are usually to be found condensed 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 1 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 1 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 applying 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 }
    Methylene green }  in 0.5 per cent. aqueous solutions.
    Vesuvin,        }

_Negative Stain_ (Burri).--By this method of demonstration 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       1 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 1 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.

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 1 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 on 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, b).

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

[Illustration: FIG. 69.--Spreading negative film.]

8. Place a drop of immersion oil on the centre of the film, lower the
1/12-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.

4. Obtain a small quantity of the material it is desired to examine, in
the manner detailed above (pages 74-76, steps 2 to 11 must be followed
in their entirety and with the strictest exactitude whenever tube
contents are being handled), and mix it with the drop of water on the
cover-slip.

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

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

7. Examine microscopically.

The examination of a "fresh" specimen or a "hanging-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 hanging-drop specimen some
form of movement can practically 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 particles of solid matter
(including bacteria), when suspended in a fluid, will always show a
vibratory movement 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.)

(b) 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 demonstrate the nature of this motion.

(c) Locomotive movement, or ~true motility~, is determined by observing
some one particular bacillus changing 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).

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 it 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 11) 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 1 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 Bunsen 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:

1. Absolute alcohol, for five to fifteen minutes.

                         { equal parts, for five to thirty
    2. Absolute alcohol, { minutes (e. g., for blood or
    Ether,               { milk).

3. Osmic acid, 1 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           0.75 gramme
      Hydrarg. perchloride     12.00 grammes
      Acetic acid               5.00 grammes
      In distilled water      100.00 c.c.
    Filter.

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

    Absolute alcohol                                            25.0 c.c.
    Ether                                                       25.0 c.c.
    Corrosive sublimate, 20 per cent. alcoholic solution         0.4 c.c.

7. Formalin 10 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.)

If the film has been prepared from blood or bloodstained 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
hæmoglobin 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; (b) chemical means--e. g., by the
addition 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 solution of aniline oil in
water.

      (d) Thionine blue.
      (e) Bismarck brown.
      (f) Neutral red.
    (B) Acid:
      (a) Eosin, aqueous yellowish.
      (b) Safranine.

These dyes are kept in 1 per cent. aqueous solution to which is added 5
per cent. of alcohol, as a preservative. They are generally used in this
form.

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

_Decolourisation._--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, 1 per cent.
4. Alcohol.
5. Alcohol absolute,         }   equal parts.
   Acetic acid, 1 per cent., }

                             {Hydrochloric, 1 per cent. aqueous solution.
                             {Hydrochloric, 1 per cent. Alcoholic
                             {  (90 per cent.) solution.
6. Mineral acids:            {Sulphuric, 25 per cent. aqueous solution.
                             {Nitric, 33 per cent. aqueous solution.

_Counterstaining._--Use colours which will contrast with the first
stain; e. g.,

Vesuvin,                     }
Neutral red,                 }for films stained by methylene-blue or
Eosin,                       }Gram's method.
Fuchsin,                     }

Methylene-blue,              }for films stained by fuchsin.
Gentian violet,              }

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 1 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
cultivation. 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
vertical, grasp the cover-slip with the forceps and remove it from the
plate. Re-cover the plate.

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

6. Fix by immersing in one of the fixing fluids previously 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 operations with extreme gentleness.

~Microscopical Examination of the Unstained Specimens.~--

1. Place the body tube of the microscope in the vertical position.

2. Arrange the hanging-drop slide on the microscope 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 1/6-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 illumination.

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

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

Some difficulty is often experienced at first in finding the hanging
drop, and if the first attempt is unsuccessful, the student must not on
any account, whilst still applying his eye to the ocular, rack the body
tube down (for by so doing there is every likelihood of the front lens
of the objective being forced through the cover-glass, and not only
spoiling the specimen, but also contaminating the objective); but, on
the 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 2/3 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 Liliput 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 interposed between
light and microscope); and adjust illuminant 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 accurately 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 1 glass slips of less than
1 mm. thickness, and No. 1 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 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 2/3-inch objective by the 1/12-inch 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
preparation 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 substage 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 illuminated bacteria will stand out in vivid contrast to
the dark background.

[Illustration: FIG. 70.--Immersion oil bottle.]

~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 accidentally overturned) and a dust cap of boxwood fitted
     with a wooden rod with which the drop of oil is applied to
     the cover-glass or lens.

3. Use the 1/12-inch oil immersion lens of the microscope. Rack down the
body tube till the front lens of the objective is in contact with the
oil and nearly touching the cover-slip.

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

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

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

7. Focus the condenser accurately on the film.

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




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 convenient 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 vigourously for a few moments every day.
Filter.

2. _Saturated Alcoholic Solution._

Weigh out

    Methylene-blue          1.5 grammes

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

    Alcohol, 90 per cent         100.0 c.c.

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

3. _Carbolic Methylene-blue_ (Kuehne).

Weigh out

    Methylene-blue       1.5 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, 0.1 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.

7. _Carbolic Gentian Violet_ (Nicollé).

Measure out and mix

    Gentian violet, saturated alcoholic solution   10.0 c.c.
    Carbolic acid, 1 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  11 c.c.

Filter.

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

~Thionine Blue (or Lauth's Violet).~--

9. _Carbolic Thionine Blue_ (Nicollé).

Weigh out

    Thionine blue            1.0 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._).

11. _Saturated Alcoholic Solution._

Weigh out

    Basic fuchsin   3.5 grammes

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

12. _Carbolic Fuchsin_ (Ziehl).

Weigh out

    Basic fuchsin     1.0 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 varieties, of which two only are useful for bacteriological
work:

    Eosin, aqueous yellowish.
    Eosin, aqueous bluish.

13. _Eosin Aqueous Solution_ (Yellowish or Bluish Shade), 1 per cent.

Weigh out

    Eosin, aqueous   1.0 gramme

dissolve in

    Distilled water   100.0 c.c.

and add

    Absolute alcohol   5.0 c.c.

Filter.

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. _Aqueous Solution._

Weigh out.

    Safranine                  0.5 gramme

and dissolve in

    Distilled water      100.0 c.c.

Filter.

~Neutral Red.~--

16. _Aqueous Solution._

Weigh out

    Neutral red      1.0 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.


TISSUE STAINS.


~Aniline Gentian Violet~ (For Weigert's Fibrin Stain).--

Weigh out

    Gentian violet      1.0 gramme

and dissolve in

    Absolute alcohol         15.0 c.c.
    Distilled water          80.0 c.c.

then add

    Aniline oil               3.0 c.c.

Shake well and filter before use.


~Hæmatoxylin~ (Ehrlich).--

1. Weigh out

    Hæmatoxylin               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         100.0 c.c.

3. Mix 1 and 2, allow the mixture to stand forty-eight hours, then
filter.

4. Add

    Glycerine                85.0 c.c.
    Acetic acid, glacial     10.0 c.c.

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


~Hæmatin~ (Mayer's).--

A. Weigh out

    Hæmatin                   1.0 gramme

and dissolve in

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

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 simultaneously and slowly into the funnel to mix
thoroughly. Store for future use.

     NOTE.--If acid hæmatin 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      1.0 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 boiling 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

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
Romanowsky to demonstrate malarial parasites, but various modifications
are now employed for staining blood films generally, and also for
bacteria and protozoa. The best modifications of the original Romanowsky
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 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.


~Leishman's Stain.~--

_A._ Weigh out:

    Methylene blue (medicinal)      1 gramme

Dissolve in

    Sodium carbonate, 0.5 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.      0.1 gramme

Dissolve in

    Distilled water      100 c.c.

1. Mix the two solutions A and B in equal volumes, 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 intensify 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 (00 crystals)      1.5 grammes

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.

     12. Clear in xylol and mount in xylol balsam.

     ~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 gentian 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., 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 relative 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                       5 c.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.

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 distilled 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 contact with
the film two minutes.

7. Wash well in water and dry carefully.

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

9. Wash well in water.

10. Dry and mount.

     ~2. "Pitfield" Original Method.~--

     (a) _Mordant._--

    Tannic acid     1 gramme
    Water          10 c.c.

     (b) _Stain._--

    Saturated aqueous solution of alum              10 c.c.
    Saturated alcoholic solution of gentian violet   1 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, 0.1 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.
    Hæmatoxylin solution                            3 c.c.
    Carbolic acid, 1 per cent. aqueous solution     4 c.c.

     This solution must be freshly prepared.

     _Hæmatoxylin solution_ is prepared by boiling 1 gramme
     logwood

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

    _Alternative Mordant_ (Bunge's Mordant).--

    Tannic acid, 20 per cent. aqueous solution       10 c.c.
    Ferrous sulphate, saturated aqueous solution      5 c.c.
    Fuchsin, saturated alcoholic solution             1 c.c.

    (b) _Stain._--

    Weigh out
        Methylene-blue        }
        Or methylene-violet   } 4 grammes
        Or fuchsin            }

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, Loeffler recommends the addition of sodium hydrate, 1 per
cent. aqueous solution, 1 c.c.; or an equal quantity of an exactly
comparable solution of sulphuric 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                             1 c.c.

     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
     preceding method, steps 1 to 4.

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

     3. Wash well in water.

     4. Wash in absolute alcohol.

     5. Wash in distilled water.

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

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

     8. Without washing, again treat the film with the
     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.

3. Wash thoroughly in water.

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

5. Wash in water.

6. Differentiate in ferric ammonia sulphate solution for 1-1/2-2
minutes, examining wet under microscope 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, 1 per cent., 1 part.
(This removes the stain from everything but the spores.)

5. Wash in water.

6. Mount the cover-slip in water and examine microscopically with the
1/6-inch objective. (Spores should be red, and the rest of the film
colourless or a very light pink.) If satisfactory, pass on to section 7;
if unsatisfactory, repeat steps 2 to 5.

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

8. Wash in water.

9. Dry and mount.

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

Sulphuric acid, 1 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.

     5. Wash thoroughly in water.

     6. Counterstain in eosin, 1 per cent. aqueous solution.

     7. Wash.

     8. Dry and mount.

     (Spores blue, bacilli red.)


DIFFERENTIAL METHODS OF STAINING.

~Gram's Method.~--This method depends upon the fact that the protoplasm of
some bacteria permits aniline gentian violet and Lugol's iodine
solution, when applied consecutively, to enter into a chemical
combination which results in the formation of a new blue-black pigment,
only very sparingly soluble in 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 of 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
     vigourously 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 process until the film ceases to darken with the
fresh application of iodine solution.

Lugol's solution is prepared by dissolving

    Iodine                  1 gramme
    Iodide of potassium     3 grammes
    In distilled water    300 c.c.

5. Wash in water.

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

The following mixture may be substituted for absolute 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, 1 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 with xylol.

7. Mount in xylol balsam.


~Gram-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, 1 part, xylol, 2 parts, until no more colour is
discharged.

8. Wash in xylol.

9. Mount in xylol balsam.


~Modified Gram-Weigert Method.~--(To demonstrate trichophyta in 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 15 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 1/6-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 everything 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.)

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 corallin (Rosolic acid) solution.

    Corallin                             1 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 diphtheroid bacilli.)

_Stain I._--

Measure out and mix

    Methylene-blue, saturated alcoholic solution     4.0 c.c.
    Acetic acid, 5 per cent. aqueous solution       96.0 c.c.

Filter.

_Stain II._--

Weigh out

    Neutral red                2.5 grammes

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 (?
metachromatic granules) stained by the acid methylene-blue.

     ~Wheal and Chown (Oxford) Method.~--(To demonstrate
     actinomyces.)

     1. Stain briefly with Ehrlich's hæmatoxylin (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
     repeatedly examined microscopically with the 1/6-inch
     objective.

     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 unaltered, 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; corrosive 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.")

(b) Hardening; that is, by rendering the tissue of sufficient
consistency to admit of thin slices or "sections" 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.


FREEZING METHOD.

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

2. ~Hardening.~ Remove the alcohol (no longer absolute, as it has taken up
water from the tissues) from the bottle and replace it with fresh
absolute alcohol. Allow the tissues to remain therein for twenty-four
hours.

[Illustration: FIG. 71.--Washing tissues.]

     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 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      1 gramme
    Boric acid      1 gramme
    Water         100 c.c.

5. Place the tissue on the plate of a freezing microtome (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 containing 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; 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
     carefully 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.

[Illustration: FIG. 72.--~L~-shaped brass moulds.]

[Illustration: FIG. 73.--Paraffin kettle.]

3. ~Hardening.~ Place the tissues in each of the following 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 chloroform. On the
surface 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).

(b) 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 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
removal of the paraffin block.

[Illustration: FIG. 74.--Paraffin mould.]

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.

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:

[Illustration: FIG. 75.--Section rack.]


~Mayer's Albumin.~--

    Weigh out
          Salicylate of soda    1 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.


_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 subsequently 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 subsequent 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 employed, 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 1 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                          1 c.c.
    Alcohol (90 per cent.)                   160 c.c.

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.

[Illustration: FIG. 76.--Staining pot.]

11. Wash with aniline oil, 2 parts, xylol, 1 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 staining in the usual
manner.

2. Stain in alum carmine for about fifteen minutes.

3. Wash thoroughly in water.

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

5. Wash thoroughly in water.

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

7. Wash thoroughly in water.

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

9. Wash thoroughly with xylol.

10. Decolourise and dehydrate rapidly with absolute 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.   }
    Glycerine    20.0 c.c.   } and add to the stain.

~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. _Friedländer's Method._--

Stain.--

    Gentian violet, saturated alcoholic solution     50 c.c.
    Acetic acid, glacial                             10 c.c.
    Distilled water                                 100 c.c.

     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, 1 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 hæmatin solution ten to twenty seconds, 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 Spirochætes in Tissues.~

~Piridin Method (Levaditi).~--

1. Cut slices of tissue 1 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 1 per cent., and 10 grammes pyridin 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.

    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 spirochætes 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 1 in 1500 aqueous solution.
    Caustic soda 1 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 (1 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 obtained (as seen with the 1/6-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 torulæ).
    ~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 accidentally contaminate his future
preparations.

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

(a) Nutritive or submerged.

(b) Reproductive or aerial.

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

~Reproduction.~--Apical spore formation--asexual;
                               zoospores--sexual.

~Mucorinæ.~--_Mucor_ (Fig. 77).--Note the branching filaments--"mycelium"
(a), "hyphæ" (b).

Note the asexual reproduction.

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).

[Illustration: FIG. 77.--Mucor mucedo.]

[Illustration: FIG. 78.--Aspergillus]

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

Finally the membrane ruptures and spores escape.

~Perisporaceæ.~--_Aspergillus_ (Fig. 78).--Note the branching
filaments--"mycelium" (a).

[Illustration: FIG. 79.--Penicillium.]

Note the asexual reproduction.

1. A filament (b) grows upward, its termination becomes clubbed; on
the clubbed extremity flask-shaped cells appear--"sterigmata" (c).

2. At free end of each sterigma is formed an oval body--a spore or
"gonidium" (d), which, when ripe, is thrown off from the sterigma. 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.

[Illustration: FIG. 80.--Oïdium.]

~Ascomycetæ.~--_Oïdium_ (Fig. 80).--(This family is 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,
"oïdia," and behave as spores.

Note the asexual reproduction. From the pseudomycelium arise true hyphæ
(b), each of which in turn ends in a chain of spores (c).


~MORPHOLOGY OF THE BLASTOMYCETES.~

The blastomycetes are composed of spherical or oval cells (8 to 9.5µ 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 hæmatoxylin (see page 105).

During their growth and multiplication the blastomycetes split up
solutions containing sugar into alcohol and CO_{2}.

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

~Reproduction.~--Budding; ascospores--asexual.

Note the asexual _reproduction_.

1. "Gemmation"--that is, the budding out of daughter cells (b) from
various parts of the gradually enlarging mother cell. These are
eventually cast off and in turn become mother cells and form fresh
groups of buds.

[Illustration: FIG. 81.--Saccharomyces with ascospores.]

[Illustration: FIG. 82.--Torula.]

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

~Torulæ~ (Fig. 82).--Torulæ, 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, subdivided according to the
arrangement of the individuals after fission, into--

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

_Tetrads_, _Merismopedia_, or _Pediococci_, where division takes place
alternately in two planes at right angles to each other, and the
individuals remain attached in flat tablets of four, or its multiples.

[Illustration: FIG. 83.--Types of bacteria--cocci: 1, Diagram of sphere
indicating planes of fission; 2, diplococci; 3, streptococci; 4,
tetrads; 5, sarcinæ; 6, staphylococci.]

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

[Illustration: FIG. 84.--Types of bacteria--bacilli, etc.: 1, Bacilli;
2, diplobacilli; 3 streptobacilli; 4, spirilla; 5, vibrios; 6,
spirochætæ.]

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

~2. Bacilli~ (Fig. 84, 1 to 3).--Rod-shaped cells. A bacillus, however
short, can usually be distinguished from a coccus in that two sides are
parallel. Some bacilli after fission retain a characteristic arrangement
and may be spoken of as _Diplobacilli_ or _Streptobacilli_.

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
leptothriciæ (_vide infra_).

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

    Spirillum.
    Vibrio (comma).
    Spirochæta.

Many Spirochætes appear to belong to the animal kingdom and are grouped
under protozoa; other organisms to which this name has been given are
undoubtedly bacteria.

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

(a) Differentiation between base and apex;

(b) Growth apparently apical;

(c) Exaggerated pleomorphism;

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

    1. Beggiotoa.    }   Free swimming forms, which
    2. Thiothrix.    }   contain sulphur granules.

    3. Crenothrix.   }
    4. Cladothrix.   }  These forms do not contain
    5. Leptothrix.   }  sulphur granules.

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

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

For example, under one set of conditions the examination of a pure
cultivation of a bacillus may show a short oval rod as the predominant
form, whilst another culture of the same bacillus, but grown under
different conditions, may consist almost entirely of long filaments 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
connection with each.


~ANATOMY.~

1. _Capsule_ (Fig. 85, b).--A gelatinous envelope (probably akin to
mucin in composition) surrounding each individual organism, and
preventing absolute contact between any two. In some species the capsule
(e. g., B. pneumoniæ) 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 mesenteroides). In
some species colouring matter or ferric oxide is stored in the capsule.

2. _Cell Wall_ (Fig. 85, c).--A protective differentiation 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
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.

[Illustration: FIG. 85.--Dragrammatic sketch of composite bacterium to
illustrate details of anatomical structure.]

[Illustration: FIG. 86.--Plasmolysis.]

3. _Cell Contents._--Protoplasm (mycoprotein) contains a high percentage
of nitrogen, but is said to differ from proteid in that it is not
precipitated by C_{2}H_{6}O. 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 appearance due to plasmolysis, on the other.

The cell contents may sometimes be differentiated into a parietal layer,
and a central body (e. g., beggiotoa) when stained by hæmatoxylin.

4. _Nucleus._--This structure has not been conclusively 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.
diphtheriæ).

5. _Flagella_ (Organs of Locomotion, Fig. 85, a).--These are
gelatinous elongations of the cell protoplasm (or more probably of the
capsule), occurring either at one pole, at both poles, or scattered
around the entire periphery. Flagella are not pseudopodia. The
possession of flagella was at one time suggested as a basis for a system
of classification, when the following types of ciliation were
differentiated (Fig. 87):

[Illustration: FIG. 87.--Types of ciliation.]

1. Polar: (a) _Monotrichous_ (a single flagellum situated 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 place midway between
these aggregations, and a septum is formed in the interior of the cell
at right angles to its length.

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

[Illustration: FIG. 88.--Fission of cocci.]

[Illustration: 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 growing in the same medium 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. diphtheriæ on potato four to six days old.

[Illustration: FIG. 90.--Involution forms.]

They are of two classes, viz.:

(a) Involution forms characterised by alterations of shape (Fig. 90).
(Not necessarily dead.)

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

_Resting Stage._--Spore Formation.--Conditions influencing 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.

(b) The medium became toxic from the accumulation 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 "germinate," nor is their resistance very marked, as they
fail to initiate new cultures, after having been exposed to a
temperature of 80° C. for ten minutes.

(B) Endogenous: The cell protoplasm becomes differentiated and condensed
into a spherical or oval mass (very rarely cylindrical). After further
contraction the outer layers of the mass become still more highly
differentiated and form a distinct spore membrane, 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 unaltered, 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).

(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.

[Illustration: FIG. 91--Types of spore-bearing bacilli.]

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

4. _Capitate_ (Fig. 91, e): Rods with a spherical enlargement at one
pole; drumstick-shaped; e. g., B. tetani.

The endo-spores 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  } of the spore
    (c) Low permeability           } membrane.

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

~Germination.~--When transplanted to suitable media and placed under
favourable conditions, the spores germinate, usually within twenty-four
to thirty-six hours, and successively undergo the following changes
which may be followed in hanging-drop cultures on a warm stage:

1. Swell up slowly and enlarge, through the absorption of water.

2. Lose their refrangibility.

3. At this stage one of three processes (but the particular 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. megatherium.

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.

[Illustration: FIG. 92. Simple.]

[Illustration: FIG. 93. Solution.]

[Illustration: FIG. 94. Polar.]

[Illustration: FIG. 95. Bipolar.]

[Illustration: FIG. 96. Equatorial.]


~Food Stuffs.~--1. _Organic Foods._--

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

(b) Both saprophytic and facultative parasitic bacteria 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 cultivated; 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. 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)                 0° C.   15° C.   30°C.
2. Mesophilic bacteria
   (includes pathogenic 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 Production._--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 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 production 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 clotting 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
enzymic 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 no means has been devised for their separation or
extraction.

_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._).




X. NUTRIENT MEDIA.


In order that the life and growth of bacteria may be accurately observed
in the laboratory, it is necessary--

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 carbohydrate 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 bacterial growth diffuses itself through a
liquid, it is impossible to study therein the characteristics of
individual organisms. Many such media are, therefore, subsequently
rendered solid by the addition of substances like gelatine or agar, in
varying proportions, the proportions of such added material being
generally mentioned when referring to the media; e. g., 10 per cent.
gelatine, 2 per cent. agar. Gelatine is employed for the solidification
of those media it is intended to use in the cultivation of bacteria at
the room temperature or in the "cold" incubator. In the percentages
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 general use.

Media, on the other hand which have been solidified by the addition of
agar, only become liquid when exposed 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 apparatus, vessels, funnels, etc.,
employed in the preparation of media; although in the preliminary stages
of 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 concentrated 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, 2-1/2
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 ten minutes. (This precipitates some
of the albumins, the hæmoglobin, 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 substituted for the beef;
or the meat extract may be prepared 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 prepared is acid in its
reaction, owing to the presence of acid phosphates of potassium and
sodium, weak acids of the glycolic series, and organic compounds in
which the acid character predominates. Owing to the nature of the
substances from which it derives its reaction, the total acidity of meat
extract can only be estimated accurately when the solution is at the
boiling-point.

Moreover, it has been observed that prolonged boiling (such as is
involved in the preparation of nutrient media) causes it to undergo
hydrolytic changes which increase its acidity, and ~the meat extract only
becomes stable in this respect after it has been maintained at the
boiling-point for forty-five minutes~.

Although meat extract always reacts acid to phenolphthalein, it
occasionally reacts neutral or even alkaline 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 phenolphthalein.

2. Dibasic sodium phosphate which is formed during the process of
neutralisation is a salt which reacts alkaline to litmus, but neutral to
phenolphthalein. In order, therefore, to obtain an accurate estimation
of the reaction of any given sample of meat extract, it is essential
that--

1. The meat extract be previously exposed to a temperature of 100° C.
for forty-five minutes.

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

3. Phenolphthalein be used as the indicator.

The estimation is carried out by means of titration 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           1. 10N NaOH, accurately
in tenths of a centimetre.             standardised.

2. 1 c.c. pipette graduated in         2. n/1 NaOH, accurately
hundredths, and provided               standardised
with rubber tube, pinch-cock,
and delivery nozzle.

3. 25 c.c. measure (cylinder or        3. n/10 NaOH, accurately
pipette, calibrated for                standardised.
98°C.--_not_ 15°C).

4. Several 60 c.c. conical             4. 0.5 per cent. solution of
beakers or Erlenmeyer                  phenolphthalein in 50 per
flasks.                                cent. alcohol.

5. White porcelain evaporating basin, filled with boiling water and
arranged over a gas flame as a water-bath.

6. Bohemian glass flask, fitted as a wash-bottle, and filled
with distilled water, which is kept boiling on a tripod stand.

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

(A) 1. Fill the burette with n/10 NaOH.

2. Fill the pipette with n/1 NaOH.

[Illustration: 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 boiling
distilled water from the wash-bottle, and then add this rinse water to
the meat extract already in the beaker.

4. Run in about 0.5 c.c. of the phenolphthalein solution and immerse the
beaker in the water-bath, and raise to the boil.

5. To the medium in the beaker run in n/10 NaOH cautiously from the
burette until the end-point is reached, as indicated by the development
of a pinkish tinge, shown in figure 98 (b). 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.

[Illustration: 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 n/10 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 1.0 c.c. n/10
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 n/1 NaOH from the pipette, just short of the equivalent of the
amount of _deci_-normal soda solution required to neutralise the 25 c.c.
of medium. (For example, if in the first estimation 5 c.c. of n/10 NaOH
were required to render 25 c.c. of medium neutral to phenolphthalein,
only add 0.48 c.c. of n/1 NaOH.) Immerse the beaker in the water-bath.

3. Complete the titration by the aid of the n/10 NaOH.

4. Note the amount of n/10 NaOH solution required to complete the
titration, and add it to the equivalent of the n/1 NaOH solution
previously run in. Take the total as the correct estimation.


_Method of Expressing the Reaction._--

The reaction or _titre_ of meat extract, medium, or any solution
estimated in the foregoing manner, is most conveniently expressed by
indicating the number of cubic centimetres of normal alkali (or normal
acid) that would be required to render _one litre_ of the solution
exactly neutral to phenolphthalein.

[Illustration: FIG. 99.--Stock bottle for dekanormal soda solution.]

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

For example, "meat extract + 10," indicates a sample of meat extract
which reacts acid to phenolphthalein, 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 experiments we find that
25 c.c. of meat extract require the addition of 5 c.c. n/10 NaOH to
neutralise, then 1000 c.c. of meat extract will require the addition of
200 c.c. n/10 NaOH = 20 c.c. n/1 NaOH.

And this last figure, 20, preceded by the sign + (i. e., +20), to
signify that it is acid, indicates the reaction of the meat extract.

     NOTE.--The standard soda solutions should be prepared by
     accurate measuring operations, controlled by titrations,
     from a stock solution of 10N 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 CO_{2}. This may be
     done by first leading it over H_{2}SO_{4} and soda lime, or
     soda lime alone, by some such arrangement as is shown in
     figure 99, which also shows a constant burette arrangement
     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 +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.

Briefly, the method of standardising a litre of media to +10 consists in
subtracting 10 from the initial _titre_ 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. n/10 NaOH to neutralise.

Two controls give the following results:

2. 25 c.c. require the addition of 5.70 c.c. n/10 NaOH to neutralise.

3. 25 c.c. require the addition of 5.60 c.c. n/10 NaOH to neutralise.

Averaging these two controls, 25 c.c. require the addition of 5.65 c.c.
n/10 NaOH to neutralise, and therefore 1000 c.c. require the addition of
226 c.c. n/10 NaOH, or 22.60 c.c. n/1 NaOH, or 2.26 c.c. n/10 NaOH.

Initial _titre_ of the bouillon = +22.6, and as such requires the
addition of (22.6 c.c. - 10 c.c.) = 12.6 c.c. of n/1 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
n/1 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 x = 11.65 c.c. n/1 NaOH.

Whenever possible, however, the required reaction is produced by the
addition of dekanormal soda solution, 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 0.01 c.c. it is possible to deliver 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. dekanormal NaOH. The first being too bulky a
quantity, and the second inconveniently small for exact measurement, the
total weight of soda is obtained by substituting 1.16 c.c. dekanormal
soda solution, and either 0.05 c.c. of normal soda solution or 0.5 c.c.
of decinormal 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 Swedish 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
ordinary "quadrant" shape, as by this means a large surface is available
for filtration and a smaller area in contact with the glass funnel
supporting it.

To fold the filter proceed thus:

1. Take a circular piece of filter paper and fold it exactly through its
centre to form a semicircle (Fig. 100, a).

2. Fold the semicircle exactly in half to form a quadrant; make the
crease 2, distinct by running the thumbnail 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 3a, thus formed and
again open out to a semicircle.

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

5. Fold the point 1 over to the point 3, and 1a to 3a, to form the
creases 4 and 4a, indicated in the diagram by the light lines. Fold
point 1 over to 3a, and 1a to 3, to form the creases 5 and 5a.

[Illustration: FIG. 100.--Filter folding: a, Filter folded in half,
showing creases; b, appearance of filter on completion of folding;
c, filter opened out ready for use.]

6. Thus far the creases have all been made on the same side of the
paper. Now subdivide each of the eight sectors by a crease through its
centre on the opposite side of the paper, indicated by the 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.

When opened out the filter assumes the shape represented 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.

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

[Illustration: FIG. 101.--Hot-water filter funnel and ring burner.]

Gelatine, when properly made, filters through this paper as quickly as
bouillon does through the Swedish filter paper, and does _not_ require
the use of the hot-water funnel.

Agar, likewise, if properly made, filters readily, although not at so
rapid a rate as gelatine. If badly "egged," and also during the winter
months, it is necessary to surround the glass funnel, in which the
filtration of the agar is carried on, by a hot-water jacket. This is
done by placing the glass funnel inside a double-walled copper
funnel--the space between the walls being filled with water at about
90° C.--and supporting the 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 the flask and fill in with melted paraffin wax to
the level of the mouth. When the wax has set the flasks are stored in a
cool dark cupboard for future use.

[Illustration: FIG. 102.--Rubber cap closing store bottle. a, before,
and b, after sterilizing.]

This plan is not absolutely satisfactory, although very generally
employed on occasion, and it is preferable 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.[3] 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,
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 valvular 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, b). 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 definite 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 communicates with a
small graduated tube (capacity 20 c.c. and graduated in cubic
centimetres) attached to the side. The shape of this piece of apparatus,
known as Treskow's funnel, renders it particularly liable to damage. It
is better, therefore, to arrange a less 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
perforated 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 graduated 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 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.

[Illustration: FIG. 103.--Separatory funnel and three-way tap arranged
for tubing media.]

[Illustration: FIG. 104.--Gas tube (Durham).]

~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 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 provided 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.

[Illustration: FIG. 105.--Medium box, showing alternative partitions A
and B.]

FOOTNOTES:

[3] This rubber cap has been made for me by the Holborn Surgical
Instrument Co., Thavies Inn, London, W. C.




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 Witté's peptone, 10 grammes (= 1 per cent.), salt, 5
grammes (= 0.5 per cent.), and mix into a smooth paste with 200 c.c. of
distilled water previously heated to 60° C. (Be careful to leave no
unbroken globular masses of peptone.)

3. Add the peptone emulsion to the meat extract in the flask and heat 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 +10 (_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 discontinuous method (_vide_ page 35).

     NOTE.--As an alternative method when neither fresh nor
     frozen meat is available nutrient bouillon may be prepared
     from a commercial 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
     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 Witté's peptone (10 grammes), salt (5 grammes).
     Mix into smooth paste with 100 c.c. distilled water
     previously heated to 60°C.

     4. 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.

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

~Nutrient Gelatine.~--

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

[Illustration: FIG. 106.--Trip balance.]

An extremely useful counterpoise is a small sheet-brass cylinder about
38 mm. high and 38 mm. in diameter, with a funnel-shaped top and
provided with a side tube by which its contents, fine "dust" shot, may
be emptied out (Fig. 107).

[Illustration: FIG. 107.--Counterpoise; weight when empty, 35 grammes;
when full of dust shot, 200 grammes.]

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.

[Illustration: FIG. 108.--Arrangement of steam can and water-bath for
the preparation of media.]

4. Arrange a 5-litre 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).

5. Keep the water in the steam can vigourously boiling, and so steam at
100°C, bubbling through the medium mass, for ten minutes, by which time
complete solution of the gelatine is effected. 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 meat 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 (1115[4] grammes + weight of
the flask) minus (weight of the flask and its contents) equals the
weight of water required to make up the bulk to 1 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) together 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
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 counterpoise 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 (1035[5] 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 1 litre. Add the
requisite amount (see preparation of gelatine, page 166, step 6).

7. Titrate, and estimate the reaction of the medium mass; control the
result. Calculate the amount of soda solution required to make the
reaction of the medium mass + 10 (i. e., calculated for 1000 c.c.,
less the quantity used for the titrations).

8. Add the necessary amount of soda solution and 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 Chardin, 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 slaughterhouse,
otherwise the serum will be stained with hæmoglobin.)

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.

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.)

[Illustration: FIG. 109.--Blood-serum jar with wicker basket for
transport.]

The serum inspissator (Fig. 110) 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 thermo-regulator. 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 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.

[Illustration: FIG. 110.--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
     minutes. (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. 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 sterile capillary pipette (Fig. 13, c); 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 ventricle 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.

11. 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 1 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 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 1 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 reaction of the milk (normal
cows' milk averages +17). If of higher acidity than +20, or lower than
+10, reject 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 remainder for future use.


~Litmus Milk.~--

1. Prepare milk as described above, sections 1 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).

1. Collect 250 c.c. perfectly fresh ox serum (_vide_ Blood Serum, page
168, steps 1 to 5) and add to it 450 c.c. sterile distilled water.

2. Weigh out agar powder, 20 grammes, and emulsify it with 250 c.c. of
the cold serum water.

3. Weigh out

    Witté'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 1 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 solution (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 minutes on each of
three successive days--i. e., by the discontinuous method for three
days.


~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
     (sufficient to give a definite pink colour), or a few drops
     of waterproof Chinese ink added to the medium at this stage
     facilitates the subsequent "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 incubator 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.~--

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

2. Peel and take out the eyes.

3. Remove cylinders from the longest diameter of each potato by means of
an apple-corer or a large cork-borer (i. e., one of about 1.4 cm.
diameter).

The reaction of the fresh potato is strongly acid to phenolphthalein.
If, therefore, the potatoes are required to approximate +10, as for the
cultivation of some of the vibrios, the cylinders should be soaked in a
1 per cent. solution of sodium carbonate for thirty minutes.

4. Cut each cylinder obliquely from end to end, forming two wedge-shaped
portions.

5. Place a small piece of sterilised cotton-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. 111).

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

[Illustration: FIG. 111.--Potato tube.]

     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.

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 interfered with.

     NOTE.--It is sometimes more convenient to obtain
     "_unhopped_"[6] 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.

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 Witté'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 substances are the following, arranged in accordance with
their chemical constitution:

    _Monosaccharides_       Dextrose (glucose), lævulose, galactose,
                              mannose, arabinose, xylose.
    _Disaccharides_         Maltose, lactose, saccharose.
    _Trisaccharides_        Raffinose (mellitose).
    _Polysaccharides_       Dextrin, inulin, starch, glycogen, amidon.
    _Glucosides_            Amygdalin, coniferin, salicin,
                              helicin, phlorrhizin.
    _Polyatomic alcohols_   _Trihydric_, Glycerin.
                            _Tetrahydric_, Erythrite.
                            _Pentahydric_, Adonite.
                            _Hexahydric_, Dulcite, (dulcitol or
                              melampirite), isodulcite (rhamnose),
                              mannite (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 exemplified 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 n/1 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 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, prepared 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 CO_{2} through the solution until reaction is definitely
acid.

11. 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.

~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
commonly used for this purpose. The principal of these are as follows:

     ~Bile Salt Broth (MacConkey).~--

     1. Weigh out Witté'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 1 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 1 to
     7) and measure out 1000 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).~--

     1. Prepare nutrient agar (_vide_ page 167, sections 1 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 Bouillon (Weyl).~--

     1. Measure out nutrient bouillon (_vide_ page 163, sections
     1 to 6 1000 c.c.).

     2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium
     sulphindigotate, 1 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 1 to
     7). Measure out 1000 c.c.

     2. Weigh out glucose, 20 grammes (= 2 per cent.), and sodium
     sulphindigotate, 1 gramme (= 0.1 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 1 to 8).
     Measure out 1000 c.c.

     2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium
     sulphindigotate, 1 gramme (= 0.1 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
     decolourised to a light yellow.

FOOTNOTES:

[4] This figure is obtained by adding together 1 litre water, 1000
grammes; 10 per cent. gelatine, 100 grammes; 1 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 alterations of the figures. The average
weight of a measured litre of 10 per cent. nutrient gelatine when
prepared in this way _after filtration_ is 1080 grammes.

[5] This figure is obtained by adding together 1 litre of water (meat
extract), 1000 grammes; 2 per cent. agar, 20 grammes; 1 per cent.
peptone, 10 grammes; 0.5 per cent. salt, 5 grammes--total 1035 grammes.
Modifications of the process as to quantities or percentages will
necessitate corresponding 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.

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




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 requirements 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 intended for work with
intestinal bacteria, have been included; but beyond the fact that the
author's modification of the Drigalski-Conradi medium has been included
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 rarely given in full; the
routine method of exposure in the steam steriliser at 100° C. (without
pressure) for twenty minutes 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 departed from are further details given.

_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                          0.1 gramme
    Acid potassium phosphate (KH_{2}PO_{4})   1.0 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, (Na_{2}HPO_{4}) 12OH   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 advantageously employed in the solid condition.


~Proteid Free Broth (Uschinsky).~--

1. Weigh out and mix
    Calcium chloride                           0.1 gramme
    Magnesium sulphate                         0.2 gramme
    Acid potassium phosphate (KH_{2}PO_{4})    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 Biochemical 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 bouillon (+10), 1000 c.c. (_vide_, page 161), from
the meat extract, and store in 1-litre flask.

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 condition; but if acid
or gas is present, again make the bouillon in the flask +10, reinoculate
with one or other of the above-mentioned bacteria, and incubate; then
test again. Repeat this till neither acid nor gas appears in the medium
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 percentage of
gelatine or agar respectively and completing the medium according to
directions given on pages 166 and 167. The most important application of
inosite-free bouillon is its use in the preparation of sugar bouillons,
whether glucose, maltose, lactose, or saccharose, of exact percentage
composition.


~Sugar (Dextrose) Bouillon.~--

1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1
to 6) or sugar-free bouillon (_vide supra_).

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 1 to 7). Measure
out 1000 c.c.

2. Weigh out glucose, 20 grammes (= 2 per cent.), and dissolve in the
hot gelatine.

3. Filter through papier Chardin.

4. Tube, and sterilise as for nutrient gelatine.


~Sugar Agar.~--

1. Prepare nutrient agar (_vide_ page 167, sections 1 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 1
to 6).

2. Weigh out ferric tartrate, 1 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 1
to 6).

2. Weigh out lead acetate, 1 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 1
to 6).

2. Weigh out potassium nitrate, 5 grammes (= 0.5 per cent.), and
dissolve it in the bouillon.

3. Tube, and sterilise as for bouillon.

     NOTE.--The nitrate of sodium or ammonium may be substituted
     for that of potassium, or the salt may be added in the
     proportion of from 0.1 to 1 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, 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 0.1 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 0.1 c.c.
of a 1 per cent. neutral aqueous solution of lead acetate.


~Nitrate Peptone Solution (Pakes).~--

1. Weigh out Witté'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, 1 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 1
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.~--

1. Weigh out and mix

    Sodium chloride            2.0 grammes
    Magnesium sulphate         0.1 gramme
    Calcium chloride           0.2 gramme
    Monopotassium phosphate    2.0 grammes

2. Dissolve in water 1000 c.c. in a 2-litre flask

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    1 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 1010, 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.


~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 Witté's peptone, 1 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 1.5 per cent. or 2 per cent. powdered agar, and add it to
the urine.

4. Heat in the steamer at 100° C. for ninety 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. 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 170)    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.

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.

Lævulose, 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_).--

1. Weigh out and mix

    Potassium phosphate   4.0  grammes
    Magnesium sulphate    2.0  grammes
    Ammonium sulphate     4.0  grammes
    Sodium chloride       0.25 gramme

2. Dissolve in distilled water 4000 c.c.

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 100° C.
for thirty minutes on each of three consecutive days.

(A solid medium of the colour of _café 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.)


_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.

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 gelatine 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 (KH_{2}PO_{4})  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 (K_{2}HPO_{4})  1.0 gramme
    Magnesium sulphate                          0.2 gramme
    Calcium chloride                            0.1 gramme
    Ammonium tartrate                          10.0 grammes

and dissolve in

    Distilled water                        1000 c.c.

2. Tube or flask; sterilise as for nutrient bouillon.


~Plaster-of-Paris Discs.~--

1. Take large corks, 2.5 cm. diameter, and roll a piece of stiff
note-paper round each, so that about a 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.

[Illustration: Fig. 112.--Cork and paper mould for plaster-of-Paris
disc.]

5. Remove the sterile discs from the oven by means of sterile forceps,
place each inside a sterile capsule, and moisten with a little sterile
water.

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


~Gypsum Blocks (Engel and Hansen).~--

These are in the form of truncated cones and for their preparation 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 distilled 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 × 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 1 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.


~Wheat Bouillon (Gasperini).~--

1. Weigh out and mix wheat flour, 150 grammes; magnesium sulphate, 0.5
gramme; potassium nitrate, 1 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 Witté'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 to employ an incubating
temperature of 30°C., 10 per cent. gelatine and 0.5 per cent. agar must
be dissolved in the meat extract before the addition of the 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 Witté'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 calculated 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.


~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. Weigh 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
168).

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 Berkefeld 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).~--

1. Measure out into a flask

    Fresh milk                                    1000 c.c.

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 n/1 caustic soda solution and
boil. Whey now cloudy and acid again.

5. Again neutralise to litmus by addition of n/10 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. Weigh out

    Peptone                 10 grammes

and emulsify in a few cubic centimeters of the whey and return to the
flask.

5. Weigh 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 figure 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.c.

2. Weigh out

    Extractum malti (malt extract)        25 grammes

and add to distilled water in flask.

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 figure 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 1 gramme potassium hydrogen phosphate, 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 1 c.c. of a one per thousand aqueous solution of ferrous
sulphate.

4. Add 1 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.


~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      1.0 gramme
    Magnesium sulphate       0.5 gramme
    Calcium chloride         0.01 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        1 gramme
    Potassium sulphate       1 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         0.01 gramme

and dissolve in

    Distilled water         50 c.c.

Label--Solution A.

2. Weigh out and mix

    Potassium phosphate      0.10 gramme
    Sodium carbonate         0.60 gramme

and dissolve in

    Distilled water         50 c.c.

Label--Solution B.

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 Witté'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.


_Media for the Study of Plant Bacteria._

    ~Beetroot.~--  }
    ~Carrot.~--    } are prepared tubes and sterilised in a manner
    ~Turnip.~--    } precisely similar to that described for potato.
    ~Parsnip.~--   }


~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 1 c.c. of a 1 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 100 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 1 c.c. of 1 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.

9. Cool to 60°C., clarify with egg and filter.

10. Weigh out 20 grammes saccharose and add to the contents of the
flask.

11. Tube, and sterilise as for nutrient agar.


~Wood Ash Agar.~--

1. 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.

6. Add 10 c.c. sterile neutral red, 1 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 0.1, 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 contaminated
tubes. Store the remainder for future use.

~Carbolised Bouillon.~--

1. Prepare nutrient bouillon (_vide_ page 163, sections 1 to 6). Measure
out 1000 c.c.

2. Weigh out carbolic acid, 1 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 1 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 1 to 8). Measure out
1000 c.c.

2. Weigh out 1 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 1 to 8).

2. Add sterile litmus solution, sufficient to tint the medium a deep
lavender colour.

3. Tube, and sterilise as for nutrient gelatine.


~Lactose Litmus Bouillon (Lakmus Molke).~--

1. Weigh 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 completely
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 1 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 1 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 1 kilo of potatoes, wash thoroughly in water, peel, and grate
finely on a bread-grater.

2. Weigh the potato gratings, place them in a 2-litre flask, and add
distilled water in the proportion of 1 c.c. for every gramme weight of
potato. Allow the flask to stand in the ice-chest for twelve hours.

3. Strain the mixture through butter muslin and filter through Swedish
filter paper into a graduated cylinder. Note the amount of the filtrate.

4. Place the filtrate in a flask, add an equal quantity of distilled
water, and heat in the steam steriliser for sixty minutes.

5. Add glycerine, 4 per cent., mix thoroughly, and again filter.

6. Tube and sterilise as for nutrient bouillon.

~Potato Gelatine (Elsner).~--

1. Take 1 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 1 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 1 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               1.0 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 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 (= 1 per cent.), and dissolve it in the
agar.

If desired, add 5 c.c. of a 1 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 extract 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.

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) 1 per thousand aqueous solution     6.5 c.c.

4. Measure out and add to the medium

    Picric acid (Gruebler), 1 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.

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, 1 per thousand aqueous solution     20 c.c.

and mix thoroughly with the fluid agar.

9. Measure out and add to the medium

    Picric acid, 1 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, 0.1 per cent. aqueous solution     16 c.c.
        for ~"weak"~ medium.

_4a._ 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.

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. Diphtheriæ._

~Glycerine Blood-serum.~--

1. Prepare blood-serum as described, page 168, sections 1 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 1 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 1 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 1 to 4), as free from
hæmoglobin as possible.

2. Weigh out 0.15 per cent. sodium hydrate and dissolve it in the fluid
(or add 0.375 c.c. of dekanormal soda solution for every 100 c.c. of
serum).

3. Tube, and stiffen at 100° C. in the serum inspissator.

4. Incubate at 37° C. for forty-eight hours to eliminate any
contaminated tubes. Store the remainder for future use.


~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 exhaust 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.

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 vide_ page 174) 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 between steps 4 and 5.


~Glycerine Bouillon.~--

1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1
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 1 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 Blood-serum.~--

1. Prepare blood-serum as described, page 168, sections 1 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.


~Glycerinated Potato.~--

1. Prepare ordinary potato wedges (_vide_ page 174, sections 1 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.


~Animal Tissue Media (Frugoni).~--

1. Take a number of sterile test-tubes 16 × 3 or 4 cm., plugged with
cotton wool, and into each insert a 2 cm. length of stout glass tubing
(about 1 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 successive 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 3 × 1.5 × 1 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 preliminary
sterilisation by boiling or autoclaving.


_Media for the Study of Special Cocci._

_Diplococcus Gonorrhoeæ._


~Ascitic Bouillon (Serum Bouillon).~--

1. Collect ascitic fluid (pleuritic fluid, hydrocele fluid, etc.), by
aspiration directly into sterile flasks, under strictly aseptic
precautions.

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 contaminated
tubes. Store the remainder for future use.


~Serum Agar (Heiman).~--

1. 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._

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 (= 1 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.


~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 0 (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 effusion
(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. of dekanormal NaOH solution per 100 c.c. of
the mixture).

_3a._ Glucose to the extent of 1 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 solution for ten
days. (After this time the white becomes firm like gelatine.)

2. Carefully remove the shell and cut the egg into fine slices.

3. Wash for two hours in running water.

4. Place the egg slices in a large beaker and sterilise in the steamer
at 100° C. for one hour.

5. Transfer each slice of egg by means of a pair of sterilised forceps
to a Petri dish or large capsule.

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


~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 n/10-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 contaminated
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.


_Diplococcus Pneumoniæ._

~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 1 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 spirochætes (cultivations must be grown
anaerobically)._

1. Plug and sterilise test-tubes 20 × 2 cm.

2. Kill a small rabbit with chloroform vapour. Open the abdomen 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 jar, 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.


[Illustration: FIG. 113.--Incubator.]

An incubator (Fig. 113) consists essentially of a chamber for the
reception of cultivations, etc., surrounded by a water jacket, the walls
of which are of metal, usually copper, and outside all an asbestos or
felt jacket, or 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
me[7] some years ago is of the greatest practical utility. Here the
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 × 12 cm.
wide × 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.

[Illustration: FIG. 114.--Cellular incubator.]

Two incubators at least are required in the laboratory, 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 most essential portion
of the incubator, as upon its efficient working depends the maintenance
of a constant temperature in the cultivation chamber. It is also used in
the fitting up of water and paraffin baths, and for many other purposes.

[Illustration: FIG. 115.--Reichert's thermo-regulator.]

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 metallic capsule
containing a fluid, the boiling-point of which corresponds to the
temperature at which the incubator is intended to act. They are:

(a) _Reichert's_ (Fig. 115), consists of a bulb containing mercury
which is to be suspended in the medium, 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 reopens the narrow tube C. By means of a thumbscrew D
(which mechanically raises or lowers the column of mercury irrespective
of the temperature) and the aid of a thermometer the apparatus can be
set to keep the incubator at any desired temperature. With this form a
special gas burner is required, with separate supply of gas to a pilot
jet at the side.

(b) _Hearson's capsule regulator_ consists of a metal capsule
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 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 116. 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.

[Illustration: FIG. 116.--Capsule thermo-regulator.]

The construction of the valve is such that, whenever 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 the screw needle S, hence the gas
flame below the incubator is never extinguished.

The expansion of the metal walls of the capsule, which takes place upon
the boiling of its contents, provides 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 temperature, the
lever will only be acted upon when the critical temperature is reached,
no sensible effect being produced at even 1° C. below that at which the
capsule is destined to act.

W is a weight sliding on the lever rod D; by increasing the distance
between the weight and the fulcrum 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.

FOOTNOTES:

[7] Made by the firm of Chas. Hearson & Co., 235 Regent St., London, W.




XIV. METHODS OF CULTIVATION.


Cultivations of micro-organisms are usually prepared 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 indifferent gas, such as hydrogen, nitrogen, or carbon
dioxide).

With regard to the temperature at which the cultivations are grown, it
may be stated as a general rule that all media rendered solid by the
addition of gelatine are incubated at 20°C., or at any rate at a
temperature not exceeding 22° C. (that is, in the "cold" incubator);
whilst fluid media and all other solid media are incubated at 37° C.
(that is, in the "hot" 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 employed for all media.

Tube cultivations are usually packed in the incubator in small tin
cylinders, such as those in which American cigarettes are sold, 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 incubator and each
accommodating two rows of tubes, are also exceedingly useful.


~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:

[Illustration: FIG. 117.--Inoculating tubes, seen from the front.]

~1. Fluid Media~ (e. g., Nutrient Bouillon).--

1. Flame the cotton-wool plug of the tube containing the cultivation and
also that of the tube of sterile bouillon.

2. Hold the two tubes, side by side, between the left thumb and the
first and third fingers, allowing the sealed ends to rest on the dorsum
of the hand, and separating the mouths of the tubes (which are pointed
to the right) by the tip of the second finger. Keep the tubes as nearly
horizontal as is possible without allowing the fluid in the bouillon
tube to reach the cotton-wool plug (Fig. 117).

3. Sterilise the platinum loop and allow it to cool.[8]

4. Grasp the plug of the tube containing the cultivation 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. Oblique 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
bottom of the tube almost to its mouth.

This is employed for "streak" or "smear" cultivations (_Strichcultur_).

2. Straight tube (Fig. 119), in which the medium forms a cylindrical
mass in the lower portion of the tube and presents an upper surface
which is at right angles to the long axis of the tube.

This is employed for "stab" or "stick" cultivations (_Stichcultur_), or
by inoculating the medium whilst fluid, and allowing to solidify in this
position, for "shake" cultivations.


_Streak Culture._--

1. Flame the plugs, sterilise the 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, terminating the "streak" over the thin layer of medium near the
mouth of the tube.

3. Replug the tubes, sterilise the platinum loop.

4. Label the newly inoculated tube and incubate.

_Smear Culture._--Proceed generally as in streak culture, but rub the
infected loop all over the surface of the medium, instead of restricting
the inoculation to a narrow line.

     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 substance 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.

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.

[Illustration: FIG. 118.--Sloped or slanted medium for streak or smear
culture.]

[Illustration: 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.

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.

     [Illustration: FIG. 120. 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 1 per cent.
     gelatine or 1 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 liquefied gelatine, agar,
or other similar medium, and the infected 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 organism becomes visible to the naked eye
as a "colony." This is the principle upon which the method of plate
cultivation is based and its practice enables the bacteriologist to
study the particular manner of development affected by each species of
microbe when growing (a) unrestricted upon the surface 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.

[Illustration: FIG. 121.--Handy form of water-bath for melting tubes of
agar and gelatine previous to slanting them; or to making shake cultures
or pouring plates.]


Method of "Pouring" Plates.--

1. Place the glass dish on the levelling tripod (Figs. 122, 123); 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.

This leveling is an important matter since the development 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 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.

[Illustration: FIG. 122.--Plate-levelling stand.]

3. Place three sterile Petri dishes in a row on the surface of the glass
plate and number them 1, 2, and 3, from left to right.

[Illustration: FIG. 123.--Plate-levelling stand, side view.]

4. Number the previously liquefied tubes of nutrient media 1, 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. 1, treating the
liquefied medium as bouillon. After replugging, 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 inoculum throughout the
medium; avoid jerky movements, as these cause bubbles of air to form in
the medium.

[Illustration: 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).

[Illustration: 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. 1, remove it, then flame the lips of the
tube; slightly raise the cover of Petri dish No. 1, introduce the mouth
of the tube; then, 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. 1, 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 usually 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 to 45° C. or 42° 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. 1 (for the first
     dilution) rarely gives a plate that is of any practical
     value it is frequently replaced by a tube of bouillon or
     sterile salt solution, and in such case plate No. 1 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
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 derivatives 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 allow it to solidify.
Then turn each plate and its cover upside down.

[Illustration: FIG. 126.--Surface plate spreader.]

3. When quite cold raise the bottom of plate 1, revert it and deposit a
drop of the inoculum (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 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.

[Illustration: FIG. 127.--Spreading surface plate.]

After incubation, plate No. 1 will probably yield an enormous number of
colonies; plate 2 will show fewer colonies, since only those bacteria
adhering to the rod after rubbing over plate 1 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 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.

[Illustration: FIG. 128.--Drying surface plate of agar.]

5. Inoculate the plates in series of three, as described for gelatine
surface plates 3-8.


Hanging-drop Cultivation.

    ~Apparatus Required.~--

    Hanging-drop slides.
    Cover-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 thoroughly in water and
drying, then plunging it into a beaker of absolute alcohol, draining off
the greater part of the spirit, grasping the slide in a pair of forceps,
and burning off the remainder of the alcohol in the flame.

3. Place the hanging-drop slide on a piece of blotting paper moistened
with 2 per cent. lysol solution and cover it with a small bell glass
that has been rinsed out with the same solution and _not dried_.

4. Raise the bell glass slightly and smear sterile vaseline around the
rim of the metal cell by means of a sterile spatula of stout platinum
wire.

5. Remove a clean cover-slip from the alcohol pot with sterile forceps
and burn off the alcohol; again raise the bell glass and place the
sterile cover-slip on the blotting paper by the side of the hanging-drop
slide.

6. Remove a drop of the broth from the second dilution tube with a large
platinum loop; raise the bell glass and deposit the drop on the centre
of the cover-slip. Sterilise the loop.

7. Raise the bell glass sufficiently to allow of the cover-slip being
grasped with forceps, inverted, and adjusted over the cell of the
hanging-drop slide. Remove 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 technique, 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
cultivation 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
rectangular 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~ (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 forceps, and with
the help of a second pair of forceps lower it carefully on the
inoculated surface of the agar (avoiding 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.


ANAEROBIC CULTIVATIONS.

Numerous methods have been devised for the cultivation 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 vacuo_.

(c) ~Absorption of oxygen~ from the air in contact with the cultivation
by means of pyrogallic acid rendered alkaline with caustic soda--i. e.,
cultivation in an atmosphere of nitrogen.

(d) ~Displacement of air~ by an indifferent gas, such as hydrogen or coal
gas--i. e., cultivation in an atmosphere 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 employed in any of the
processes should contain some easily oxidisable substance, such as
sodium formate (0.4 per cent.) or sodium sulphindigotate (0.1 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 1 part, olive oil 4 parts.


(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), 1
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 excluding 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 capillary 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, 1 or 2 cm. deep, of broth cultivation of a vigourous
aerobe--e. g., B. aquatilis sulcatus or B. prodigiosus--upon the
surface of the medium; or an equal depth of liquefied gelatine, which is
then inoculated with the aerobic organism.

3. Incubate.

The growth of the aerobe will use up all the oxygen that reaches it and
will not allow any to pass through to the medium below, which will
consequently remain in an anaerobic condition.


(B) ~Method V.~--

1. 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.

[Illustration: FIG. 129.--Vacuum culture.]

3. Connect the tube leading from the culture vessel with a water or air
pump, interposing a Wulff's bottle fitted as a wash-bottle and
containing sulphuric acid.

4. Exhaust the air from the culture vessel.

5. Before disconnecting the apparatus, seal the glass tube from the
culture vessel at the constriction, using the blowpipe flame.

6. Incubate.


(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 1
     gram.

     Dekanormal solution of caustic soda.

METHOD.--

1. Prepare the tube cultivation in the usual way.

2. Moisten the india-rubber stopper of the Buchner's tube with water and
see that it fits the mouth of the tube accurately.

3. Remove the stopper from the caustic soda bottle.

4. Drop one of the pyrogallic acid tablets[9] into the Buchner's tube
(roughly, use 1 gramme pyrogallic acid for every 100 c.c. air capacity
of the receiving vessel).

5. Add about 1 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 hurriedly.

7. Fit the india-rubber stopper tightly into the mouth of the Buchner's
tube.

[Illustration: FIG. 130.--Buchner's tube.]

The pyrogallic acid tablet dissolves slowly in the soda solution and its
oxidation 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 support, and incubate.


~Method VII~ (Wright's Method).--

1. Prepare tube cultivation in the usual way.

2. Cut off that portion of the cotton-wool plug projecting above the
mouth of the tube with scissors, then push the plug into the tube for a
distance of 2 or 3 cm.

3. By means of a pipette drop about 1 c.c. of pyrogallic 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.

[Illustration: FIG. 131.--McLeod's anaerobic plate base with half petri
dish inverted _in situ_]


~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).

    Plasticine.
    Pyrogallic acid (1 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.--

1. Roll out a long cylinder of plasticine and fit it into the groove on
the upper surface of the earthenware base.

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.

[Illustration: FIG. 132.--Kipp's hydrogen apparatus, (a) connected up
to two washing bottles containing (b) lead acetate 10 per cent.
solution, to remove H_{2}S and (c) silver nitrate solution to remove
AsH_{3}. A third washing bottle containing pyrogallic acid 10 per cent.
solution, rendered alkaline, to remove any trace of oxygen, is sometimes
introduced.]

[Illustration: 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 (b).]

5. Connect up the india-rubber tubing on one of the side tubes with a
cylinder of compressed hydrogen (or the delivery tube of a Kipp's 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-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 outside arms (Fig. 134).

6. Fill the glass dish with glycerine or metallic mercury to a depth of
about 5 cm.

[Illustration: 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.

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.

[Illustration: FIG. 135.--Novy's jar for plate cultivations.]

[Illustration: 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.
    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, containing
      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., 1 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 ointment and apply the
jar firmly to the glass slab, covering the cultivations--so arranged
that the long tube passes with its lower end into the glass dish at a
point directly opposite to the pyrogallic acid 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 strokes of the
air pump, aspirate a small quantity 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 pyrogallic acid
converting the latter into alkaline pyro. and so bringing its latent
capacity for oxygen into action).

[Illustration: FIG. 137.--Bulloch's jar.]

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 transfer the apparatus to the incubator.

This last method is the most satisfactory for anaerobic cultivations, as
by its means complete anaerobiosis can be obtained with the least
expenditure of time and trouble.

FOOTNOTES:

[8] See also method of opening and closing culture tubes, pages 74-76.

[9] 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.




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 progeny of a single cell is termed a
"pure culture"; one which contains representatives of two or more
species of bacteria is spoken of as an "impure," or "mixed"
"cultivation," and it now becomes necessary to indicate the chief
methods by which one or more organisms may be isolated in a state of
purity from a mixture; whether that mixture exists as an impure
laboratory cultivation, or is contained in pus and other morbid
exudations, infected tissues, or water or food-stuffs.

[Illustration: FIG. 138.--Hæmatocytometer cell, showing, a, section
through the centre of the cell, and b, a magnified image of the cell
rulings.]

Before the introduction of solid media the only method of obtaining pure
cultivations was by "dilution"--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 hæmatocytometer, Fig. 138), and diluting the fluid by the
addition of sterile water or bouillon until a given volume (usually 1
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
occasionally happened that the resulting growth was the product of one
individual microbe. A method so uncertain is now fortunately replaced by
many others, more reliable and convenient, and in those methods selected
for description here, the segregation and isolation of the required
bacteria may be effected--

A. ~By Mechanical Separation.~

1. 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 circumstances, and often a combination of two
or more will ensure a quicker and more reliable result than a rigid
adherence to any one method. Experience is the only reliable guide, but
as a general rule the use of either the first or the third method will
be found most convenient, affording as each of them does an opportunity
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) _Agar_ (_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 1, 2, and 3, and place
them, with three tubes of sterile fluid serum, also labelled 1a, 2a,
and 3a, 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. 1a and agar tube No. 1. 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. 1 _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._--

1. Melt a tube of sterile agar and pour it into a sterile plate; let it
set.

2. Collect a few drops of human blood, under all aseptic conditions, in
a sterile capillary 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.)

(f) _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. 1, 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, inoculate 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. 1.

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, discrete 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 shallow pit of liquefied gelatine, fourteen flat irridescent
filmy colonies, and four raised white slimy colonies. A film preparation
(stained Gram) from each variety examined microscopically showed that
the yellow liquefying 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 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 following
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
1-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 1, and the central bottom portion 8 (Fig. 139).
Revert the dish. The numbers 1 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 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 microscopically, using the oil immersion lens and note the
numbers of those cover-slips which morphologically and by Gram results
appear to be composed of different species of bacteria.

[Illustration: FIG. 139.--Diagram for stock plate.]


(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 results of
incubation. (If any of the subcultures fail, further material can be
obtained from the corresponding 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 torulæ 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 containing 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 process 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 1 per cent. will inhibit the growth of practically everything
but the Bacillus coli communis.


~5. Differential Incubation.~--

In isolating certain bacteria, advantage is taken of the fact that
different species vary in their optimum temperature. A mixture
containing the Bacillus typhosus and the Bacillus aquatilis sulcatus,
for example, may be planted on two slanted agar tubes, the one incubated
at 40°C., and the other at 12° C. After twenty-four hours' incubation
the first will show a pure cultivation of the Bacillus typhosus, whilst
the second will be an almost pure culture of the Bacillus aquatilis.


6. Differential Sterilisation.--

(a) _Non-sporing Bacteria._--Similarly, advantage may be taken of the
varying thermal death-points of bacteria. From a mixture of two
organisms whose thermal 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 heating 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 resulting growth will
be found to consist entirely of the B. mesentericus.

(b) _Sporing 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 cultivations made from the
mixture will yield a growth resulting from the germination of the spores
only.

Differential sterilisation at 80° C. is most conveniently 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
provided 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 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.

[Illustration: FIG. 140.--Benzole bath.]


7. Differential Atmosphere Cultivation.--

(a) By adapting the atmospheric conditions to the particular organism
it is desired to isolate, it is comparatively easy to separate a strict
aerobe from a strict anaerobe, and _vice versa_. In the first case,
however, it is important that the cultivations should be made upon
solid media, for if carried out in fluid media the aerobes multiplying
in the upper layers of fluid render the depths completely anaerobic, and
under these conditions the growth of the anaerobes will continue
unchecked.

(b) When it is desired to separate a facultative anaerobe from a
strict anaerobe, it is generally sufficient 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 subcultures, 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 suitable temperature. Pick
off the colonies of the required organism when the growth appears, and
transfer to tubes of the various media.

Incubate under suitable conditions as to 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 account 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 examinations duly recorded.

It must be stated definitely that no micro-organism can be identified by
any _one_ character or property, whether microscopical, biological or
chemical, but that on the contrary its entire life history must be
carefully studied and then its identity established from a consideration
of the sum total of these observations.

In order to give to the recorded results their maximum 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 Examination
     of Cultivation, page 261.)

    Gelatine plates, }
    Gelatine streak, } at 20°C.
    Gelatine stab,   }
    Gelatine shake,  }

    Agar plates,             }
    Agar streak or smear,    }
    Agar stab,               }
    Inspissated blood-serum, } at 20° C. and 37°C.
    Bouillon,                }
    Litmus milk,             }
    Potato,                  }

     Special media for the purpose of demonstrating
     characteristic 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.

    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 appearances 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 liquefaction of the
surrounding medium. If liquefaction is present, note shape and character
(_vide_ page 269, "stab" cultures).

_Agar._--No liquefaction takes place in this medium. The liquid found on
the surface of the agar (or at the bottom of the tube in agar tube
cultures) is merely water which has been expressed during the rapid
solidification of the medium and has subsequently condensed.

_Gelatine and Agar._--Examine the colonies at intervals of twenty-four
hours.

(a) With the naked eye.

(b) With a hand lens or watchmaker's glass.

(c) Under a low power (1 inch) of the microscope, or by means of a small
dissecting microscope.

Distinguish superficial from deep colonies and note the characters of
the individual colonies.

(A) ~Size.~--The diameter in millimetres, at the various ages.

(B) ~Shape.~--

Punctiform: Dimensions too slight for defining form by naked eye;
minute, raised, hemispherical.

Round: Of a more or less circular outline.

Elliptical: Of a more or less oval outline.

Irregular: 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).

[Illustration: FIG. 141.--Types of colonies: a, Cochleate; b,
amoeboid; c, mycelioid.]

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 character (Fig. 142, b).

Conglomerate: An aggregate of colonies of similar size and form (Fig.
142, c).

Toruloid: An aggregate of colonies, like the budding of the yeast plant
(Fig. 142, d).

Rosulate: Shaped like a rosette.

[Illustration: FIG. 142.--Types of colonies: a, Filamentous; b,
rhizoid; c, conglomerate; d, toruloid.]

(C) ~Surface Elevation.~--

1. _General Character of Surface as a Whole_:

Flat: Thin, leafy, spreading over the surface (Fig. 143, a).

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.
143, c).

Pulvinate: Surface the segment of a circle, but decidedly convex (Fig.
143, d).

Capitate: Surface hemispherical (Fig. 143, e).

Umbilicate: Having a central pit or depression (Fig. 143, f).

Conical: Cone with rounded apex (Fig. 143, g).

Umbonate: Having a central convex nipple-like elevation (Fig. 143, h).

2. _Detailed Characters of Surface_:

Smooth: Surface even, without any of the following distinctive
characters.

Alveolate: Marked by depressions separated by thin walls so as to
resemble 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 with minute vesicles due to gas
formation; more minute than bullate.

[Illustration: FIG. 143.--Surface elevation of colonies: a, Flat; b,
raised; c, convex; d, pulvinate; e, capitate; f, umbilicate;
g, conical; h, umbonate.]

[Illustration: FIG. 144.--Types of colonies--alveolate.]

Verrucose: Wart-like, bearing wart-like prominences.

Squamose: Scaly, covered with scales.

Echinate: Beset with pointed prominences.

Papillate: Beset with nipple or mamma-like processes.

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.

[Illustration: FIG. 145.--Types of colonies: a, Grumose; b,
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 appearance, and
particles in clustered grains (Fig. 145, a).

Moruloid: Having the character of a mulberry, segmented, 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).

[Illustration: 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, irregular 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.

[Illustration: FIG. 147.--Types of colonies--curled.]

4. _Filamentous Colonies:_

Filamentous: As already defined.

Floccose: Composed of filaments, densely placed.

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).

[Illustration: FIG. 148.--Edges of colonies: a, Entire; b, undulate;
c, repand; d, erose.]

Lobate.

Lobulate: Minutely lobate (Fig. 149, e).

Auriculate: With ear-like lobes (Fig. 149, f).

Lacerate: Irregularly cleft, as if torn (Fig. 149, g).

Fimbriate: Fringed (Fig. 149, h).

Ciliate: Hair-like extensions, radiately placed (Fig. 149, j).

Tufted.

Filamentous: As already defined.

Curled: As already defined.

[Illustration: FIG. 149.--Edges of colonies: e, Lobar-lobulate; f,
auriculate; g, lacerate; h, fimbriate; i, ciliate.]

(F) ~Optical Characters~ (after Shuttleworth).--

1. _General Characters_:

Transparent: Transmitting light.

Vitreous: Transparent and colourless.

Oleaginous: Transparent and yellow; olive to linseed-oil coloured.

Resinous: Transparent and brown, varnish or resin-coloured.

Translucent: Faintly transparent.

Porcelaneous: Translucent and white.

Opalescent: Translucent; greyish-white by reflected light.

Nacreous: Translucent, greyish-white, with pearly lustre.

Sebaceous: Translucent, yellowish or greyish-white.

Butyrous: Translucent and yellow.

Ceraceous: Translucent and wax-coloured.

Opaque.

Cretaceous: Opaque and white, chalky.

Dull: Without lustre.

Glistening: Shining.

Fluorescent.

Iridescent.

2. _Chromogenicity_:

Colour of pigment.

Pigment restricted to colonies.

Pigment restricted to medium surrounding colonies.

Pigment present in colonies and in medium.


~Streak or Smear Cultures.~--

_Gelatine and Agar._--Note general points as indicated under plate
cultivations.

_Inspissated Blood-serum._--Note the presence or absence of liquefaction
of the medium. (The presence of condensation water at the bottom of the
tube must not be confounded with liquefaction of the medium.)

_All Oblique Tube Cultures._--

1. Colonies Discrete: Size, shape, etc., as for plate cultivations
(_vide_ page 261).

2. Colonies Confluent: Surface elevation and character 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).

[Illustration: 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 characters (Fig. 150, a).

Nodose: Consisting of closely aggregated colonies.

Beaded: Consisting of loosely placed or disjointed colonies (Fig. 150,
b).

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.

[Illustration: FIG. 151.--Stab cultivations--types of growth: f,
Crateriform; g, saccate; h, infundibuliform; j, napiform; k,
fusiform; l, 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,
f).

Saccate: Shape of an elongated sack, tubular cylindrical (Fig. 151,
g).

Infundibuliform: Shape of a funnel, conical (Fig. 151, h).

Napiform: Shape of a turnip (Fig. 151, j).

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, l).

(D) _Character of the Liquefied Gelatine._--

1. Pellicle on surface.

2. Uniformly turbid.

3. Granular.

4. Mainly clear, but containing flocculi.

5. Deposit at apex of liquefied portion.


(E) _Production of Gas Bubbles._


~Shake Cultures.~--

1. Presence or absence of liquefaction.

2. Production of gas bubbles.

3. Bulk of growth at the surface--aerobic.

4. Bulk of growth in depths--anaerobic.


~Fluid Media.~


~1. Surface of the Liquid.~--

Presence or absence of froth due to gas bubbles.

Presence or absence of pellicle formation.

Character of pellicle.


~2. Body of the Liquid.~--

Uniformly turbid.

Flocculi in suspension.

Granules in suspension.

Clear, with precipitate at bottom of tube.

Colouration of fluid, presence or absence of.


~3. Precipitate.~--

Character.

Amount.

Colour.


~Carbohydrate Media.~--

Growth.

Reaction.

Gas formation.

Coagulation or not of serum albumen (when serum water media are
employed).


~Litmus Milk Cultivations.~--


                    {Unaltered.
    1. Reaction:    {Acid.
                    {Alkaline.
    2. Odour.

    3. Formation of gas.

                      {Unaltered.
    4. Consistency:   {Peptonised (character of solution).
                      {Coagulated.

                         {hard: solid.
    5. Clot: Character   {soft: floculent.
                         {ragged and broken up by gas
                         {bubbles.

(a) Coagulum undissolved.

(b) Coagulum finally peptonised, completely: incompletely.

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 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 _motility_ 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 conditions should form the basis of an opinion as to the
absence of true locomotion.

_Size._--In the case of non-motile or sluggishly motile organisms,
endeavour to measure several individuals in each hanging drop by means
of the eyepiece micrometer 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 cultivations (_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 microscope; if necessary,
using a warm stage.

Arrange illumination, etc., and select a solitary bacillus for
observation, by the help of the 1/6-inch lens.

Substitute the 1/12-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 cultivations from old
cultivations in which no living vegetative 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, remove 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 decolourise 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 temperature 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.

(c) Measure (_vide_ page 66) some twenty-five individuals 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.

(b) 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.

~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        } 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--H_{2}S, CO_{2}, H_{2}. Estimate the
ratio between the last two gases.

Prepare all cultivations for these methods of examination under
_optimum_ conditions, previously determined for each of the organisms it
is intended to investigate, as to

    (a) Reaction of medium;
    (b) Incubation temperature;
    (c) Atmospheric environment;

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 1 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.

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 ammonium sulphate.

Pink colour = peptone.

b. Boil with Millon's reagent.

Red colour = tyrosine.

c. Add glyoxylic acid solution and run in concentrated 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 solution.

METHOD.--

1. Prepare tube cultivation and incubate.

2. Prepare a thin starch paste and add 2 per cent. thymol to it.

3. Mix equal parts of the cultivation to be tested and the starch paste,
and place in the incubator at 37°C. for six to eight hours.

4. Filter.

Test the filtrate for sugar.

Boil some of the Fehling's solution in a test-tube.

Add the filtrate drop by drop until, if necessary, a quantity has been
added equal in amount to the Fehling's solution employed, keeping the
mixture at the boiling-point during the process.

Yellow or orange precipitate = sugar.

(C) _Invertase._--(Convert saccharose into a mixture of dextrose and
lævulose 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 solution 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 preceding section.

(D) _Rennin and "Lab" Enzymes._--(Coagulate milk independently of the
action of acids; e. g., B. prodigiosus.)

    _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.

_Media Required_:

Peptone water containing various percentages (generally 2 per cent.) of
each of the substances referred to under "sugar" media (page 177), also
tubes of peptone water containing 1 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 _titre_ 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 Berkefeld filter candle previous
     to step 2, and the filtrate employed in the titration.

    (b) _Qualitative_ (of all the organic acids present).--

    _Medium Required_:
    Sugar (glucose or lactose) bouillon as in quantitative examination.

    _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, 1 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.

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.

[Illustration: 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.

7. Acidify 250 c.c. with 20 c.c. concentrated phosphoric 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.)
                                      ¦
                                      ¦
                   1. 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                                RESIDUE
                  ¦                                       ¦
                  ¦                                       ¦
   may contain barium propionate,         may contain barium acetate,
   barium butyrate.                       barium formate, barium benzoate.
                  ¦                                       ¦
                  ¦                                       ¦
   1. Evaporate to dryness.               1. Evaporate off alcohol and
                                          dissolve up the residue on
   2. Dissolve residue in 150             the filter in hot water and
   c.c. water.                            neutralise.

   3. Acidify with phosphoric             2. Divide the solution into
   acid and distil.                       four portions:

   4. Saturate distillate with            (a) Add ferric chloride solution.
   calcium chloride and distill
   over a few c.c.                            ~Brown~ colour = _acetic_ or
                                              _formic_ acids.
   5. Test distillate for butyric
   acid:                                      ~Buff ppt.~ = _benzoic_ acid
                                              (see ether soluble acids).
   Add 3 c.c. alcohol and 4 drops
   concentrated sulphuric acid.           (b) Add silver nitrate
                                          solution; then add one drop
      ~Smell of pineapple~ = _butyric_    ammonia water, and boil.
      acid.
                                            ~Black~ precipitate of metallic
      Propionic acid in small               silver = _formic_ acid.
      quantities cannot be
      distinguished from butyric         (c) Evaporate to dryness; mix
      acid by tests within the           with equal quantity of
      scope of the bacteriological       arsenious oxide and heat
      laboratory.                        on platinum foil.

                                             Unpleasant ~smell of cacodyl~
                                             = _acetic_ acid.

                                         (d) Add a few drops of
                                         mercuric chloride solution
                                         in test-tube, and heat to
                                         70° C.

                                             ~Precipitate~ of mercurous
                                             chloride which is slowly
                                             reduced to mercury =
                                             _formic_ acid.

8. If the distillation of "B" is continued as long as acid comes 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 excess 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 characteristic 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.

Buff precipitate = benzoic acid, and other acids related 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 magnesia, then connect up
with condensers and distil.

3. Collect 50 c.c. distillate, from each, in a Nessler glass.

4. Add 1 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.

1. 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.

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
acetaldehyde 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 addition of
ammonium sulphate a deep permanganate colour, which takes ten minutes to
reach its full intensity. 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-benzaldehyde 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 incubated at 37°C., it must be allowed
to cool to the room temperature before applying the test.)

1. Remove 2 c.c. of the cultivation by means of a sterile pipette and
transfer to a clean tube, then,

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 1
     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 0.01 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, 1 per cent. aqueous solution.

METHOD.--

1. Prepare cultivation in a Bohemian flask containing 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.

5. Add to (b) about 0.5 c.c. ferric chloride solution. Violet colour =
phenol.

(If the distillate be acid the reaction will be negative.)

6. Add to (c) bromine water. Crystalline white ppt. of tribromo-phenol
= phenol.

     NOTE.--If both indol and phenol appear to be present in
     cultivations of the same organism, it is well to separate
     them before testing. This may be done in the following
     manner:

1. Prepare inosite-free bouillon cultivation, say 200 or 300 c.c., in a
flask as before.

2. 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 temperature (at 37° C. and at 20°C.),
atmosphere (aerobic and anaerobic), and light (exposure to and
protection from).

Note the conditions most favorable to pigment formation.

2. Note the solubility of the pigment in various solvents, such as water
(hot and cold), alcohol, ether, chloroform, benzol, carbon bisulphide.

3. Note the effect of acids and alkalies respectively upon the pigmented
cultivation, or upon solutions of the pigment.

4. Note spectroscopic reactions.


~7. Reducing Agent Formation.~--

(a) _Colour Destruction._--

1. Prepare tube cultivations in nutrient bouillon tinted with litmus,
rosolic acid, neutral red, and incubate.

2. Examine the cultures each day and note whether any colour change
occurs.

(b) _Nitrates to Nitrites._--

    _Medium Required_:

    Nitrate bouillon (_vide_ page 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 opinion 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 (glucose, lactose, etc.). The medium should be
     prepared from inosite-free bouillon (_vide_ page 183).

     _Reagent Required_:

     n/2 caustic soda.

METHOD.--

1. Inoculate the surface of the medium in the bulb of a fermentation
tube and incubate.

2. Mark the level of the fluid in the closed branch of the fermentation
tube, at intervals of twenty-four hours, and when the evolution of gas
has ceased, measure the length of the column of gas with the millimetre
scale.

Express this column of gas as a percentage of the entire length of the
closed branch.

3. To analyse the gas and to determine roughly the relative proportions
of CO_{2} and H_{2}, 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 H_{2}S will cause the yellowish-white precipitate
to darken to a brownish-black, or jet black, the depth of the colour
being proportionate to the amount of sulphuretted hydrogen present.

Quantitative: For exact quantitative analyses of the gases produced by
bacteria from certain media of definite composition, the methods devised
by Pakes must be employed, as follows:

[Illustration: FIG. 153.--Gas-collecting apparatus.]

     _Apparatus Required_:

     Bohemian flask (300 to 1500 c.c. capacity) containing from
     100 to 400 c.c. of the medium. The mouth of the flask is
     fitted with a perforated rubber stopper, 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 horizontal arm being 10 cm. in length. A
     three-way tap is let horizontally into the vertical tube
     just above its junction with the bell jar.

     An iron cylinder just large enough to contain the bell jar.

     About 15 kilos of metallic mercury.

     Melted paraffin.

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 CO_{2}, O_{2}, H_{2}, and N_{2}.

METHOD.--

1. 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 thoroughly flamed before and after the operation.

[Illustration: FIG. 154.--Orsat-Lunge gas analysis apparatus.]

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 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 communication 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.

11. 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 commencement 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 perforated rubber
stopper carrying an ~L~-shaped piece of manometer tubing, each arm 5 cm.
in length.

2. Prepare a second ~L~-shaped piece of tubing, the short arm 5 cm. and
the long arm 20 cm., and connect its short arm to the horizontal arm of
the tube in the culture flask by means of a length of pressure tubing,
provided with a screw clamp.

3. Fill the culture flask completely with boiling medium and pass the
long piece of tubing through the plug of an Erlenmeyer flask (150 c.c.
capacity) which contains 100 c.c. of the same medium.

4. Sterilise these coupled flasks by the discontinuous method, in the
usual manner.

Immediately the last sterilisation is completed, screw up the clamp on
the pressure tubing which connects 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 tubing to allow 4 or 5
c.c. of the culture to enter the flask.

7. Clamp the rubber tube tightly; remove the bent glass tube from the
culture tube and plunge it into a flask containing recently boiled and
quickly cooled distilled water.

8. Release the clamp again and wash in the remains of the cultivation
until the culture flask and tubing are completely filled with water.

9. Clamp the rubber tubing tightly and take away the long-armed glass
tubing.

10. Prepare the gas receiver as in the previous method (in this case,
however, the mercury should be warmed slightly) and fill the horizontal
arm of the receiver with hot water.

11. Connect up the culture flask with the horizontal arm of the gas
receiver.

12. Remove the screw clamp from the rubber tubing, adjust the three-way
tap, seal all joints with melted wax, and incubate.

13. Complete the investigation as described for the previous method.


BY PHYSICAL METHODS.

Examine cultivations of the organism with reference to 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 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 respectively. This method,
however, is not sufficiently accurate for the present purpose, and the
examination of an organism with respect to its behaviour in the absence
of oxygen is carried out as follows:

     _Apparatus Required:_

    Buchner's tubes.
    Bulloch's apparatus.
    Exhaust pump.
    Pyrogallic acid.
    Dekanormal caustic soda.

     _Media Required:_

    Glucose formate agar.
    Glucose formate gelatine.
    Glucose formate bouillon.

METHOD.--

1. Prepare four sets of cultivations:

(A) Sloped glucose formate agar, and incubate aerobically at 37° C.

Sloped glucose formate gelatine, and incubate aerobically at 20° C.

(B) Sloped glucose agar to incubate anaerobically at 37° C.

Sloped glucose formate gelatine to incubate anaerobically at 20° C.

(C) Sloped glucose formate agar to incubate anaerobically at 37° C.

Glucose formate bouillon to incubate anaerobically at 37° C.

(D) Sloped glucose formate gelatine to incubate anaerobically at 20° C.

Glucose formate bouillon to incubate anaerobically at 20° C.

2. Seal the cultures forming set B in Buchner's tubes (_vide_ page 239).

3. Seal the cultures forming set C in Bulloch's apparatus; exhaust the
air by means of a vacuum pump, and provide for the absorption of any
residual oxygen by the introduction of pyrogallic acid and caustic soda
in solution (_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 (SO_{2}, N_{2}O, NO,
      CO_{2}, 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 replaced 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 temperature.

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 apparatus each day. If no
growth is visible, incubate the tube under optimum conditions as to
temperature _and_ atmosphere, and in this way determine the length of
exposure to the action of the gas necessary to kill the organisms under
observation.

7. Control these results.

~II. Temperature.~--

(A) _Range._--

1. Prepare a series of ten tube cultivations, in fluid media, of optimum
reaction.

2. Arrange a series of incubators at fixed temperatures, 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 1° 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 _t. d. p._ here is that ~temperature~ which with certainty kills a
watery suspension of the organisms in question after an exposure of ~10
minutes~.

[Illustration: FIG. 155.--Hearson's water-bath.]

     _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 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.

METHOD.--

1. Prepare tube cultivations on solid media of optimum reaction;
incubate forty-eight hours under optimum conditions as to temperature
and atmosphere.

2. Examine preparations from the cultivation microscopically 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 suspension, 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.

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.

16. 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:

     (a) Thermometer to 120° C., its bulb immersed in the normal
     saline.

     (b) Straight entry tube, reaching to the bottom of the
     flask, the upper end plugged with cotton-wool.

     (c) 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.

     (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.

[Illustration: FIG. 156.--Apparatus arranged for the determination of
the death-point of spores.]

METHOD.--

1. Prepare twelve tube cultivations upon the surface (or two cultures in
large flat culture bottles--_vide_ page 5) of nutrient agar and
incubate under the optimum conditions (previously determined), for the
formation of spores.

Examine preparations from the cultures microscopically to determine the
presence of spores.

2. Pipette 5 c.c. sterile normal saline into each culture 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 centimeter of suspension that syphons
over (i. e., the contents 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.

     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 sterile 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, varying 5° C. between 60° C. and 120° C.

4. Remove each capsule from the oven with crucible tongs immediately
after 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.--

1. Prepare a sloped agar tube cultivation and incubate under optimum
conditions as to spore formations.

2. Pipette 5 c.c. sterile normal saline into the culture tube and
emulsify the entire surface growth in it. Examine 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 incubate, under
optimum conditions as to temperature and atmosphere, for twenty-four
hours.

2. Pipette 0.1 c.c. of the cultivation into a sterile 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, -30.

4. Inoculate each of the bouillon tubes with 0.1 c.c. of the diluted
cultivation by means of a sterile graduated 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 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, 1 instead of 5) for five points on either side
of the previously observed optimum. For example, the optimum reaction
observed in the set of experiments to determine the range was +10. Now
plant tubes having reactions of +15, +14, +13, +12, +11, +10, +9, +8,
+7, + 6, +5, and observe as before.


~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 (b). 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. 158).

METHOD.--

1. Prepare a surface cultivation on nutrient agar in a culture bottle
and incubate under optimum conditions for forty-eight hours.

2. Examine preparations from the cultivation, microscopically, 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.

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.

[Illustration: 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. Ten minutes later connect up the desiccator
to a sulphuric acid wash-bottle interposing an air filter so that only
dry sterile air enters.

[Illustration: FIG. 158.--Petri dish for drying cultivations.]

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. Reseal the
desiccator and again exhaust, and subsequently admit dry sterile air as
before.

8. Incubate the culture flask under optimum conditions 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:

1. Prepare a tube cultivation in nutrient bouillon, and incubate under
optimum conditions, for forty-eight hours.

[Illustration: 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 0.1 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).

5. Expose these plates to the action of diffuse daylight (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 observation until the end of
seven days.

8. Control the results.

(b) Direct Sunlight:

1. Prepare plate cultivations precisely as in the former experiments and
place the two controls in the incubators.

2. Arrange the remaining plates upon a platform in the direct rays of
the sun.

3. On the top of each plate stand a small glass dish 14 cm. in diameter
and 5 cm. deep.

4. Fill a solution of potash alum (2 per cent. in distilled water) into
each dish to the depth of 2 cm. to absorb the heat of the sun's rays and
so eliminate possible effects of temperature on the cultivations.

5. After exposures for periods similar to those employed 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 contains 2 per cent. of either of the
following aniline dyes, as may be necessary:

    Chrysoidin (for red).
    Malachite green (for green).
    Eosin, bluish (for blue).
    Methyl violet (for violet).

4. Expose the plates, thus prepared, to bright daylight (but not direct
sunlight) for varying periods, and complete the observations as in the
preceding experiments. 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
     laboratory 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 exposure_ necessary
to kill _vegetative forms_ of the bacterium suspended in water at 20° to
25° C, in which the disinfectant is present in _medium_ concentration
(concentration insufficient to cause plasmolysis). And if the bacterium
is one which forms spores,

(C) ~Superior lethal coefficient~--i. e., the _time exposure_ 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;
    Formaldehyde;
    Carbolic acid;

investigated with regard to B. anthracis, but the technique 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, 1 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 1 to 6. Inoculate each from
the stock cultivation of B. anthracis and at once add varying
quantities[10] of the carbolic acid solution, viz.:

    To tube 1 add 2.0 c.c. (= 1:100)
    To tube 2 add 1.0 c.c. (= 1:200)
    To tube 3 add 0.6 c.c. (= 1:300)
    To tube 4 add 0.5 c.c. (= 1:400)
    To tube 5 add 0.4 c.c. (= 1:500)
    To tube 6 add 0.2 c.c. (= 1:1,000)

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 0.1   (= 1:1,000)
    To tube  8 add 0.05  (= 1:2,000)
    To tube  9 add 0.03  (= 1:3,000)
    To tube 10 add 0.025 (= 1:4,000)
    To tube 11 add 0.02  (= 1:5,000)
    To tube 12 add 0.01  (= 1:10,000)


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 1.0   c.c. (= 1:1,000)
    To tube No. 14 add 0.4   c.c. (= 1:2,500)
    To tube No. 15 add 0.2   c.c. (= 1:5,000)
    To tube No. 16 add 0.1   c.c. (= 1:10,000)
    To tube No. 17 add 0.075 c.c. (= 1:15,000)
    To tube No. 18 add 0.05  c.c. (= 1:20,000)

4. Incubate all three sets of cultivations under optimum 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 subcultivations upon suitable
media, and ascertain that the organism causing the growth is the one
originally 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
     concentrated 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, 1 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
microscopically to determine the absence of spores.

2. Prepare solutions of different percentages of each disinfectant.

3. Make a series of hanging-drop preparations from the agar culture,
using a loopful of disinfectant solution 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[11] 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.~--

1. Prepare surface cultivations of the "test" organisms upon nutrient
agar in a culture bottle, and incubate 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 disinfectant determined in
the previous experiment, and complete the investigations as detailed
therein, steps 7 to 14, increasing the interval between planting the
subcultivations to two, three, or five hours if considered advisable.

     NOTE.--Where it is necessary to leave the organisms in
     contact with a strong solution of the disinfectant for
     lengthy periods, some means must be adopted to remove every
     trace of the disinfectant from the 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 germicidal 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 hundredth 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 neutralise 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 throughout the fluid, and transfer the suspension to
     a litre flask.

     4. Make up to a litre by the addition of sterile water;
     filter the suspension through a sterile porcelain candle.

     5. Emulsify the bacterial residue with 5 c.c. sterile
     bouillon.

     6. Prepare the necessary subcultivations from this emulsion.


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 incubate under optimum
conditions.

2. After seven days' incubation inject that amount of the culture
corresponding to 1 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 35° to 37° C.,

1. Prepare cultivations in nutrient broth and incubate 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 1 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 complete post-mortem
examination.

If death follows shortly after the injection of cultivations of
bacteria, the inoculation experiments should be repeated two or three
times. Then, if the organism under observation invariably exhibits
pathogenic 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 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. l. 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, observing the usual precautions against
contamination, and emulsify it evenly with the bouillon in the first
capsule. Each cubic centimetre of the emulsion will now contain
one-tenth of the organisms contained in the original loopful (written
shortly 0.1 loop).

4. Remove 0.1 c.c. of the emulsion in the first capsule 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 capsule to 10 c.c., and
therefore every cubic centimetre of bouillon in capsule II contains
0.001 loop.

5. Similarly, 0.1 c.c. of the mixture is transferred from capsule II to
capsule III (1 c.c. of bouillon in capsule III contains 0.00001 loop),
and then from capsule III to capsule IV (1 c.c. of bouillon in capsule
IV contains 0.0000001 loop).

The dilutions thus prepared may be summarised in a table;

Capsule   I = 1 loopful + 10 c.c. water             [.'.] 1 c.c.=0.1 loop.
Capsule  II = 0.1 c.c. capsule I + 9.9 c.c. water   [.'.] 1 c.c.=0.001 loop.
Capsule III = 0.1 c.c. capsule II + 9.9 c.c. water  [.'.] 1 c.c.=0.00001 loop.
Capsule  IV = 0.1 c.c. capsule III + 9.9 c.c. water
      [.'.] 1 c.c. = 0.0000001 loop.

6. With sterile graduated pipettes remove the necessary 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 1 c.c.

7. Multiples of the loop are prepared by emulsifying 1, 2, 5, or 10
loops each with 1 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 containing the smallest dose, then from
the capsule containing 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 0.0000001 loop to 0.01 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.

_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.

[Illustration: FIG. 160.--Apparatus arrange for toxin filtration.]

3. Inject intraveneously that amount of the cultivation corresponding to
1 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 examination.

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:

1. Filter the cultivation through a porcelain filter candle (Berkefeld)
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 corresponding to 1 per cent. of the body-weight
of each respectively, and observe, if necessary, until the completion 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 complete 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. l. d._ of a toxin is carried out on lines
similar to those laid down for living bacteria (_vide_ page 316) merely
substituting 1 c.c. of toxin as the unit in place of the unit "loopful"
of living culture.

It frequently happens, during the course of casual investigations 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 quantities of
fluid, is invaluable. This consists of a narrow filter flask just large
enough to accommodate an ordinary 18 × 2 cm. test-tube. The mouth of the
tubular Chamberland candle 15 × 1.5 cm. is closed by a perforated 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 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 inoculation of pure cultivations of the
organism into highly susceptible animals, and passing it as rapidly as
possible from animal to animal, always selecting that method of
inoculation-e. g., intraperitoneal--which places the organism under
the most favorable conditions for its growth and multiplication.

[Illustration: FIG. 161--Martin's filtering apparatus for small
quantities of fluid.]

2. _Virus Plus Virulent Organisms._--The inoculation of pure
cultivations of the organism together with pure cultivations of some
other microbe which in itself is sufficiently virulent to ensure the
death of the experimental animal, either into the same situation or into
some other part of the body. By this association the organism of low
virulence will frequently acquire a higher degree of virulence, which
may be still further raised by means of "passages" (_vide supra_).

3. _Virus Plus Toxins._--The inoculation of pure cultivations of the
organism into some selected situation, together with the subcutaneous,
intraperitoneal, or intravenous injection of a toxin--e. g., one of
those elaborated by the proteus group--either simultaneously with,
before, or immediately after, the injection of the feeble virus. By
this means the natural resistance of the animal is lowered, and the
organism inoculated is enabled to multiply and produce its pathogenic
effect, its virulence being subsequently exalted by means of "passages."

~Attenuating the Virulence of an Organism.~--Attenuating 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 unfavorable 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
remembered 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., 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 necessary).

     B. Combined Active and Passive Immunisation:

     IV. By the inoculation of toxin-antitoxin mixtures.


ACTIVE IMMUNISATION.

The immunisation of the rabbit against the Diplococcus pneumoniæ 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 experiments). 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 pneumoniæ.

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 37° 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 37° 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.

This will prevent evaporation, and cultures thus sealed will remain
unaltered in virulence for a considerable 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
316).

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 cultivation 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 pneumoniæ 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.

A slight local reaction will probably be observed as well as the weight
and temperature reactions.

11. 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
antibodies.

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 formation of antibody, specific to the diplococcus
pneumoniac, 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 experimental work a sufficient amount of any required antibody
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 experiment has been the preparation of agglutinin the
inoculations 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 injection 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 Hæmolysin, 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 hæmoglobin, 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 hæmolysin.

_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
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.

_Hæmolysin_ (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 hæmolysis.

Hæmolysin 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.

Hæmolysin 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 _hæmolytic
system_ (that is hæmolysin, corresponding erythrocyte solution and
complement) to certain laboratory methods having for their object either
the identification 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 hæmolytic 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 instead,
and although the following method is directed to the preparation of
human hæmolysin the same procedure serves in all cases.


THE PREPARATION OF HÆMOLYTIC SERUM.

_Apparatus Required:_

    Small centrifuge, preferably electrically driven, with two
      receptacles for tubes, and enclosed in a safety shield (Fig. 162).
    Sterile centrifuge tubes (10 c.c. capacity), Fig. 163.
    Sterile pipettes (10 c.c. graduated) in case.
    Sterile glass capsules (in case).
    Sterile test-tubes.
    Sterile all glass syringe (5 c.c. or 10 c.c. capacity)
      and needle.

[Illustration: FIG. 162.--Small electrical centrifuge.]

[Illustration: FIG. 163.--Centrifuge tube.]

_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 intraperitoneal 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.

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 followed, within a few
     minutes, by the death of the rabbit from pulmonary embolism.

9. Allow five days to elapse, then collect a preliminary sample of
blood, say about 2 c.c., from the rabbit's ear. Allow it to clot,
separate off the serum and transfer 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 hæmolytic properties in the
following manner:


THE TITRATION OF HÆMOLYTIC SERUM.

_Apparatus Required:_

    Electrical centrifuge.
    Sterile centrifuge tubes.
    Water-bath regulated at 56°C.
    Sterilised pipettes 10 c.c. graduated in tenths.
    Sterilised pipettes 1 c.c. graduated in tenths.
    Sterile test-tubes, 16 × 2 cm.
    Small sterile test-tubes, 9 × 1 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. Hæmolytic 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 1 and 2, and pipette into each 9
c.c. of normal saline solution.

2. Add 1 c.c. of hæmolytic rabbit serum to tube No. 1 and mix
thoroughly: take up 1 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 1 to 10.

    4. Pipette into tube No. 1 0.5 c.c. = 0.5 c.c.}
    hæmolytic serum                               }    From tube
    Pipette into tube No. 2 0.1 c.c. = 0.1 c.c.   }     H. S.
    hæmolytic serum                               }

    Pipette into tube No. 3 0.5 c.c. = 0.05 c.c.  }
    hæmolytic serum                               }
    Pipette into tube No. 4 0.3 c.c. = 0.03 c.c.  }
    hæmolytic serum                               }    From
    Pipette into tube No. 5 0.2 c.c. = 0.02 c.c.  }    tube 1.
    hæmolytic serum                               }
    pipette into tube No. 6 0.1 c.c. = 0.01 c.c.  }
    hæmolytic serum                               }

    Pipette into tube No. 7 0.5 c.c. = 0.005 c.c.  }
    hæmolytic serum                                }
    Pipette into tube No. 8 0.3 c.c. = 0.003 c.c.  }
    hæmolytic serum                                } From
    Pipette into tube No. 9 0.2 c.c. = 0.002 c.c.  } tube 2.
    hæmolytic serum                                }
    Pipette into tube No. 10 0.1 c.c. = 0.001 c.c. }
    hæmolytic serum                                }

5. To each tube add 1 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 0.1
c.c. complement (guinea-pig's serum) and replace tubes in incubator at
37° C. for further period of one hour.

9. Remove the tubes from the incubator, and if complete hæmolysis 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 hæmolysis is indicated by a clear red solution,
     with no deposit of red cells at the bottom of the test-tube.

     Absence of hæmolysis 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 hæmolytic serum that has caused complete
hæmolysis is known as the minimal hæmolytic dose (_M. H. D._) and if
hæmolysis has occurred in all the tubes down to No. 7--the m. h. d. of
this particular serum is .005 c.c. = 200 minimal hæmolytic doses per
cubic centimetre. Such a serum is strong enough for experimental work;
indeed, for many purposes, complete hæmolysis 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
hæmolysed, this is an indication that the rabbit should receive further
injections in order to raise the hæmolytic power to a sufficiently high
level.


STORAGE OF HÆMOLYSIN.

If, and when the hæmolysin 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 centrifuge 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 1 c.c., into
small glass ampoules or pipettes, and hermetically seal in the blowpipe
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 hæmolytic power.

Place the ampoules in a closed metal box and store in the ice chest for
future use.

FOOTNOTES:

[10] 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.

[11] See Percentage Formula, Appendix, page 496.




XVII. EXPERIMENTAL INOCULATION OF ANIMALS.


The use of living animals for inoculation experiments may become a
necessary procedure in the Bacteriological Laboratory for some one or
more of the following reasons:

A. ~Determination of Pathogenetic Properties of Bacteria 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 pneumococcus, blood agar, is
also an excellent pabulum for the 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, existing between certain
pathogenetic bacteria are in some cases so great as to completely
overwhelm the differences; 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 septicæmia, and
cultures from the rabbit's blood gave a pure growth of a typical
(capsulated) 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-à-vis with the infecting bacterium
that it becomes possible 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 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 immunity 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 Government, 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 permission to experiment
upon animals under general anæsthesia, provided that after the
experiment is completed the animal must be destroyed before regaining
consciousness. 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
anæsthesia must be obtained _in addition to the license_; whilst if the
inoculation entails more extensive operative procedures, 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
certificate removes the compulsion to destroy the animal whilst under
the anæsthetic. Further special certificates 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 laboratory inoculations are
subject to infection by various animal and vegetable parasites, and in
some instances such infection presents no symptoms which are obvious to
the casual examination; the sex should be noted, the weight recorded,
and the rectal temperature taken. The remaining items of importance are
the time of the inoculation, the material that is inoculated, 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 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).

[Illustration: Fig. 164.--Front of inoculation card.]

~Preliminary Inspection and Examination.~--The preliminary examination
should comprise observation 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 examination of the body and
limbs, and palpation of the groins and abdomen; and in many cases the
microscopical examination of fresh and stained blood-films.

Some of the commoner forms of naturally acquired infection may be
briefly mentioned, without however touching upon the various fleas, lice
and ticks which at times infect the ordinary laboratory animals.

[Illustration: FIG. 165.--Back of inoculation card.]

~The Rabbit~, particularly in captivity, is subject to 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. _cuniculi_) gives rise to the
ordinary 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 potassæ. 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, fæces, 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 containing 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.

Infection by _Coccidium oviforme_ is very common, without however
presenting any symptoms by which the infection may be recognised.
Usually the condition is only noted post-mortem, when the liver is found
to be studded with numerous cascating 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 _Tænia 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 frequently the animal house of a laboratory
is decimated by an infective septicæmia due to _B. cuniculicida_.

The ~Mouse~ and ~Rat~ suffer from septicæmia, and from the cysticercus form
of _Tænia 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 1 c.c. Old Tuberculin on arrival in the laboratory. The tissues of
the monkey also serve as the habitat for a Nematode worm parasitic in
cattle (_Oesophagostoma inflatum_) resembling the Anchylostomum, and
this parasite frequently bores through the intestinal wall, and
provokes the formation of small cysts in the immediately adjacent
mesentery. The presence of these cysts may give rise to considerable
speculation at the post-mortem.

The ~Pigeon~ may be infected by _Hæmosporidia_, 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
septicæmia--infections due to members of the hæmorrhagic septicæmia
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. 166). Mice and rats are weighed in a modification of
the letter balance, weighing to 250 grammes, which has a conical wire
cage, (carefully counterpoised) substituted for its original pan (Fig.
167).

[Illustration: FIG. 166.--Rabbit scales.]

~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, and then push it on gently and steadily
until the entire bulb and part of the stem, as far as the constriction,
have passed into the rectum. Three to five minutes later, the time
varying of course with the sensibility of the thermometer used, withdraw
the instrument and take the reading. The thermometers employed for
recording temperature should be verified from time to time by comparison
with a standard Kew certified Thermometer kept in the laboratory for
that purpose.

[Illustration: FIG. 167.--Mouse scales]

~Cages.~--During the period which elapses between inoculation and death,
or complete recovery, the experimental animals must be kept in suitable
receptacles which can easily be kept clean and readily disinfected.

The _mouse_ is usually stored in a glass jar (Fig. 168) 11 cm. high and
11 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 similar, but larger,
to that used for the mouse.

[Illustration: FIG. 168.--Mouse jar.]

[Illustration: 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 contamination 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 sides and top and bottom
are of woven wire work; beneath the cage is a movable metal tray filled
with sawdust, for the reception of the excreta. The cage as a whole is
raised from the ground on short legs. The sides, 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.

[Illustration: FIG. 170.--Metal rabbit rage.]

A plentiful supply of straw should be provided for bedding and the food
should consist of fresh vegetables, 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 autoclaving or spraying
with formalin.

As ~animal inoculation~ is purely a surgical operation, the necessary
instruments will be similar to those employed 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
carefully disinfected.

The list of apparatus used in animal inoculations given below comprises
practically everything needed for any inoculation. Needless to remark,
all the apparatus will never be required for any one inoculation.

[Illustration: FIG. 171.--Hypodermic syringe with finger rests.]

     Apparatus Required for Animal Inoculation:

     1. 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.

     2. Injection syringe. The best form is one of the ordinary
     hypodermic pattern, 1 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 accidental 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 after the disinfection is
     completed.

     Scissors, probe and sharp-pointed.

     Dissecting forceps of various patterns.

     Pressure forceps.

     Retractors (small self retaining Fig. 172).

     Aneurism needles, sharp and blunt.

     Scalpels, } Keratomes, } with metal handles. Trephines, }

     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 oesophageal bougies with connection to fit
     syringe.

[Illustration: FIG. 172. Small self retaining retractors.]

5. Anæsthetic.

(a) General: The safest general anæsthetic for animals is an A. C. E.
mixture, freshly prepared, containing by volume alcohol 1 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.

(b) Local:

    1. Cocaine hydrochloride, 2 per cent. in adrenalin 1 per mille
       solution.
    2. Beta-eucaine, 2 per cent. in adrenalin, 1 per mille solution.
    3. Ethyl chloride jet.

6. Sterile glass capsules of various sizes.

7. Cases of sterile pipettes { 10 c.c. (in tenths of a cubic centimetre).
                             {  1 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.

15. Razor.

16. Small pot of warm water.

17. Liquid soap. Liquid soap is prepared as follows: Measure out 100
grammes of soft soap and add to 500 c.c. of 2 per cent. lysol solution
in a large glass beaker; dissolve by heating in a water-bath at about
90° C. Bottle and label "Liquid Soap."

18. In place of the liquid soap and razor it is sometimes convenient to
use a Depilatory powder.

    Barium sulphide     1 part
    Rice starch         3 parts

Dust the powder thickly over the area to be denuded of hair, sprinkle
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 inoculated 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 excretions, solid
tissues).

~The Preparation of the Inoculum.~--

(a) _Cultivations in Fluid Media._--

1. Flame the plug of the culture tube.

2. Remove the plug and flame the mouth of the tube.

3. Slightly raise the lid of a sterile capsule, insert the mouth of the
culture tube into the aperture and pour some of the cultivation into the
capsule.

4. Remove the mouth of the culture tube from the capsule, replace the
lid of the latter, flame the mouth of the tube, and replug.

5. Remove the syringe from the steriliser, squirt out the water from its
interior, and allow to cool.

6. Raise the lid of the capsule sufficiently to admit the needle of the
syringe and draw the required amount of the cultivation into the barrel
of the syringe.

(Or, remove a definite measured quantity of the cultivation 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.)

[Illustration: FIG. 173.--Conical separatory funnel, fitted for
injection of fluid cultivations.]

If it is necessary to introduce a large bulk of fluid into the animal,
the cultivation should be transferred with aseptic precautions, to a
sterile separatory funnel, preferably of the shape shown in figure 173,
and graduated 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 operation 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
injection in.

(b) _Cultivations on Solid Media (e. g., Sloped Agar)._--

1. By means of a sterile graduated pipette introduce a suitable small
quantity of sterile bouillon (or sterile normal saline solution) into
the culture tube.

[Illustration: FIG. 174.--Arrangement of pressure injection apparatus.]

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.

(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, excretions, 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.

[Illustration: FIG. 175.--Holding rabbit for shaving.]

If it is desired to inoculate tissue _en masse_, remove from the
material a small cube of 1 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 anæsthetic 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 cylinder, one end of
which is turned over a ring of stout copper wire, and from this open end
a slot is cut extending about half way along one side of the cylinder.
The opposite end is closed by a "pull-off" cap and is perforated around
its edge by a row of ventilating holes, which correspond with holes cut
in the rim of the cap. In the event of the animal resisting attempts to
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.

[Illustration: FIG. 176.--Taking guinea-pig's temperature.]

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 be entirely of metal, and the wire cage detachable and
easily renewed.

[Illustration: FIG. 177.--Voge's holder.]

When the animal is anæsthetised, it is more convenient to secure it
firmly to some simple form of operating table, such as Tatin's (Fig.
179), which will accommodate rabbits, guinea-pigs, and rats: or to the
more elaborate table devised by the author (Fig. 180).

[Illustration: FIG. 178.--Mouse holder.]

[Illustration: FIG. 179.--Tatin's operation table.]

~Operation Table.~--This is a table of the "aseptic" type, composed of
steel tubing, nickel-plated or enamelled. The table-top frame is
sufficiently large to accommodate rabbits, dogs and monkeys; and is
supported 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
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, 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.

[Illustration: FIG. 180.--Author's operating table[12].]


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.~--(_Anæsthetic, none._)

1. Have the animal firmly held by an assistant (or secured to the
operating table).

2. Apply the liquid soap to the fur, over the area selected for
inoculation, with a wad of cotton-wool, and lather freely by the aid of
warm water; shave carefully and thoroughly; 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 incisions 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._--(_Anæsthetic, 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 hypodermic 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, unless large in amount, will be absorbed within a very
short time.

7. Label, etc.

(b) _Solid Inoculum.--(Anæsthetic, 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
underlying 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 introduce a small piece of
the inoculum into this pocket and deposit it as far as possible from the
point of entrance.

[Illustration: 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.
Withdraw 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.

~3. Intramuscular.~--

(a) _Fluid Inoculum.--(Anæsthetic, 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.

(b) _Solid Inoculum.--(Anæsthetic, A. C. E.)_

1. Secure the animal to the operation table and anæsthetise.

2. Shave and disinfect the skin at the seat of operation.

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.--(Anæsthetic, 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 thickness 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.

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).

[Illustration: FIG. 182.--Intraperitoneal inoculation--fluid.]

8. Inject the fluid from the syringe.

9. Label, etc.

[Illustration: FIG. 183.--Section of abdominal wall, etc., showing point
of needle lying free in the peritoneal cavity above the coils of
intestine.]

Second Method:

Steps 1-4. As in the first method.

5. Anæsthetise a small selected area of skin by spraying it with ethyl
chloride.

6. Heat platinum searing wire (0.5 mm. wire, twisted to the shape
indicated in figure 184, mounted in an aluminium handle) to redness, and
with it burn a hole through the anæsthetic 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.

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.

[Illustration: FIG. 184.--Platinum wire for burning hole through
parietes.]

(b) _Solid Inoculum_ (or the implantation of capsules containing fluid
cultivations).--(_Anæsthetic, A. C. E._)

1. Anæsthetise 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 sutures.

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.~

1. 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.

2. Dip the tube, with its coating of collodion, alternately into a
beaker of alcohol and one of water. This loosens the collodion and
allows it to be peeled off in the shape of a small test-tube.

3. Take a 20 cm. length of glass tubing, of about the diameter of the
test-tube used in forming the sac, and insert one end into the open
mouth of the sac.

4. Suspend the glass tube with attached sac, inside a larger test-tube,
by packing cotton-wool in the mouth of the test-tube around the glass
tubing, and place in the incubator at 37° C. for twenty-four hours. When
removed from the incubator, the sac will be firmly adherent to the
extremity of the glass tubing.

5. Plug the open end of the glass tubing with cotton-wool, and sterilise
the test-tube and its contents in the hot-air oven.

To use the sac, remove the plug from the glass tubing, partly fill the
sac with cultivation to be inoculated, by means of a sterile capillary
pipette, and replug the tubing. When the abdominal cavity has been
opened, remove the tubing and attached sac from the protecting
test-tube, close the sac by tying a sterilised silk thread tightly
around it a little below the end of the glass tubing, and separate it
from the tubing by cutting through the collodion above the ligature, and
the sac is ready for insertion in the peritoneal cavity.

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 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.

[Illustration: FIG. 185.--Making celloidin capsules.]

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
blowpipe 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 capsule, and the line of junction of the capsule 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.

7. Remove the solution of gelatine from 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 1 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.

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 experimental animal.

~5. Intracranial.~--(_Anæsthetic, A. C. E._)

[Illustration: FIG. 186.--Guarded trephine.]

_Trephines and Surgical Engine._--The most useful instrument for
intracranial operations upon animals is the small nasal trephine
(Curtis) having a tooth cutting circle of 7 mm. The addition of an
adjustable collar guard--secured by a screw--prevents accidental
laceration of the dura mater or brain substance[13] (Fig. 186). 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 electrical Dental engine[14] (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.



(a) _Subdural._

1. Anæsthetise the animal and secure it to the operating table, dorsum
uppermost.

2. Shave a portion of the scalp immediately in front of the ears.

[Illustration: FIG. 187.--Guy's electrical dental engine.]

3. Mark out with a sharp scalpel a crescentic flap 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 periosteum 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 intersection of the median line and a line joining the
posterior canthi (Fig. 188).

6. Introduce the inoculum by means of a hypodermic 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.

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 substance of one or other cerebral hemispheres
before the contents are ejected.

[Illustration: FIG. 188.--Intracranial inoculation of rabbit. The circle
indicates the situation of the trephine hole.]

~6. Intraocular.~--

(a) _Fluid Inoculum._--(_Anæsthetic, cocaine._)

1. Instil a few drops of a sterile solution of cocaine, and repeat the
instillation in two minutes.

2. Five minutes later have the animal firmly held by an assistant as in
intravenous 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
inoculum 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._--(_Anæsthetic, A. C. E._)

1. Anæsthetise the animal and secure it firmly to the operating table.

2. Irrigate the conjunctival sac thoroughly with sterile saline
solution.

3. Make an incision through the upper quadrant of the cornea into the
anterior chamber by means of a triangular keratome.

4. 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 cornea.

6. Release the animal from the operating table.

7. Label, etc.

~7. Intrapulmonary.~--

_Fluid Inoculum._--(_Anæsthetic, 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._--(_Anæsthetic, 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 anæsthetic 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
distended 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 position, 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 inoculum 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 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.

[Illustration: FIG. 189.--Intravenous inoculation.]

~9. Inhalation.~--

(a) _Fluid Inoculum._--(_Anæsthetic, 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 attached the bellows, spray the inoculum into
the interior of the box.

4. On the completion of the spraying, open the box, spray the animal
thoroughly with a 10 per cent. solution of formaldehyde (to destroy any
of the virus that may be adhering to fur or feathers).

5. Transfer the animal to its cage.

6. Label, etc.

7. Thoroughly disinfect the inhalation chamber.

(b) _Fluid or Powdered Inoculum._--_Anæsthetic, A. C. E._

1. Anæsthetise the animal and secure it firmly to the operating table.

[Illustration: 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 oesophageal
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 containing 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. 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 semi-fluid inoculum.
(Anæsthetic 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 English scale, or No.
18 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 centimetre 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)._

1. 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 to the left hand of an assistant who secures the animal's tail
between the fingers grasping the handle of the forceps. (See Fig. 191.)

[Illustration: FIG. 191.--Intragastric inoculation of rat.]

(b) _Mouse._--An assistant grasps the loose skin between the ears as far
forwards as possible between the forefinger and thumb of the left hand.
He now 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.

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 interference.

~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.

FOOTNOTES:

[12] This table is made by Messrs. Down Bros., St. Thomas's Street,
London, S. E.

[13] This modification is made for the author by Messrs. Down Bros., St.
Thomas's Street, London, S. E.

[14] Manufactured by Messrs. Francis Lepper, 56, Great Marlborough
Street, London, W.




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 successful
invasion of the tissues of the experimental animal by the
micro-organisms inoculated and by their multiplication 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 pathological 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 subcultivations 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 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 animal 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,
naturally 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 regarded as normal for
man (37.5° C.) is not the normal average temperature of any of the
lower animals save the rat and mouse. The accompanying table of normal
averages for the animals usually employed in bacteriological research
may be of use in preventing the erroneous assumption that pyrexia is
present in an animal, which merely shows its own normal temperature.

                  NORMAL AVERAGES.
----------------------------------------------------
                |  Rectal  | Pulse. | Respirations.
    Animal.     | Temp. °C.|------------------------
                |          |    Rate per minute.
----------------------------------------------------
                |          |        |
Frog            | 8.9-17.2 |   80   |      12
Mouse           |   37.4   |  120   |     ...
Rat             |   37.5   |  ...   |     210
Guinea pig      |   38.6   |  150   |      80
Rabbit          |   38.7   |  135   |      55
Cat             |   38.7   |  130   |      24
Dog             |   38.6   |   95   |      15
Goat            |   40.0   |   75   |      16
Ox              |   38.8   |   45   |      ..
Horse           |   37.9   |   38   |      11
Monkey (Rhesus) |   38.4   |  100   |      19
Pigeon          |   40.9   |  136   |      30
Fowl            |   41.6   |  140   |      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 examined 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 examined both
microscopically and culturally.

Fluid secretions and excretions, such as pus or serous exudates when
accessible are collected direct from the body in sterile capillary
pipettes (_vide_ Fig. 13a,) in the following manner:

1. Open the case containing the pipettes, grasp one by the plugged end,
remove it from the case, and replace the lid of the latter.

2. 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 13b), 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 cover-slip
preparations, 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
hæmatologists, some notes on the normal characters of the blood 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 HÆMOCYTOLOGY OF LABORATORY ANIMALS.

--------------------------------------------------------------------
       |       Totals    |                Percentages
       |------------------------------------------------------------
Animal |          |      | Hb, |Lympho-|Large |Poly- |Eosin-| Mast
       |Red cells |White | per | cytes,|monos,|morph,| oph, |cells,
       |          | cells|cent.|  per  | per  | per  | per  | 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 | 1.4  |  0.1
Rat    | 9,000,000| 9,000|  85 |   54  |  7.0 | 37.5 | 1.3  |  0.2
Guinea-|          |      |     |       |      |      |      |
  pig  | 5,700,000|10,000|  99 |   55  |  9.0 | 32.8 | 3.0  |  0.2
Rabbit | 6,000,000| 7,000|  70 |   50  |  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 | 1.25 |  0.75
Fowl   | 3,500,000|30,000| 100 |   49  |  3.0 | 42.0 | 1.0  |  5.0
Pigeon | 3,500,000|20,000| 101 |   43  |  9.0 | 43.0 | 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- |(20-30)| (4-8)|(55-  |(3-5) |(0.5-2)
limits.| millions.| thou-| 101)|       |      |  68) |      |
       |          |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 (20-25µ by 12-15µ)
discs, the nucleus relatively small and irregularly elongated or oval,
about 10µ in length. Many primitive and developing forms are usually
observed--also free nuclei and many cells in various stages of
degeneration. Hæmoglobin estimation is difficult owing to turbidity of
the blood after dilution with water. The _polymorphonuclear_ leucocytes
are large cells, about 20µ; 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 _polymorphonuclear_
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 basophilia of _red cells_ are
very commonly observed--nucleated red cells are also frequent. The large
_mononuclear_ 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 4.5µ
diameter, not much more than half that of the human red cell. The
_polymorphonuclear_ leucocytes have only a few very minute
coccal-shaped oxyphile granules, the nucleus is polymorphous. The
_eosinophile_ cells are large cells up to 20µ, the cytoplasm 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 12µ by 6µ, the
nucleus being relatively small (about 4µ 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 basophile 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 _polymorphonuclear_ leucocytes are rod-shaped, but smaller and finer
than in the fowl, and do not show clubbed appearances. 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:

1. 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 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 1 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 1 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 (see page 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 injected 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.

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           0.75 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 animals, 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 a state of purity
apart from the blood serum, consequently special methods have been
elaborated to permit of their recognition. In every instance the
behaviour 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 "normal"
serum. In view of minor differences in constitution 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 drop 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 hyperæmic and the vein prominent.

3. Remove the lysol with ether dropped from a drop bottle, and allow the
ether to evaporate.

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 end at the bunsen flame to expel some of the
contained air; then seal the clean point in the flame.

[Illustration: FIG. 192.--Collecting blood from rabbit.]

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.

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 graduated capillary pipettes to contain 10 c. mm. (Fig. 17).
Sterile graduated capillary pipettes to contain 90 c. mm. (Fig. 17).
Small sterile test-tubes 5 × 0.5 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.

METHOD.--

1. Take three sterile test-tubes and number them 1, 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 0.1 c.c. of the serum from the blood pipette tube, and mix it
thoroughly with the fluid in tube No. 1; and label ~s.s.~, (specific
serum), 10 per cent.

5. Remove 0.1 c.c. of the solution from tube No. 1 by means of a fresh
pipette, and mix it with the contents of tube No. 2; and label ~s.s.~, 1
per cent.

6. Remove 0.1 c.c. of the solution from tube No. 2 by means of a fresh
pipette, and mix it with the contents of tube No. 3; and label ~s.s.~, 0.1
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
      centrifugalisation.
    Capsule of diluting fluid--normal saline solution.
    Supply of Pasteur pipettes (Fig. 13a).
    India-rubber teats.
    Small test-tubes.
    A block of plasticine to act as a test-tube stand.
    Grease pencil.

METHOD:

1. Mark three small test-tubes 10 per cent., 1 per cent. and 0.1 per
cent. respectively, and stand them upright in the plasticine block.

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.

[Illustration: 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 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.

11. 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 repeatedly 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."

16. Eject the contents of this pipette into the second tube marked 1 per
cent. and mix thoroughly as before.

17. In similar fashion make the 0.1 per cent. solution and transfer to
the third tube.

18. Further dilutions in multiples of ten can be prepared 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 solution may be sufficient to entirely vitiate
     the subsequent observations--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 slide), 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, 1, and 0.1 per cent. respectively.

     Pipette end with pooled normal serum labelled ~p.s.~

METHOD.--

1. 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 1 per cent. serum;
label 0.5 per cent.

(e) One loopful bouillon culture + one loopful 0.1 per cent. serum;
label 0.05 per cent.

Mount these two cover-slips on a double-celled slide.

2. Note the time: Examine the control to 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 microscopically.

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
     hæmatocytometer (intended for the collection of the specimen
     of blood required for the enumeration of leucocytes), giving
     a dilution of 1 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 permit 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.

     Three test-tubes containing the 10, 1, and 0.1 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 17) 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 quantity 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 leucocytes. 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.

     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.

     3 × 1 slides.

     Piece of light rubber tubing.

     Rectangular 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
     opsonising 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 approximately 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) 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 distribute 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 centrifugalise 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 near to one end.

_Bacterial Emulsion._--

1. Take an 18- to 24-hour culture of the required bacterium (e. g.,
Diplococcus pneumoniæ) 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
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 treated as already described (see page
379), and the portions of the blood pipettes containing them are
arranged in the remaining space in plasticine block.

[Illustration: FIG. 194.--Plasticine block with materials arranged for
opsonin estimations.]

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).

[Illustration: FIG. 195. Opsonin pipette.]

3. Mix thoroughly on a 3 by 1 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 central portion of the capillary stem.

4. Seal the point of the pipette in the peep flame of the bunsen burner
and remove teat.

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 aspirate 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 15 minutes (or start the stop watch).

8. At the expiration of the incubation time remove the pipettes from the
incubator.

9. Cut off the sealed end of the specific serum preparation. Mix its
contents thoroughly as in step 3, and then divide the mixture between
two 3 by 1 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 minutes to fix.

Transfer to the second trough containing the diluted stain for ten
minutes.

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.

[Illustration: FIG. 196. Glass staining trough for blood films.]

13. Examine specific serum slide microscopically with 1/12 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
encountered is marked down. (The _average_ number of bacilli ingested
per leucocyte = the "_phagocytic index_.")

14. In precisely similar manner enumerate the bacteria present in the
first 50 cells of the control preparation. This number is recorded as
the denominator 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 particular 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 innocuous, and ultimately destroyed. The
presence of the immune body in the serum can be demonstrated _in vitro_
by the reaction elaborated by Bordet and Gengou, known as the complement
fixation test, the existence or the absence of the phenomenon of
complement fixation being rendered obvious macroscopically by the
absence or presence of hæmolysis on the subsequent addition of
"sensitised" red blood corpuscles, (e. g., a mixture of crythrocyte
solution and the appropriate hæmolysin--two of the three essentials in
the hæmolytic system, _vide_ page 326).


     _Apparatus Required:_

     Sterile pipettes 1 c.c., (graduated in tenths).

     16 × 2 cm. test-tubes.

     9 × 1 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.

     Hæmolytic 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.

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 sterilise by heating to 60° C. in a
water-bath for one hour.

METHOD.--

1. Take five small test-tubes, and number them 1 to 5 with a grease
pencil.

2. Into tubes Nos. 1, 3, 4 and 5 pipette 0.1 c.c. of complement.

3. Into tubes Nos. 1 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. 1, 2, 3 and 4 pipette 1 c.c. of the bacterial
emulsion which forms the antigen.

6. Place the whole set of tubes in the incubator at 37° C. for a period
of one hour.

7. Remove the tubes from the incubator and pipette 1 c.c. erythrocyte
solution and 4 minimal hæmolytic doses of the corresponding hæmolysin
into each tube.

8. Mix thoroughly and return the tubes to the incubator 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 hæmolysis; tube 2 should give no
evidence whatever of hæmolysis.

These tubes form the controls to the first tube, which contains the
serum to be tested.

In tube No. 1 the absence of hæmolysis 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 hæmolytic system; on the
other hand the presence of hæmolysis would show that no appreciable
amount of antibody has yet been formed in response to the inoculations.
In other words, there is an absence of infection, since the complement
remained unfixed at the time of the addition of the erythrocyte solution
and hæmolytic serum, and was ready to combine with those reagents to
complete the hæmolytic system.

The method may be shown diagramatically as under using the symbols
already indicated

                       Test-tubes.

     1              2               3               4               5

0.1 c.c. C.      ........       0.1 c.c. C.     0.1 c.c. C.    0.1 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.
--------------------------------------------------------------------------

1 c.c. E.       1. c.c. E.        1 c.c. E.        1 c.c. E.     1 c.c. E.

 H.S.^{4}         H.S.^{4}         H.S.^{4}         H.S.^{4}      H.S.^{4}
--------------------------------------------------------------------------
                 Incubate at 37° C. for one hour.
--------------------------------------------------------------------------
(?)            No hæmolysis.          |__________________________________|

                                                   Hæmolysis.

     NOTE.--It is sometimes more convenient to _sensitise_ the
     erythrocytes 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 hæmolytic doses of hæmolysin,
     add to the erythrocyte solution on the test-tube.

     3. Allow the erythrocyte and hæmolysin to remain in contact
     for fifteen minutes at room temperature. The red cells are
     then sensitised 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 1 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 exhaustive 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.

                           { Scalpels.
    Surgical instruments:  { Scissors.
                           { Forceps.
                           { 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.

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.

[Illustration: FIG. 197.--Apparatus for post-mortem examination, animal
on board.]

3. Drench the fur (or feathers) with lysol solution, 2 per cent. This
serves the twofold purpose of preventing 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.

[Illustration: FIG. 198.--Searing iron.]

4. Examine the cadaver carefully. Recollect that laboratory animals are
not always hardy; death may be 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 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 axillæ and groins, and reflect the skin
in two lateral flaps. (Place the now infected instruments 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 lesion is visible, sear
its exposed surface and with the platinum 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.

12. 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.

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 antibodies.


~Peritoneal Cavity.~--

16. Sear a broad track in the middle line of the abdominal 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.

18. 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 capsule. Later, sear the
surface of this organ; plunge the spear-headed spatula through the
centre of the seared area, twist it round between the finger and thumb,
and remove it from the organ. Sufficient material will be brought away
in the eye in its head to make cultivations. 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 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
     n/10 sodic hydrate solution using phenolphthalein as the
     indicator. The initial reaction is usually + 0.1 to + 0.5;
     add the calculated amount of n/10 sodic hydrate solution to
     neutralise.

25. Place a loopful of fresh desiccating fluid on a 3 × 1 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.~--

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 abdominal 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 scapulæ,
another at the level of the costal margin and the last across the upper
level of the pelvis. Reflect these flaps of skin.

31. 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
examination. 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.

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 specimens (_vide_ page 404).

35. Gather up all the infected instruments, return them to the
steriliser, and disinfect by boiling for ten minutes.

[Illustration: FIG. 199.--Spear-headed platinum spatula (actual size.)]

36. Sprinkle dry sawdust 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 for indicating the
position of the chief lesions in the cadaver of any of the laboratory
animals.

AUTOPSY CARD       Laboratory No. _________

Date ________

Animal ______  No. in Series ______ [Symbols: male female] Weight ________
+------------------------------------------------------------------------+
Died (or killed) _____ o'clock ____ m.  Autopsy made _____ o'clock ____ m.
+------------------------------------------------------------------------+
Notes on Post Mortem Examinations.

_General._

A. Seat of Inoculation.

B. Thoracic Cavity.

C. Abdominal Cavity.

D. Cranial Cavity.

+-------------------+---- -------------+--------------------------+
_Bacteriological_   |  _Histological_  |  _Organs Preserved._     |
    _Examination._  |  _Examination._  |                          |
A.                  |                  |                          |
                    |                  |                          |
B.                  |                  |                          |
                    |                  |                          |
C.                  |                  |                          |
                    |                  |                          |
D.                  |                  |                          |

[Illustration: FIG. 200.--Front of post-mortem card.]

41. Finally, the results of the action of the organism or organisms
isolated may be correlated with the symptoms observed during life and
the observations summarised under the following headings:

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    }   Acute
                      reactions.                     }    or
                  (b) Degeneration and necrosis.     }   chronic.

    2. General (i. e., produced at a distance from the bacteria, by
       absorption of toxins):

         (a) In special tissues--e. g., nerve cells and fibres, secreting
             cells, vessel walls, etc.

         (b) General effects of malnutrition, etc.

    Symptoms:

        (a) Associated with known tissue changes.

        (b) Without known tissue changes.

[Illustration: FIG. 201.--Back of post-mortem card.]


~Permanent Preparations--Museum Specimens.~--

_I. Tissues._--The naked-eye appearances of morbid tissues may be
preserved by the following method:

1. Remove the tissue or organ from the cadaver as soon after death as
possible, using great care to avoid distortion or injury.

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 carefully. When the
natural colours show in their original tints, average time three to six
hours, remove the tissues 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.

Equally good but much cheaper is Frost's mounting solution:

    Potassium acetate               160 grammes.
    Sodium fluoride                  80 grammes.
    Chloral hydrate                  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 1 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 searing iron).

11. Paint a narrow band of Japan black to finish off, round the joint,
overlapping on to the cover-plate.

_II. 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. diameter, 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 condensation 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 chamber after the lapse of a week,
and dry the exterior of each.

[Illustration: FIG. 202.--Bulloch's tubes.]

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 descriptions of the results of such observations.
He is, therefore, now entrusted with pure cultivations of the various
pathogenic bacteria, in order that he may study the life-history of each
and record the results of his own observations--to be subsequently
corrected or amplified by the demonstrator. In this way he is rendered
independent of text-book descriptions, the statements in which he is
otherwise too liable to take for granted, without personally attempting
to verify their accuracy.

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 following pages the grouping in use in the author's
elementary 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 necessary familiarity
with the cultural characters of important 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 pathogenic bacteria with
the Organisms of Suppuration. This is a large group, for all the
pathogenic bacteria possess the power, under certain conditions, of
initiating 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 rôle.

The organisms included in this group are:

    1. Staphylococcus pyogenes albus.
    2. Staphylococcus pyogenes aureus.
    3. Staphylococcus pyogenes citreus.
    4. Streptococcus pyogenes longus.
    5. Micrococcus tetragenus.
    6. Bacillus pyocyaneus.
    7. Bacillus pneumoniæ.

and in certain special tissues

    8. Micrococcus gonorrhoeæ.
    9. Micrococcus intracellularis meningitidis (Meningococcus).
    10. Micrococcus catarrhalis.
    11. Bacillus ægypticus (Koch-Weeks Bacillus).

The group may with advantage be subdivided as indicated in the following
pages:

I. _Pyogenic cocci._

    Staphylococcus pyogenes albus.
    Staphylococcus pyogenes aureus.
    Staphylococcus pyogenes citreus.
        to contrast with
    Micrococcus candicans.
    Micrococcus agilis.

1. Prepare subcultivations from each:

    Bouillon,         }
    Agar streak,      }
    Blood serum,      }
    Litmus milk.      }  and incubate at 37°C.
    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' incubation. 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 Pitfield'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 prepared from the three days' growth--such cocci are dead.

5. Stain section of kidney tissue provided (showing abscess formation
by Staphylococcus aureus) by Gram's method, and counterstain with cosin.

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 cosin.

7. Inoculate[15] 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. _Pyogenic cocci._

    Micrococcus gonorrhoeæ.
    Micrococcus intracellularis meningitidis (meningococcus).
    Micrococcus catarrhalis.
    Micrococcus tetragenus.
    Micrococcus paratetragenus.

III. _Pyogenic cocci._

    Streptococcus pyogenes longus.
    Streptococcus of bovine mastitis.
    Streptococcus lanceolatus (Diplococcus pneumoniæ or pneumococcus).
        to contrast with
    Streptococcus brevis.
    Streptococcus lebensis.

IV. _Pyogenic bacilli._

    Bacillus pneumoniæ (Friedlaender).
    Bacillus rhinoscleromatis.
    Bacillus lactis aerogenes.

V. _Pyogenic bacilli._

     Bacillus pyocyaneus.
        to contrast with
    Bacillus fluorescens liquefaciens.
    Bacillus fluorescens non-liquefaciens.

VI. _Pneumonia group._

    Streptococcus lanceolatus (pneumococcus).
    Bacillus pneumoniæ (Friedlaender).
    Streptococcus pyogenes longus.

VII. _Diphtheroid group._

    Bacillus diphtheriæ (Klebs-Loeffler).
    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. cloacæ.

X. _Gaertner group._

    Bacillus enteritidis (Gaertner).
    B. paratyphosus A.
    B. paratyphosus B.
    Bacillus choleræ suum (Hog Cholera).
    B. psittacosis.

XI. _Eberth group._

    B. typhosus (Eberth).
    B. dysenteriæ (Shiga).
    B. dysenteriæ (Flexner).
    B. fæcalis alcaligines.

XII. _Spirillum group._

    Vibrio choleræ.
    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 Rabinowitch.

XV. _Plague group._

    Bacillus pestis.
    B. septicæmiæ hæmorrhagicæ.
    B. suipestifer.

XVI. _Influenzæ group._

    B. influenzæ.
    Bacillus ægypticus (Koch-Weeks).
    Bacillus pertussis.

XVII. _Miscellaneous._

    Bacillus lepræ.
    Bacillus mallei.
    Micrococcus melitensis.

XVIII. _Streptothrix group._

    Streptothrix actinomycotica.
    Streptothrix maduræ.
        to contrast with
    Cladothrix nivea.

XIX. _Tetanus group._

    Bacillus tetani.
    Bacillus oedematis maligni.
    Bacillus chauvei (symptomatic anthrax).

XX. _Enteritidis sporogenes group._

    Bacillus enteritidis sporogenes.
    B. botulinus.
    B. butyricus.
    B. cadaveris.

FOOTNOTES:

[15] See note on Vivisection License, page 334.




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
calculated 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 technique there set out will be found to be
capable of expansion and adaptation to any circumstance or set of
circumstances which may confront the student.

~Controls.~--The necessity for the existence of adequate 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 presumably
"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 uninoculated
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 excremental
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 torulæ, and
the development of these will be best secured by plating in wort
gelatine and incubating 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 overhanging glass stoppers (to prevent contamination of
the bottle necks by falling dust). These must be carefully sterilised in
the hot-air steriliser (_vide_ page 31).

(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~, 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.

[Illustration: FIG. 203.--Esmarch's collecting bottle for water
samples.]

(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
designed 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 pull on the cord is released and the tension 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 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.

[Illustration: FIG. 204.--Thresh's deep water sampling bottle.]

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.
diameter (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 material 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 is no pull upon
the rubber tube, but which, however, will be easily jerked off when a
sharp pull is given to the suspending cord.

Now wind heavy lead tubing about 1 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 suspending cord, which detaches the rubber
tube and so opens the two glass tubes. Water enters through the 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.

[Illustration: FIG. 205.--Ice-box for transmission of water samples,
etc.]

_Transport of Sample._--If the examination of the sample cannot be
commenced immediately, steps must be taken to prevent the multiplication
of the bacteria contained 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 essential. 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 bolted
together by thumb screws through the overhanging 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 examination consists in adding
measured quantities of the water sample to several tubes of nutrient
media previously liquefied by heat, pouring plate cultivations from each
of these tubes, incubating at a suitable temperature, 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, 1 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, 1 to 6; the gelatine tubes,
consecutively, 1 to 6, and the wort tubes, 1, 2, and 3. Flame the plugs
and see that they are not adherent to the lips of the tubes.

3. Place the agar tubes in boiling water until the medium is melted,
then transfer them to the water-bath regulated at 42° C. Liquefy the
nutrient gelatine and wort gelatine tubes by immersing them in the same
water-bath.

4. Remove the bottle containing the water sample from the ice-box,
distribute the bacterial contents evenly throughout the water by
shaking, cut the string securing the stopper, and loosen the stopper,
but do not take it out.

[Illustration: FIG. 206.--Withdrawing water from water sample bottle.]

5. Remove one of the 1 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, holding its point well
below the surface of the water (Fig. 206). Suck up rather more than 1
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 1 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 1 c.c. of the sample to the contents of the
fourth tube.

9. Similarly, add 0.5 c.c. and 0.1 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
quantity of water sample it contains, and (c) the date.

12. 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 apparatus 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.

16. Add 0.1 c.c. of the water sample to the 9.9 c.c. sterile water in
the capsule. This will give a dilution of 1 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 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 Jeffery's or Pakes' 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 Jeffery's counter (Fig. 207), each subdivision has
an area of 1 square centimetre; in Pakes' counter (Fig. 208), radii
divide the circle into sixteen equal sectors, and counting is
facilitated by concentric circles equidistant from the centre.

[Illustration: FIG. 207.--Jeffery's disc, reduced.]

[Illustration: FIG. 208.--Pakes' disc, reduced.]

(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; Jeffery's disc for the latter. It should
be recollected however that unless the plates have been carefully
leveled 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 0.1 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 1 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.

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 110 c.c. It is collected 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 indicative 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) necessitates 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 members of the coli-typhoid group; and on occasion the
question of the presence or absence of Vibrio choleræ or (more rarely)
such bacteria as B. anthracis or B. tetani, may need investigation.

When pathogenic or excremental bacteria are present 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-pathogenic 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 concentrated 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
quantity of sterile bouillon.

A. ~Enrichment Method.~

(Dealing with the demonstration of bacteria of intestinal origin.)

_Apparatus Required_ (_Preliminary Stage_):

    Incubator running at 42° C.
    Case of sterile pipettes, 1 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).
    Aluminium 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
1a-5a.

2. Number one flask 7 and another 8.

3. To Tubes No. 1 and 1a add 0.1 c.c. water sample.

To Tubes No. 2 and 2a add 1 c.c. water sample.

To Tubes No. 3 and 3a 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 incubate 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 multiplication of intestinal
bacteria, and also their fermentative activity. The temperature 42° C.
destroys ordinary 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.

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 hanging-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 macroscopically 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. Practice 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.

16. 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 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.
    Lævulose 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. cloacæ.

II. The Gærtner Group.

    Bacillus enteritidis (of Gærtner).
    B. paratyphosus A.
    B. paratyphosus B.
    Bacillus choleræ suum.

III. The Eberth Group.

    B. typhosus.
    B. dysenteriæ (Shiga).
    B. dysenteriæ (Flexner).
    B. fæcalis 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 0.1 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 differential details are set out in tabular form.

ANALYTICAL SCHEME FOR ISOLATION OF MEMBERS OF THE COLI AND TYPHOID
GROUPS.

                           Nutrose agar.
                                  |
               -----------------------------------
               |                                  |
         Red colonies.                       Blue colonies.
        Escherich group.                   Gaertner and Eberth groups.
               ||                                 |
               ====================---------------
                                  ||
                        Lactose peptone solution.
                                  ||
               ====================---------------
               ||                                |
              Gas.                            No gas.
               ||                                |
B. coli communis and its allies.                 |
               ||                   Gaertner and Eberth groups.
Acid and gas in glucose peptone solution.        |
Acid and coagulation in milk.                    |
General turbidity and indol in bouillon.   Glucose peptone solution.
                                                 |
               ==================================|
               ||                                |
               ||                                |
              Gas.                            No gas.
               ||                                |
          Gaertner group.                  Eberth group.
               ||                                |
      ===================                ----------------
      ||                ||               |               |
      ||                ||               |               |
Litmus milk.   Peptone solution.   Litmus milk.     Peptone solution.
      ||                ||               |               |
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 16.

20. Differentiate the streptococci isolated into members of the
saprophytic group of short-chained cocci, or members of the parasitic
(pathogenic) group of long-chained cocci, by means of their cultural
characters, and record their numerical frequency in the manner indicated
for the members of the coli-typhoid group.

DIFFERENTIAL TABLE OF COLI-TYPHOID GROUP

Transcriber's note: Table split to fit 80 spaces.

+-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+
|                         | M | D   | L   | G   | M   | L   | S   | R   | D   |
|A = acid reaction        | o | e   | æ   | a   | a   | a   | a   | a   | e   |
|G = gas formation        | t | x   | v   | l   | l   | c   | c   | f   | t   |
|                         | i | t   | u   | a   | t   | t   | c   | f   | r   |
|                         | l | r   | l   | c   | o   | o   | h   | i   | i   |
|                         | i | o   | o   | t   | s   | s   | a   | n   | n   |
|                         | t | s   | s   | o   | e   | e   | r   | o   |     |
|                         | y | e   | e   | s   |     |     | o   | s   |     |
|                         |   |     |     | e   |     |     | s   | e   |     |
|                         |   |     |     |     |     |     | e   |     |     |
|                         |   +-----+-----+-----+-----+-----+-----+-----+-----+
|                         |   | A G | A G | A G | A G | A G | A G | A G | A G |
+-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+
|_The Escherich Group._   |   |     |     |     |     |     |     |     |     |
| B. coli communis        | + | + + | + + | + + | + + | + + |  O  | + + | + + |
| B. coli communior       | + | + + | + + | + + | + + | + + | + + | + + | + + |
| B. lactis aerogenes     | - | + + | + + | + + | + + | + + |  O  |  O  | + + |
| B. acidi lactici        | - | + + | + + | + + | + + | + + |  O  |  O  |  O  |
| B. pneumoniæ            | - | + + | + + | + + | + + | + + | + + | + + | + + |
| B cloaceæ(A)            | + | + + | + + | + + | + + | + + | + + | + + | + + |
|                         |   |     |     |     |     |     |     |     |     |
|_The Gærtner Group._     |   |     |     |     |     |     |     |     |     |
| B. enteritidis          | + | + + | + + | + + | + + |  O  |  O  |  O  |  O  |
| B. paratyphosus A       | + | + + | + + | + + | + + |  O  |  O  |  O  |  O  |
| B. paratyphosus B       | + | + + | + + | + + | + + |  O  |  O  |  O  |  O  |
| B. choleræ suum         | + | + + | + + | + + | + + |  O  |  O  |     |  O  |
| B. suipestifer          | + | + + | + + | + + | + + |  O  |  O  |     |  O  |
|                         |   |     |     |     |     |     |     |     |     |
|_The Eberth Group._      |   |     |     |     |     |     |     |     |     |
| B. typhosus             | + | +   | +   | +   | +   | O   | O   | O   | +   |
| B. dysenteriæ (Shiga)   | - | +   | +   | +   | O   | O   | O   | O   | O   |
| B. dysenteriæ (Flexner) | - | +   | +   | +   | +   | O   | O   | ±   | O   |
| B. fæcalis alkaligines  | + | O   | O   | O   | O   | O   | O   | O   | O   |
|                         |   |     |     |     |     |     |     |     |     |
+-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+
| Table Notes:            |(B)| (C)                                           |
+-------------------------+---+-----------------------------------------------+

+-------------------------+-----+-----+-----+-----+-----+-----+---+-----------+
|                         | I   | S   | G   | D   | M   | S   | I |Litmus Milk|
|A=acid reaction          | n   | a   | l   | u   | a   | o   | n |           |
|G=gas formation          | u   | l   | y   | l   | n   | r   | d |           |
|                         | l   | i   | c   | c   | n   | b   | o +-----+-----+
|                         | i   | c   | e   | i   | i   | i   | l |Early|Late |
|                         | n   | i   | r   | t   | t   | t   |   |     |     |
|                         |     | n   | i   | e   | e   | e   |   |     |     |
|                         |     |     | n   |     |     |     |   |     |     |
|                         |     |     |     |     |     |     |   |     |     |
|                         |     |     |     |     |     |     |   |     |     |
|                         |-----+-----+-----+-----+-----+-----+   |     |     |
|                         | A G | A G | A G | A G | A G | A G |   |     |     |
+-------------------------+-----+-----+-----+-----+-----+-----+---+-----+-----+
|_The Escherich Group_    |     |     |     |     |     |     |   |     |     |
| B. coli communis        |  O  |  O  | + + | + + | + + | + + | + | +   | + C |
| B. coli communior       |  O  |  O  | + + | + + | + + | + + | + | +   | + C |
| B. lactis aerogenes     |  O  |  O  |  O  |  O  | + + | + + | - | +   | + C |
| B. acidi lactici        |  O  |  O  |  O  | + + | + + | + + | + | +   | + C |
| B. pneumoniæ            |  O  |  O  | + + | + + | + + | + + | - | +   | + C |
| B cloaceæ[A]            |  O  |  O  | + + |  O  | + + | - + | + | +   | + C |
|                         |     |     |     |     |     |     |   |     |     |
|_The Gærtner Group._     |     |     |     |     |     |     |   |     |     |
| B. enteritidis          |  O  |  O  |  O  | + + | + + | + + | - | ±   | -   |
| B. paratyphosus A       |  O  |  ±  |  O  | + + | + + | + + | - | +   | O   |
| B. paratyphosus B       |  O  |  O  |  O  | + + | + + | + + | - | +   | -   |
| B. choleræ suum         |  O  |  O  |  O  |  O  |  O  | + + | ± | +   | -   |
| B. suipestifer          |  O  |  O  |  O  | + + | + + | + + | - | +   | -   |
|                         |     |     |     |     |     |     |   |     |     |
|_The Eberth Group._      |     |     |     |     |     |     |   |     |     |
| B. typhosus             | O   | O   | O   | O   | +   | +   | - | +   | +   |
| B. dysenteriæ (Shiga)   | O   | O   | O   | O   | O   | O   | - | +   | -   |
| B. dysenteriæ (Flexner) | O   | O   | O   | O   | +   | O   | ± | +   | -   |
| B. fæcalis alkaligines  | O   | O   | O   | O   | O   | O   | - | -   | -   |
|                         |     |     |     |     |     |     |   |     |     |
+-------------------------+-----+-----+-----+-----+-----+-----+---+-----+-----+
| Table Notes:            |                                   |(D)| (E)       |
+-------------------------+-----------------------------------+---+-----------+

Table Notes:

(A) * Liquefies gelatine.

(B) + = motile. - = non-motile.

(C) + = acid or gas production. ± = slight acid production. O = no
change.

(D) + = indol production. ± = slight indol production. - = no indol
formed.

(E) + = acid production. - = alkali production. O = no change in
reaction. C = clot.

21. Determine the pathogenicity for mice (subcutaneous inoculation) and
rabbits (intravenous inoculation) 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 reception is an ordinary blue glass Winchester quart bottle,
sterilised in the hot-air oven, and over this a paper or parchment cap
fastened with string. The bottle may be packed in a wooden box or in an
ordinary wicker case. The method of collecting the sample is identical
with that described under the heading of Quantitative Examination; there
is, however, not the same imperative 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 32 cm. in length, the other 52 cm., the shorter arm
     being plugged with cotton-wool. The rubber stopper must be
     sterilised by boiling and the glass tubing by hot air,
     before use.

     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, 1 c.c. in tenths.

     Case of sterile pipettes, 1 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.

[Illustration: Sample form]]

[Illustration: 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.]

METHOD.--

1. Fit up the filtering apparatus as in the accompanying diagram (Fig.
209), interposing the wash-bottle with sulphuric acid between the
filter flask and the force-pump (in the position occupied in the diagram
by the central vertical line), and placing another screw clamp on the
rubber tubing connecting the lateral arm of the filter flask with the
wash-bottle.

[Illustration: FIG. 210. Sterile test-tube brush.]

2. Filter the entire 2000 c.c. of water through the filter candle.

3. When the nitration 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) emulsify the slimy
residue which lines the 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 1 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.

The Coli-Typhoid Group.--

1. Number nine tubes of bile salt broth (_vide_ page 180), consecutively
from 1 to 9.

2. To No 1 add 1 c.c. } of the original water sample
         2 add 2 c.c. } before the nitration is commenced.
         3 add 5 c.c. }

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   1.0   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 18).

     _Alternative Methods._--

     A few of the older methods for the isolation of the members
     of the coli-typhoid groups are referred 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 1 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. Subcultivate from any colonies that make their
     appearance, and determine their identity on the lines laid
     down in the previous method.

     (B) Parietti's Method:

     1. Take nine tubes of Parietti's bouillon (_vide_ page
     202)--i. e., three each of those containing 0.1 c.c., 0.2
     c.c., and 0.5 c.c. 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) Elsner's Method: This method simply consists in
     substituting Elsner'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 organisms (_vide_ page 257), and expose to
a temperature of 80° C. for twenty minutes.

3. Number ten tubes of litmus milk consecutively from 1 to 10.

4. Remove the test-tube from the benzole bath and shake well to
distribute the spores evenly through the fluid.

5. To each tube of litmus milk add a measured quantity 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 apparatus. If, however, whole milk has been used in making the
litmus milk the layer of cream that rises to the surface 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 bubbles in the
coagulum, and possibly masses of coagulum driven up the tube almost to
the plug.

8. Replace the tubes which show no signs of growth in the incubator for
a further period of twenty-four 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 Choleræ.~--

1. Number ten tubes of peptone water consecutively from 1 to 10.

2. To each of the tubes of peptone water add a measured quantity of the
suspension, corresponding to those amounts employed in isolating the
members of the coli group (_vide_ page 437).

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 pellicle itself--and incubate at
37° C. for twenty-four hours.

5. Test the peptone water itself for the presence of indol and nitrite
by the addition of pure concentrated H_{2}SO_{4}.

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
choleræ and subcultivate upon all the ordinary laboratory media.

7. Test the vibrio isolated against the serum of an animal immunised to
the Vibrio choleræ 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 organisms (_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 1 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 1, 2, and 3. To No. 1 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.

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 1 c.c. sterile bouillon. Observe
during life, and if the animal succumbs, make a complete post-mortem
examination.


~B. Tetani.~--

1. Proceed as detailed above in steps 1 and 2 for the isolation of the
B. anthracis.

2. Add 1 c.c. of the suspension to each of three tubes of glucose
formate broth, and incubate anaerobically in Buchner's tubes at 37° C.

3. From such of the tubes as show visible growth (with or without the
production of gas) after twenty-four hours' incubation inoculate
guinea-pigs, subcutaneously (under the skin of the abdomen), using 0.1
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 incubate anaerobically in
Bulloch's apparatus, at 37° C.

5. Subcultivate suspicious colonies on the various media, incubate
anaerobically, making control cultivations 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 (a) fore-milk, (b) mid-milk, (c) strippings, and, if possible,
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
examination 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 micro-organisms 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 1 c.c. of a 1 per cent. aqueous
solution of ortol (ortho-methyl-amino-phenol sulphate), recently
prepared 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 junction of the
two liquids = formaldehyde.

4. _Hydrogen Peroxide._

To 10 c.c. milk (diluted with equal quantities of water) in a test 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 1 per cent. aqueous solution 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 1 per cent. alcoholic solution of rosolic acid. _Brownish_
color = pure milk; _rose_ color = preserved milk.

[Illustration: FIG. 211.--Milk-collecting bottle and dipper in case.]

Quantitative.--

_Collection of Sample._--

The apparatus used for the collection of a retail mixed milk sample
consists of a cylindrical copper case, 16 cm. high and 9 cm. in
diameter, provided with a "pull-off" lid, containing a milk dipper, also
made of copper; and inside this, again, a wide-mouthed, stoppered 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 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 identification 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, 1 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.--

1. Arrange four sterile capsules in a row; number them I, II, III, and
IV.

2. Fill 9 c.c. sterile bouillon into the first, and 9.9 c.c. bouillon
into each of the three remaining capsules.

3. Remove 1 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 0.1 c.c. of the milky bouillon from capsule I, add it to the
contents of capsule II, and mix as before.

5. In like manner add 0.1 c.c. of the contents of capsule II to capsule
III; and then 0.1 c.c. of the contents of capsule III to capsule IV.

    Then 1 c.c. of dilution   I contains 0.1       c.c. milk sample.
         1 c.c. of dilution  II contains 0.001     c.c. milk sample.
         1 c.c. of dilution III contains 0.00001   c.c. milk sample.
         1 c.c. of dilution  IV contains 0.0000001 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 1 to 12.

8. Inoculate the tubes with varying quantities of the material as
follows:

    To tube No.  1 add 1.0 c.c. of the milk sample.
                 2 add 0.1 c.c. of the milk sample.
              {  3 add 1.0 c.c. from capsule I.
              {  4 add 0.1 c.c. from capsule I.
              {  5 add 1.0 c.c. from capsule II.
              {  6 add 0.1 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 0.5 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 1.0 c.c. of the
milk sample and a similar quantity of the diluted milk from capsules I,
II, and III and IV respectively.

11. Pour plates from the wort gelatine; label, and incubate at 20° C.

12. Inoculate the liquefied agar tubes as follows:

    To tube No. 1 add 0.1 c.c. of the milk sample.
                2 add 0.1 c.c. from capsule I.
                3 add 0.1 c.c. from capsule II.
                4 add 0.1 c.c. from capsule III.
                5 add 1.0 c.c. from capsule IV.  }
                6 add 0.1 c.c. from capsule IV.  }

13. Pour plates from the agar tubes; label, and incubate 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.

16. 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 examination 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 frequency.

    Members of the Coli-typhoid group.
    Vibrio choleræ.
    Streptococcus pyogenes longus.
    Micrococcus melitensis.
    Staphylococcus pyogenes aureus.
    Bacillus enteritidis sporogenes.
    Bacillus diphtheriæ.
    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 intestinal origin, the latter for the isolation of the
micro-organisms of diphtheria and tubercle. The first essential 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 considerable period of
     time. In other words, the machine should run down very
     gradually and slowly after the motive power is removed, thus
     obviating any disturbance of the relative positions of
     particulate matter in the solution that is being
     centrifugalised.

     4. The machine should preferably be driven by 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.

     (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.

[Illustration: 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).

     Case of sterile pipettes, 1 c.c. (in tenths of a cubic
     centimetre).

     Sterile teat pipettes.

     Flask of sterile normal saline solution.

METHOD.--

1. 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
     introduced into the buckets of the centrifuge before the
     glass tubes are inserted will obviate any risk of breakage
     to the latter.

[Illustration: FIG. 213.--Milk sedimenting tubes.]

[Illustration: 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 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, fæcal débris, 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 C^{1} and C^{2}.

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 D^{1}
and D^{2}.

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 (C^{1}) and deposit (D^{1}) microscopically--

(a) In hanging-drop preparations.

(b) In film preparations stained carbolic methylene-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.

11. 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 centrifugal tube (D^{2}) with 10
c.c. sterile bouillon and inoculate three tubes of bile salt broth as
follows:

    To Tube No. 1 add 2.5 c.c. milk deposit emulsion
      (=25 c.c. original milk.)
    To Tube No. 2 add 1.0 c.c. milk deposit emulsion
      (=10 c.c. original milk.)
    To Tube No. 3 add 0.5 c.c. milk deposit emulsion
      (= 5 c.c. original milk.)

2. Inoculate tube of bile salt broth No. 4 with 1 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 1.0 c.c. from capsule I.
    To tube No.  6 add 0.1 c.c. from capsule I.
    To tube No.  7 add 1.0 c.c. from capsule II.
    To tube No.  8 add 0.1 c.c. from capsule II.
    To tube No.  9 add 1.0 c.c. from capsule III.
    To tube No. 10 add 0.1 c.c. from capsule III.
    To tube No. 11 add 1.0 c.c. from capsule IV.
    To tube No. 12 add 0.1 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 examination (see pages 428 to 431).

     NOTE.--The B. coli communis, derived from the alvine
     discharges of the cow, is almost universally present in
     large or small numbers, in retail milk. Its detection,
     therefore, unless in enormous numbers, (when it indicates
     want of cleanliness), is of little value.

~2. Vibrio Choleræ.~--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 examination
as detailed under the corresponding section of water examination (see
page 439).

~3. B. Enteritidis Sporogenes.~--Inoculate tubes of litmus milk with
similar amounts to those used in the previous searches, omitting tube
No. 1 (_vide ante_ 1-3) place in the differential steriliser at 80° C.
for ten minutes and then incubate anaerobically at 37° 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. Diphtheriæ.~--

(A) 1. Plant three sets of serial cultivations, twelve tubes in each
set, from (a) cream C^{2}, (b) deposit D^{1} upon oblique
inspissated blood-serum, and incubate at 37° 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, Neisser's method, Gram's method. Subcultivate
such as appear to be composed of diphtheria bacilli in glucose peptone
solution. Note those in which acid production takes place.

5. Inoculate guinea-pigs subcutaneously with one or two cubic
centimetres forty-eight-hour-old glucose bouillon cultivation derived
from the first subcultivation of each glucose fermenter, and observe the
result.

6. If death, apparently from diphtheritic toxæmia, 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-Loeffler bacillus becomes absolute.

7. Inoculate guinea-pigs subcutaneously with filtered glucose bouillon
cultivations (toxins?) and observe the result.

(B) 1. Emulsify the remainder of the deposit with 5 c.c. sterile
bouillon and inoculate two guinea-pigs, thus: guinea-pig a,
subcutaneously with 1 c.c. emulsion; guinea-pig b, subcutaneously with
2 c.c. emulsion; and observe the result.

2. If either or both of the inoculated animals succumb, make complete
post-mortem examination and endeavour to isolate the pathogenic
organisms from the local lesion. Confirm their identity as in A5 and 6
(_vide supra_).

~5. Bacillus Tuberculosis.~--

(A) 1. Inoculate each of three guinea-pigs (previously tested with
tuberculin, to prove their freedom from spontaneous tuberculosis)
subcutaneously at the inner aspect of the bend of the left knee, with 1
c.c. of the deposit emulsion remaining in one or other tube (D^{1} or
D^{2}).

2. Introduce a small quantity of the cream into a subcutaneous pocket
prepared at the inner aspect of the bend of the right knee of each of
these three animals. Place a sealed dressing on the wound.

3. Observe carefully, and weigh accurately each day.

4. Kill one guinea-pig at the end of the second week and make a
complete post-mortem examination.

5. If the result of the examination is negative or inconclusive, kill a
second guinea-pig at the end of the third week and examine carefully.

[Illustration: FIG. 215.--Cadaver of guinea-pig experimentally infected
with B. tuberculosis.]

6. If still negative or inconclusive, kill the third guinea-pig at the
end of the _sixth_ week. Make a careful 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) 1. 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, intraperitoneally, 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 microscopical examination
and cultivations upon Dorset's egg medium.

     NOTE.--No value whatever attaches to the result of a
     microscopical examination for the presence of the B.
     tuberculosis unless confirmed by the result of inoculation
     experiments.

~6. Streptococcus Pyogenes Longus.~--

(A) 1. 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 resemble those of the
streptococcus, make subcultivations upon agar and in bouillon, in the
first instance, and study carefully.

(B) 1. Plant a large loopful of the deposit D^{2} into each of three
tubes of glucose formate bouillon, and incubate anaerobically (in
Buchner's tubes) for twenty-four hours at 37° C.

2. If the resulting growth resembles that of the streptococcus, make
subcultivations upon nutrient agar.

3. Prepare subcultivations of any suspicious colonies that appear, upon
all the ordinary media, and study carefully.

If the streptococcus is successfully isolated, inoculate 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. diphtheriæ and the serial agar cultivations to
isolate streptococci 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 organisms 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 D^{1} or D^{2}.

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 microscopically 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.

6. If apparently M. melitensis, inoculate growth from a nutrose agar
culture after three days incubation intracranially 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 examination in precisely the same manner as
described for milk (_vide_ page 443).


EXAMINATION OF CREAM AND BUTTER.

~Collection of the Sample.~--Collect, store, and transmit 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, 1 c.c. (in hundredths).
    Tubes of gelatine-agar (+10 reaction).
    Plate-levelling stand, with its water chamber filled with water at
      42° C.

METHOD.--

1. Transfer a few grammes of the sample to a sterile test-tube by means
of the sterilised spatula.

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 following quantities of
the sample by the help of a sterile pipette graduated to hundredths of a
cubic centimetre--viz.,

    To tube No. 1 add 1    c.c. liquefied butter.
                2 add 0.5  c.c. liquefied butter.
                3 add 0.3  c.c. liquefied butter.
                4 add 0.2  c.c. liquefied butter.
                5 add 0.1  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 0.01 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.

     Case of sterile pipettes, 10 c.c.

     Case of sterile graduated pipettes, 1 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 1
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 centrifugalise, 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 1/12 inch
oil-immersion lens.

11. Proceed with the examination of the deposit as in the case of milk
deposit (see pages 450 _et seq._).


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. enteritidis
of Gaertner and its allies--which are usually associated with epidemic
outbreaks of food poisoning, and such anaerobic bacteria as initiate
putrefactive changes in the food which result in the formation of
poisonous ptomaines, consequently 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, 1 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.--

1. Place the flask containing 200 c.c. sterile broth on one pan of the
scales and counterpoise accurately.

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 regulated to that temperature) for half an hour,
shaking its contents from time to time. Better results are obtained if
an electrical shaker is fitted inside the incubator and the flask kept
in motion throughout the entire thirty minutes.

Now every centimetre contains the bacteria washed out from 0.1 gramme of
the original food.

4. Inoculate tubes of liquefied gelatine as follows:

    To tube No. 1 add 1.0 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 0.2 c.c. of the extract.
                5 add 0.1 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 37° 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 apparatus at 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
vegetative 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 compelled by the growth
of liquefying colonies) enumerate the gelatine plates in the same way.

10. Subcultivate from the colonies that make their appearance and
identify the various organisms.

11. 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 examination 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. cadaveris.

The methods by which these organisms if present may be identified and
isolated have already been described 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 experimentally 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.

_Extract._--

1. Introduce various quantities of the bouillon extract 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 subcutaneously 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 centimetres (0.1 c.c. to 10 c.c.)--in the latter case the
bulk of the fluid is probably the last quantum of water ingested by the
bivalve before closing its shell. In order to obtain a working average
of the bacteriological flora of a sample, ten oysters should be taken
and the body, gastric juice and liquor should be thoroughly mixed before
examination. The examination, 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.

_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, 1 per mille.

     Bile salt broth tubes.

     Litmus milk tubes.

     Surface plates of nutrose agar.

     Case of sterile pipettes, 1 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 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 previously 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 0.1 c.c. of the mixed liquor to inseminate each of a series of
three nutrose surface plates.

11. Inoculate 0.1 c.c. of the mixed liquor into each of three tubes of
litmus milk.

12. 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
practical 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 1 c.c. of the diluted liquor, etc. from the
cylinder, and mix thoroughly. To capsule II add 1 c.c. of dilution in
capsule I and mix thoroughly. Carry over 1 c.c. of fluid from capsule
II to capsule III, afterwards adding 1 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  1 c.c. cylinder    fluid = 0.01     oyster.
    No. 4 with  1 c.c. capsule   I fluid = 0.001    oyster.
    No. 3 with  1 c.c. capsule  II fluid = 0.0001   oyster.
    No. 2 with  1 c.c. capsule III fluid = 0.00001  oyster.
    No. 1 with  1 c.c. capsule  IV fluid = 0.000001 oyster.

16. Transfer 100 c.c. cylinder fluid (= 1 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 1 the organism finally isolated, 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 15; heat to 80° C. for ten
minutes, and incubate 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 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 1 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 0.1 c.c. of the mixture
from No. I to No. II and mix thoroughly.

6. In like manner transfer 0.1 c.c. from No. II to No. III, and then 0.1
c.c. from No. III to No. IV.

Now 1 c.c. of dilution No.   I contains 0.1       c.c. of the original sewage.
    1 c.c. of dilution No.  II contains 0.001     c.c. of the original sewage.
    1 c.c. of dilution No. III contains 0.00001   c.c. of the original sewage.
    1 c.c. of dilution No.  IV contains 0.0000001 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
conducted on precisely similar lines to those already indicated (_vide_
pages 426 to 441).


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 1 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.

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.

[Illustration: 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 Erlenmeyer flask.)

7. When the aspiration is completed, disconnect the flask and remove it
from its ice packing.

8. Liquefy three tubes of nutrient gelatine and add to them 0.5 c.c.,
0.3 c.c., and 0.2 c.c., respectively, of the water from the flask, by
means of a sterile graduated 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 1 c.c.,
and then in the 50 c.c. of broth in the flask.

12. The result of air examination is usually expressed as the number of
bacteria present per cubic metre (i. e., kilolitre) of air; and as the
number of organisms present in the 50 c.c. water only represent those
contained in 10 litres of air, the resulting figure must be multiplied
by 100.

Qualitative.--

1. Proceed exactly as in the quantitative examination of air (_vide
supra_), steps 1 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 quantities 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 information of value to
the sanitarian during the progress of the process of homogenisation of
"made soil" (e. g., a dumping area for the refuse of town) and
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 suitability 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 alternations 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.

[Illustration: FIG. 217.--Soil scoop.]

The instrument used for the actual removal of the soil from its natural
position will vary according to whether we require surface samples or
soil from varying 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.

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 débris 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 transferred to the sterile capsule for
transmission.

[Illustration: 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,
however, 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 centimetres from the
drill-pointed extremity. It is provided 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.~--Four distinct investigations are included in the complete
quantitative bacteriological examination of the soil:

1. The enumeration of the aerobic organisms.

2. The enumeration of the spores of aerobes.

3. The enumeration of the anaerobic organisms (including 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, 1 c.c. (in tenths of a
     cubic centimetre).

     Flask containing 250 c.c. sterile bouillon.

     Tall cylinder containing 2 per cent. lysol solution.

     Plate-levelling stand.

     12 sterile plates.

     Tubes of nutrient gelatine.

     Tubes of wort gelatine.

     Tubes of nutrient agar.

     Tubes of glucose formate gelatine.

     Tubes of glucose formate agar.

     Water-bath regulated at 42° C.

     Bunsen burner.

     Grease pencil.

     Sterile mortar and pestle (agate).

     Sterile wide-mouthed Erlenmeyer flask (500 c.c. capacity).

     Sterile metal funnel with short wide bore delivery tube to
     just fit mouth of flask.

     Solid rubber stopper to fit the flask (sterilised by
     boiling).

     Pair of scales.

     Counterpoise (Fig. 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 Erlenmeyer 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 0.1 gramme of soil.

8. Pipette off 1 c.c. of the supernatant bouillon, termed the "soil
water," and add it to the contents of capsule I; mix thoroughly.

9. Remove 0.1 c.c. of the infected bouillon from capsule I and add it to
capsule II, and mix.

10. In like manner add 0.1 c.c. of the contents of capsule II to capsule
III, and then 0.1 c.c. of the contents of capsule III to capsule IV.

Then 1 c.c. fluid from capsule   I contains soil water
  from .01       gm. earth.
Then 1 c.c. fluid from capsule  II contains soil water
  from .0001     gm. earth.
Then 1 c.c. fluid from capsule III contains soil water
  from .000001   gm. earth.
Then 1 c.c. fluid from capsule  IV contains soil water
  from .00000001 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 0.1 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 obtained 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.

(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.

18. Place in the differential steriliser at 80° C. for ten minutes.

19. Pour two sets of four gelatine plates containing 0.1, 0.2, 0.5, and
1 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 examination 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 oedematis 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 qualitative examination of
water, and filter the soil water.

3. Suspend the bacterial residue in 5 c.c. sterile bouillon (technique
similar to that described for the water sample, _vide_ pages 434-436).

Every cubic centimetre of suspension now contains the soil water from
nearly 1 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 Tetani.~--

The methods adopted for the isolation of these organisms are identical
with those already described under water (page 437 _et seq._).

~V. Bacillus Oedematis 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 1 to 10.

2. Inoculate each tube with varying quantities of the material as
follows:

    To tube No.  1 add 1.0 c.c. of the soil water.
    To tube No.  2 add 0.1 c.c. of the soil water.
    To tube No.  3 add 1.0 c.c. from Capsule I.
    To tube No.  4 add 0.1 c.c. from Capsule I.
    To tube No.  5 add 1.0 c.c. from Capsule II.
    To tube No.  6 add 0.1 c.c. from Capsule II.
    To tube No.  7 add 1.0 c.c. from Capsule III.
    To tube No.  8 add 0.1 c.c. from Capsule III.
    To tube No.  9 add 1.0 c.c. from Capsule IV.
    To tube No. 10 add 0.1 c.c. from Capsule IV.

Label and incubate at 30° C.


~VII. The Nitric Organisms.~--

3. Take ten tubes of Winogradsky's solution No II, number them
consecutively from 1 to 10 and inoculate with quantities of soil water
similar to those enumerated 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 atmospheres 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:

_Apparatus Required_:

     Filtering apparatus: The actual filter candle that is used
     must be the one it is intended to test and must be
     previously carefully sterilised; the arrangement of the
     apparatus will naturally 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, 1 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 incubate 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 1 c.c. of the filtrate.

6. Incubate at 37° C. until, if necessary, the completion of seven days.

7. If the filtrate is not sterile, subcultivate the organism 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., Micrococcus melitensis, in
a similar manner; in this instance continuing the incubation of
cultivations from the filtrate for fourteen days.


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 convenient 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.

1. _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 account of its relative nonpathogenicity, the ease
with which it can be isolated and identified by different observers 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 bacillus is an organism which is slightly more
resistant to the lethal action of germicides than the more pathogenic
members of this group, a margin of safety is introduced 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
37° C. for 24 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 centrifuge 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 disinfectant, which may
be referred to as germicide-x, on the lines set out on page 311 to
determine its inhibition coefficient.

This constant having been fixed, prepare various solutions of
germicide-x with sterilised distilled water by accurate 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.

The following table shows the effect of different percentages of
carbolic acid upon B. coli for varying contact times, compiled from an
experiment conducted under the standard conditions referred to under
Environment. The results closely correspond to those recorded by the
Lancet Commission on Disinfectants, 1909.

---------------------+-----------------------------------
                     |        Contact time in minutes.
Percentage of phenol +------+---+---+---+---+---+---+----
                     | 2-1/2| 5 |10 | 15| 20| 25| 30| 35
---------------------+------+---+---+---+---+---+---+----
1.20                 |   -  | - | - | - | - | - | - | -
1.10                 |   -  | - | - | - | - | - | - | -
1.0                  |   +  | - | - | - | - | - | - | -
0.9                  |   +  | - | - | - | - | - | - | -
0.85                 |   +  | + | - | - | - | - | - | -
0.80                 |   +  | + | + | - | - | - | - | -
0.75                 |   +  | + | + | + | + | - | - | -
0.7                  |   +  | + | + | + | + | + | - | -
0.65                 |   +  | + | + | + | + | + | + | -
---------------------+------+---+---+---+---+---+---+----

- = No growth, i. e., bacteria killed.
+ = Growth, i. e., bacteria still living.

From this it will be seen that the following percentage solutions will
need to be prepared, namely: 1.1 per cent., 1.0 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 accurately 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 1:1000 solution of corrosive sublimate. 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 temperature at which
the multiplication of B. coli is a comparatively 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 0.1 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 insufficient 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 immediate 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 exclusion of organisms likely to contaminate the preparation, and
if possible one which affords characteristic 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 provides 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
subculture tube contains 10 c.c. of medium, but it is obvious that if
germicide-x possesses an inhibition coefficient of 0.1 per cent. the
addition of 0.1 c.c. of a 10 per cent. solution to 10 c.c. of medium
would effectually 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.

_f. 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 continued--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 necessary by plating methods, further
subcultivations upon carbohydrate media and agglutination experiments.
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, 1 c.c. (in tenths).

     Sterile graduated pipettes in case, 5 c.c. (in 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 1:1000, it will be quite safe to employ the ordinary
culture tubes containing 10 c.c. medium in the subsequent experiments.

_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 2-1/2, 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 1:300 solution.

Into tube D pipette 5 c.c. germicide-x 1:500 solution.

Into tube E pipette 5 c.c. germicide-x 1:600 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 1-10, 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 1 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 commencement of the
experiment and at subsequent intervals of 2-1/2, 5, 25 and 30 minutes.

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 0.1
c.c. of B. coli emulsion into the 1:100 solution. Then replace the
pipette and washer.

[Illustration: FIG. 219.--Test-tube rack.]

11. 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 2-1/2 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 more than 0.1 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 0.1 c.c. in the pipette.

14. At the same time, with the left hand remove Bile Salt Tube No. 1
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--replug the tube and drop it into the
wire basket. Replace 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.

16. 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 indicates strengths of
Germicide-x lethal at 2-1/2 and 30 minutes the final test can be
arranged, but if this result has not been attained, sufficient evidence
will 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 2-1/2 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 1-10, those in the back row 11-20.

4. Arrange further 20 tubes of bile salt broth numbered 21-40 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 1 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 commencement of the
experiment and subsequently at 2-1/2, 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 30-minute contact period have
been inoculated, carry out a precisely similar experiment, in which
five percentage strengths of Phenol, (e. g., 1.1, 1.0, 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 2-1/2-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 2-1/2 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 determination are set out in
the accompanying table.


TABLE 27

Organism employed, B. Coli Communis.

Culture Medium, Nutrient Agar (+10). Age, 24 hrs.
Temp. of Incubation, 37°C.

Quantities used { Culture  } Emulsion 0.1 c.c. + 5 c.c. Germicide.
                { Emulsion }

Room Temperature during Experiments, 17°C.

   Germicide   Strength          Time of exposure            Incubation
                       2-1/2   5  10  15  20  25  30  35    Time    Temp.
1 Germicide-x    4%      --   --  --  --  --  --  --  --   7 days.  37°C.
2 Germicide-x    3%      48   --  --  --  --  --  --  --   7 days.  37°C.
3 Germicide-x    2%      24   24  24  24  48  72           7 days.  37°C.
4 Germicide-x    1%      24   24  24  24  72  24  72       7 days.  37°C.
5 Germicide-x    0.5%    24   24  24  24  24  24  24  24  24 hours. 37°C.

1 Phenol         1.10%   --   --  --  --  --  --  --  --   7 days.  37°C.
2 Phenol         1.00%   24                                7 days.  37°C.
3 Phenol         0.75%   24   24  24  24  48               7 days.  37°C.
4 Phenol         0.70%   24   24  24  24  24  72           7 days.  37°C.
5 Phenol         0.65%   24   24  24  24  24  48  24  24   2 days.  37°C.


                    ((1.10/4.00) + (0.7/2.0))   0.27 + 0.35   .62
Phenol Coefficient = ------------------------ = ----------- = --- = 0.31
                                2                    2         2




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 represents 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 (_l._), 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 _milli_ = 1/1000; _centi_ = 1/100; _deci_ = 1/10; 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:~

     1 millimetre (= 1 mm.) = 1/25 of an inch.

     1 centimetre (= 1 cm.) = 2/5 of an inch.

     1 inch (1") = 25 millimetres or 2-1/2 centimetres.


     ~Mass:~

     1 milligramme (= 1 mg.) = 0.01543 grain (or approximately
     1/64 grain).

     1 gramme (= 1 g.) = 15.4323 grains.

     1 "kilo" or kilogramme (= 1 kgm.) = 2 pounds, 3-1/4 ounces
     avoirdupois.

     1 pound avoirdupois (= 1 lb.) = 453.592 grammes.

     1 ounce avoirdupois (= 1 oz.) = 28.35 grammes.

     1 grain = 0.0648 gramme or 64.8 milligrammes.


     ~Capacity:~

     1 cubic centimetre (= 1 c.c.) = 16.9 minims imperial
     measure.

     1 litre (= 1 _l._) = 35.196 fluid ounces imperial measure.

     1 fluid ounce imperial measure (= 1 [Symbol: ounce]) =
     28.42 cubic centimetres.

     1 pint imperial measure (= 1 O.) = 568.34 cubic centimetres.

     1 gallon imperial measure (= 1 C.) = 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                            × 15.432.
    To convert grammes into ounces avoirdupois                × 0.03527.
    To convert kilogrammes into pounds                        × 2.2046.
    To convert cubic centimetres into fluid ounces imperial   × 0.0352.
    To convert litres into fluid ounces imperial              × 35.2.
    To convert metres into inches                             × 39.37.
    To convert grains into grammes                            × 0.0648.
    To convert avoirdupois ounces into grammes                × 28.35.
    To convert troy ounces into grammes                       × 31.104.
    To convert fluid ounces into cubic centimetres            × 28.42.
    To convert pints into litres                              × 0.568.
    To convert inches into metres                             × 0.0254.


TABLE FOR THE CONVERSION OF DEGREES CENTIGRADE INTO DEGREES FAHRENHEIT.


_X.° C. = ((9x/5) + 32)° F._

| Cent. | Faht.  || Cent. | Faht.  || Cent. | Faht.  |
|   0   |  32.0  ||  34   |  93.2  ||  68   | 154.4  |
|   1   |  33.8  ||  35   |  95.0  ||  69   | 156.2  |
|   2   |  35.6  ||  36   |  96.8  ||  70   | 158.0  |
|   3   |  37.4  ||  37   |  98.6  ||  71   | 159.8  |
|   4   |  39.2  ||  38   | 100.4  ||  72   | 161.6  |
|   5   |  41.0  ||  39   | 102.2  ||  73   | 163.4  |
|   6   |  42.8  ||  40   | 104.0  ||  74   | 165.2  |
|   7   |  44.6  ||  41   | 105.8  ||  75   | 167.0  |
|   8   |  46.4  ||  42   | 107.6  ||  76   | 168.8  |
|   9   |  48.2  ||  43   | 109.4  ||  77   | 170.6  |
|  10   |  50.0  ||  44   | 111.2  ||  78   | 172.4  |
|  11   |  51.8  ||  45   | 113.0  ||  79   | 174.2  |
|  12   |  53.6  ||  46   | 114.8  ||  80   | 176.0  |
|  13   |  55.4  ||  47   | 116.6  ||  81   | 177.8  |
|  14   |  57.2  ||  48   | 118.4  ||  82   | 179.6  |
|  15   |  59.0  ||  49   | 120.2  ||  83   | 181.4  |
|  16   |  60.8  ||  50   | 122.0  ||  84   | 183.2  |
|  17   |  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  ||  88   | 190.4  |
|  21   |  69.8  ||  55   | 131.0  ||  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   | 140.0  ||  94   | 201.2  |
|  27   |  80.6  ||  61   | 141.8  ||  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  ||  100  | 212.0  |
|  33   |  91.4  ||  67   | 152.6  ||       |        |


TABLE FOR THE CONVERSION OF DEGREES FAHRENHEIT INTO DEGREES CENTIGRADE.


_X° F. = (5(x - 32))/9° C._

 Faht.| Cent.|| Faht.| Cent.|| Faht.|Cent. || Faht.| Cent.|| Faht.| Cent.
  32  |    0.||  68  | 20.0 ||  104 | 40.0 ||  140 | 60.0 ||  176 | 80.0
  33  |  0.6 ||  69  | 20.6 ||  105 | 40.6 ||  141 | 60.6 ||  177 | 80.6
  34  |  1.1 ||  70  | 21.1 ||  106 | 41.1 ||  142 | 61.1 ||  178 | 81.1
  35  |  1.7 ||  71  | 21.7 ||  107 | 41.7 ||  143 | 61.7 ||  179 | 81.7
  36  |  2.2 ||  72  | 22.2 ||  108 | 42.2 ||  144 | 62.2 ||  180 | 82.2
  37  |  2.8 ||  73  | 22.8 ||  109 | 42.8 ||  145 | 62.8 ||  181 | 82.8
  38  |  3.3 ||  74  | 23.3 ||  110 | 43.3 ||  146 | 63.3 ||  182 | 83.3
  39  |  3.9 ||  75  | 23.9 ||  111 | 43.9 ||  147 | 63.9 ||  183 | 83.9
  40  |  4.4 ||  76  | 24.4 ||  112 | 44.4 ||  148 | 64.4 ||  184 | 84.4
  41  |  5.0 ||  77  | 25.0 ||  113 | 45.0 ||  149 | 65.0 ||  185 | 85.0
  42  |  5.6 ||  78  | 25.6 ||  114 | 45.6 ||  150 | 65.6 ||  186 | 85.6
  43  |  6.1 ||  79  | 26.1 ||  115 | 46.1 ||  151 | 66.1 ||  187 | 86.1
  44  |  6.7 ||  80  | 26.7 ||  116 | 46.7 ||  152 | 66.7 ||  188 | 86.7
  45  |  7.2 ||  81  | 27.2 ||  117 | 47.2 ||  153 | 67.2 ||  189 | 87.2
  46  |  7.8 ||  82  | 27.8 ||  118 | 47.8 ||  154 | 67.8 ||  190 | 87.8
  47  |  8.3 ||  83  | 28.3 ||  119 | 48.3 ||  155 | 68.3 ||  191 | 88.3
  48  |  8.9 ||  84  | 28.9 ||  120 | 48.9 ||  156 | 68.9 ||  192 | 88.9
  49  |  9.4 ||  85  | 29.4 ||  121 | 49.4 ||  157 | 69.4 ||  193 | 89.4
  50  | 10.0 ||  86  | 30.0 ||  122 | 50.0 ||  158 | 70.0 ||  194 | 90.0
  51  | 10.6 ||  87  | 30.6 ||  123 | 50.6 ||  159 | 70.6 ||  195 | 90.6
  52  | 11.1 ||  88  | 31.1 ||  124 | 51.1 ||  160 | 71.1 ||  196 | 91.1
  53  | 11.7 ||  89  | 31.7 ||  125 | 51.7 ||  161 | 71.7 ||  197 | 91.7
  54  | 12.2 ||  90  | 32.2 ||  126 | 52.2 ||  162 | 72.2 ||  198 | 92.2
  55  | 12.8 ||  91  | 32.8 ||  127 | 52.8 ||  163 | 72.8 ||  199 | 92.8
  56  | 13.3 ||  92  | 33.3 ||  128 | 53.3 ||  164 | 73.3 ||  200 | 93.3
  57  | 13.9 ||  93  | 33.9 ||  129 | 53.9 ||  165 | 73.9 ||  201 | 93.9
  58  | 14.4 ||  94  | 34.4 ||  130 | 54.4 ||  166 | 74.4 ||  202 | 94.4
  59  | 15.0 ||  95  | 35.0 ||  131 | 55.0 ||  167 | 75.0 ||  203 | 95.0
  60  | 15.6 ||  96  | 35.6 ||  132 | 55.6 ||  168 | 75.6 ||  204 | 95.6
  61  | 16.1 ||  97  | 36.1 ||  133 | 56.1 ||  169 | 76.1 ||  205 | 96.1
  62  | 16.7 ||  98  | 36.7 ||  134 | 56.7 ||  170 | 76.7 ||  206 | 96.7
  63  | 17.2 ||  99  | 37.2 ||  135 | 57.2 ||  171 | 77.2 ||  207 | 97.2
  64  | 17.8 ||  100 | 37.8 ||  136 | 57.8 ||  172 | 77.8 ||  208 | 97.8
  65  | 18.3 ||  101 | 38.3 ||  137 | 58.3 ||  173 | 78.3 ||  209 | 98.3
  66  | 18.9 ||  102 | 38.9 ||  138 | 58.9 ||  174 | 78.9 ||  210 | 98.9
  67  | 19.4 ||  103 | 39.4 ||  139 | 59.4 ||  175 | 79.4 ||  211 | 99.4
      |      ||      |      ||      |      ||      |      ||  212 |100.0

~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 KNO_{3} 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.

    (_N_ + ~X~) _Y_    _A_ (~X~)
    --------------- = ----------
          100            100

_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

    (10 + ~X~) 4   25 ~X~
    ------------ = ------
         100        100

    Therefore, 40 + 4~X~ = 25~X~.

    Therefore,     21~X~ = 40.

                     ~X~ = 1.9 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
KNO_{3} makes 11.9 c.c. medium containing 4 per cent. KNO_{3}.


~TABLES FOR PREPARING DILUTIONS~

(of Serum, Disinfectants or other substances.)

In estimating the agglutinin content or _titre_ 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 increment, such for example as the
following:

From dilutions of 1:10 to 1:80 rise by increments of 5.

From dilutions of 1:80 to 1:200 rise by increments of 10.

From dilutions of 1:200 to 1:400 rise by increments of 25.

From dilutions of 1:400 to 1:500 rise by increments of 50.

From dilutions of 1:500 to 1:1000 rise by increments of 100.

From dilutions of 1: 1000 to 1:5000 rise by increments of 250.

From dilutions of 1: 5000 to 1:10,000 rise by increments of 1000.

From dilutions of 1:10,000 to 1:100,000 rise by increments of 5000.

From dilutions of 1:100,000 to 1:1,000,000 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.

1 c.c. serum + 9 c.c. normal saline solution = 10 per cent. solution or
1: 10 dilution (of which 1 c.c. contains 0.1 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:

1 gram o.s. + 10 c.c. distilled water = 10 per cent. solution, etc.

SECOND DILUTION--II.

1 c.c. first dilution + 9 c.c. normal saline solution = 1 per cent.
solution or 1: 100 dilution.

THIRD DILUTION--III.

1 c.c. second dilution + 9 c.c. normal saline solution = 1 per mille
solution or 1: 1000 dilution.

FOURTH DILUTION--IV.

1 c.c. second dilution + 9 c.c. normal saline solution = 0.1 per mille
solution or 1: 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.


TABLES FOR PREPARING DILUTIONS.

-----------------------------------+----------------------------------
                                   |
            TABLE I                |          TABLE II
    Using 10 % stock solution      |    Using 1% stock solution
    First       }                  |      Second      }
    dilution    } + Diluent        |      dilution    } + Diluent
                                   |
-----------------------------------+----------------------------------
                                   |
     1:  10 = 1 c.c. +  0 c.c.     |    1: 100  = 1 c.c. + 0 c.c.
     1:  15 = 1 c.c. +  0.5 c.c.   |    1: 110  = 1 c.c. + 0.1 c.c.
     1:  20 = 1 c.c. +  1.0 c.c.   |    1: 120  = 1 c.c.  + 0.2 c.c.
     1:  25 = 1 c.c. +  1.5 c.c.   |   [1: 125  = 1 c.c. + 0.25 c.c.]
     1:  30 = 1 c.c. +  2.0 c.c.   |    1: 130  = 1 c.c. + 0.3 c.c.
     1:  35 = 1 c.c. +  2.5 c.c.   |    1: 140  = 1 c.c. + 0.4 c.c.
     1:  40 = 1 c.c. +  3.0 c.c.   |    1: 150  = 1 c.c. + 0.5 c.c.
     1:  45 = 1 c.c. +  3.5 c.c.   |    1: 160  = 1 c.c. + 0.6 c.c.
     1:  50 = 1 c.c. +  4.0 c.c.   |    1: 170  = 1 c.c. + 0.7 c.c.
     1:  55 = 1 c.c. +  4.5 c.c.   |   [1: 175  = 1 c.c. + 0.75 c.c.]
     1:  60 = 1 c.c. +  5.0 c.c.   |    1: 180  = 1 c.c. + 0.8 c.c.
     1:  65 = 1 c.c. +  5.5 c.c.   |    1: 190  = 1 c.c. + 0.9 c.c.
     1:  70 = 1 c.c. +  6.0 c.c.   |    1: 200  = 1 c.c. + 1.0 c.c.
     1:  75 = 1 c.c. +  6.5 c.c.   +---------------------------------
     1:  80 = 1 c.c. +  7.0 c.c.   |    1: 200  = 1 c.c. + 1.0 c.c.
     ------------------------------+    1: 225  = 1 c.c. + 1.25 c.c.
     1:  80 = 1 c.c. +  7.0 c.c.   |    1: 250  = 1 c.c. + 1.5 c.c.
     1:  90 = 1 c.c. +  8.0 c.c.   |    1: 275  = 1 c.c. + 1.75 c.c.
     1: 100 = 1 c.c. +  9.00 c.c.  |    1: 300  = 1 c.c. + 2.0 c.c.
     1: 110 = 1 c.c. + 10.0 c.c.   |    1: 325  = 1 c.c. + 2.25 c.c.
     1: 120 = 1 c.c. + 11.0 c.c.   |    1: 350  = 1 c.c. + 2.5 c.c.
    [1: 125 = 1 c.c. + 11.5 c.c.]  |    1: 375  = 1 c.c. + 2.75 c.c.
     1: 130 = 1 c.c. + 12.0 c.c.   |    1: 400  = 1 c.c. + 3.0 c.c.
     1: 140 = 1 c.c. + 13.0 c.c.   +---------------------------------
     1: 150 = 1 c.c. + 14.0 c.c.   |    1: 400  = 1 c.c. + 3.0 c.c.
     1: 160 = 1 c.c. + 15.0 c.c.   |    1: 450  = 1 c.c. + 3.5 c.c.
     1: 170 = 1 c.c. + 16.0 c.c.   |    1: 500  = 1 c.c. + 4.0 c.c.
    [1: 175 = 1 c.c. +-16.5 c.c.]  +---------------------------------
     1: 180 = 1 c.c. + 17.0 c.c.   |    1: 500  = 1 c.c. + 4.0 c.c.
     1: 190 = 1 c.c. + 18.0 c.c.   |    1: 600  = 1 c.c. + 5.0 c.c.
     1: 200 = 1 c.c. + 19.0 c.c.   |    1: 700  = 1 c.c. + 6.0 c.c.
     ----------------- ------------+   [1: 750  = 1 c.c. + 6.5 c.c.]
     1: 200 = 1 c.c. + 19.0 c.c.   |    1: 800  = 1 c.c. + 7.0 c.c.
     1: 225 = 1 c.c. + 21.5 c.c.   |    1: 900  = 1 c.c. + 8.0 c.c.
     1: 250 = 1 c.c. + 24.0 c.c.   |    1: 1000 = 1 c.c. + 9.0 c.c.
     1: 275 = 1 c.c. + 26.5 c.c.   +--------------------------------
     1: 300 = 1 c.c. + 29.0 c.c.   |    1: 1000 = 1 c.c. + 9.0 c.c.
     1: 325 = 1 c.c. +-31.5 c.c.   |    1: 2000 = 1 c.c. + 19.0 c.c.
     1: 350 = 1 c.c. + 34.0 c.c.   |    1: 3000 = 1 c.c. + 29.0 c.c.
     1: 375 = 1 c.c. + 36.5 c.c.   |    1: 4000 = 1 c.c. + 39.0 c.c.
     1: 400 = 1 c.c. + 39.0 c.c.   |    1: 5000 = 1 c.c. + 49.0 c.c.
     ------------------------------+--------------------------------
     1: 400 = 1 c.c. + 39.0 c.c.   |
     1: 450 = 1 c.c. + 44.5 c.c.   |
     1: 500 = 1 c.c. + 49.0 c.c.   |

   ---------------------------------+-------------------------------
                                    |
       TABLE III                    |         TABLE IV
       Using 0.1% stock solution    |    Using 0.01% stock solution
       Third    }                   |       Fourth   }
       dilution } + Diluent         |       Dilution } + Diluent
                                    |
   ---------------------------------+-------------------------------
                                    |
    1:   1000 = 1 c.c. +  0 c.c.    |  1:   10,000 = 1 c.c. + 0 c.c.
    1:   1250 = 1 c.c. +  0.25 c.c. |  1:   15,000 = 1 c.c. + 0.5 c.c.
    1:   1500 = 1 c.c. +  0.5 c.c.  |  1:   20,000 = 1 c.c. + 1.0 c.c.
    1:   1750 = 1 c.c. +  0.75 c.c. |  1:   25,000 = 1 c.c. + 1.5 c.c.
    1:   2000 = 1 c.c. +  1.0 c.c.  |  1:   30,000 = 1 c.c. + 2.0 c.c.
    1:   2250 = 1 c.c. +  1.25 c.c. |  1:   35,000 = 1 c.c. + 2.5 c.c.
    1:   2500 = 1 c.c. +  1.5 c.c.  |  1:   40,000 = 1 c.c. + 3.0 c.c.
    1:   2750 = 1 c.c. +  1.75 c.c. |  1:   45,000 = 1 c.c. + 3.5 c.c.
    1:   3000 = 1 c.c. +  2.0 c.c.  |  1:   50,000 = 1 c.c. + 4.0 c.c.
    1:   3250 = 1 c.c. +  2.25 c.c. |  1:   55,000 = 1 c.c. + 4.5 c.c.
    1:   3500 = 1 c.c. +  2.5 c.c.  |  1:   60,000 = 1 c.c. + 5.0 c.c.
    1:   3750 = 1 c.c. +  2.75 c.c. |  1:   65,000 = 1 c.c. + 5.5 c.c.
    1:   4000 = 1 c.c. +  3.0 c.c.  |  1:   70,000 = 1 c.c. + 6.0 c.c.
    1:   4250 = 1 c.c. +  3.25 c.c. |  1:   75,000 = 1 c.c. + 6.5 c.c.
    1:   4500 = 1 c.c. +  3.5 c.c.  |  1:   80,000 = 1 c.c. + 7.0 c.c.
    1:   4750 = 1 c.c. +  3.75 c.c. |  1:   85,000 = 1 c.c. + 7.5 c.c.
    1:   5000 = 1 c.c. +  4.0 c.c.  |  1:   90,000 = 1 c.c. + 8.0 c.c.
    --------------------------------+  1:   95,000 = 1 c.c. + 8.5 c.c.
    1:   5000 = 1 c.c. +  4.0 c.c.  |  1:  100,000 = 1 c.c. + 9.0 c.c.
    1:   6000 = 1 c.c. +  5.0 c.c.  +-----------------------------------
    1:   7000 = 1 c.c. +  6.0 c.c.  |  1:  100,000 = 0.1 c.c. + 0.9 c.c.
   [1:   7500 = 1 c.c. +  6.5 c.c.] |  1:  200,000 = 0.1 c.c. + 1.9 c.c.
    1:   8000 = 1 c.c. +  7.0 c.c.  | [1:  250,000 = 0.1 c.c. + 2.4 c.c.]
    1:   9000 = 1 c.c. +  8.0 c.c.  |  1:  300,000 = 0.1 c.c. + 2.9 c.c.
    1: 10,000 = 1 c.c. +  9.0 c.c.  |  1:  400,000 = 0.1 c.c. + 3.9 c.c.
    ------------------------------- +  1:  500,000 = 0.1 c.c. + 4.9 c.c.
    1: 10,000 = 1 c.c. +  9.0 c.c.  +-----------------------------------
    1: 15,000 = 1 c.c. + 14.0 c.c.  |  1:  500,000 = 0.1 c.c. + 4.9 c.c.
    1: 20,000 = 1 c.c. + 19.0 c.c.  |  1:  600,000 = 0.1 c.c. + 5.9 c.c.
    1: 25,000 = 1 c.c. + 24.0 c.c.  |  1:  700,000 = 0.1 c.c. + 6.9 c.c.
    1: 30,000 = 1 c.c. + 29.0 c.c.  | [1:  750,000 = 0.1 c.c. + 7.4 c.c.]
    --------------------------------+  1:  800,000 = 0.1 c.c. + 7.9 c.c.
                                    |  1:  900,000 = 0.1 c.c. + 8.9 c.c.
                                    |  1:1,000,000 = 0.1 c.c. + 9.9 c.c.
                                   -+-------------------------------------


TEMPERATURE PRESSURE TABLE.

 ---------------+--------------+---------------------+-------------
    Temperature |              |  Pounds per sq. in. |
    Centigrade  |  Mm. of Hg.  |  absolute pressure  | Atmospheres
                |              |                     |
 ---------------+--------------+---------------------+-------------
                |              |                     |
     98°        |    707.1     |         13.7        |     0.93
     99°        |    733.1     |         14.2        |     0.96
    100°        |    760.0     |         14.7        |     1.00
                |              |                     |
    101°        |    787.8     |         15.2        |     1.03
    102°        |    816.0     |         15.8        |     1.07
    103°        |    845.2     |         16.3        |     1.11
    104°        |    875.4     |         16.9        |     1.15
    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     |         20.1        |     1.36
    110°        |   1075.3     |         20.8        |     1.41
                |              |                     |
    111°        |   1112.0     |         21.5        |     1.46
    112°        |   1149.8     |         22.2        |     1.51
    113°        |   1188.6     |         22.9        |     1.56
    114°        |   1228.4     |         23.7        |     1.61
    115°        |   1269.4     |         24.5        |     1.67
                |              |                     |
    116°        |   1311.4     |         25.3        |     1.72
    117°        |   1354.6     |         26.2        |     1.78
    118°        |   1399.0     |         27.0        |     1.84
    119°        |   1444.5     |         27.9        |     1.90
    120°        |   1491.2     |         28.8        |     1.96
                |              |                     |
    121°        |   1539.2     |         29.7        |     2.02
    122°        |   1588.4     |         30.7        |     2.09
    123°        |   1638.9     |         31.7        |     2.15
    124°        |   1690.7     |         32.7        |     2.22
    125°        |   1743.8     |         33.7        |     2.29
 ---------------+--------------+---------------------+-------------


TABLE FOR DESICCATION AT LOW TEMPERATURES IN VACUO.

+--------------------------+
| Temperature |            |
| Centigrade  | Mm. of Hg. |
+-------------+------------+
| 21°         | 18.4       |
| 22°         | 19.6       |
| 23°         | 20.8       |
| 24°         | 22.1       |
| 25°         | 23.5       |
|             |            |
| 26°         | 24.9       |
| 27°         | 26.4       |
| 28°         | 28.0       |
| 29°         | 29.7       |
| 30°         | 31.5       |
|             |            |
| 31°         | 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°         | 75.1       |
| 47°         | 79.0       |
| 48°         | 83.1       |
| 49°         | 87.4       |
| 50°         | 91.9       |
+-------------+------------+


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 sodæ chlorinatæ--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 antiformin.

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 homogenous 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 distribute the deposit throughout the
water. Again centrifugalise.

6. Repeat steps 4 and 5 twice more.

7. Employ one portion of the final deposit to inoculate 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
     centrifuge tube with Ligroin (a petroleum ether) in place of
     sterile distilled water in step 5 and prepare the films from
     the _surface_ of the fluid, to stain by the Ziehl-Neelsen
     process.




INDEX


Abbé'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 hæmatin, 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 bacteria, 295

Active immunisation, illustrative example, 322

Adjustable water bath, 299

Aerobic cultures, 221

Aerogenic bacteria, 131

Aesculin agar, 204

Agar gelatine (guarniari), 194
  methods of preparation, 167
  surface plates, 232

Agar-agar, preparation of, 167

Agglutination reaction, macroscopical, 386
  microscopical, 385

Agglutinin, 381

Air, analysis of, 468
  filter, 40
  pump, Geryk, 43

Albumin 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 biological method, 237
  physical method, 237
  vacuum method, 238
  Wright's method, 239

Anæsthetics, 345

Analysis of air, apparatus for, 469
  method of, 468
  qualitative bacteriological, 470
  quantitative bacteriological, 468
  of butter, qualitative bacteriological, 458
    quantitative bacteriological, 457
  of cream, qualitative bacteriological, 458
    quantitative bacteriological, 457
  of fish, 460
  of ice cream, qualitative bacteriological, 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 bacteriological, 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

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. tuberculosis, 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

Ascomycetæ, 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
  diphtheriæ in milk, 452
  enteritidis in water, 437
  sporogenes in milk, 452
    in water, 438
  oedematis 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, general considerations, 415
  examination of blood, 377

Base of microscope, 50

Basidium, 128

Beer wort, preparation of, 175

Beetroot 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 cultures, 276

Biochemistry of bacteria, 276

Biological differentiation of bacteria, 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, 11
  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

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 preparations, 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 isolating
  coli-typhoid 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, 18
  to stain, 99
  to sterilise, 31

Carbohydrate media, preparation of, 177

Carbolic acid as a germicide, 27, 481
  method of isolating coli-typhoid 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

Chromophorous 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 bacteriological 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 hæmocytology, 374

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, 31

Cream, analysis of, 457
  qualitative analysis of, 458
  quantitative analysis of, 457

Crenothrix morphology, 133

Criteria of infection, 370

Criterion of immunity, 324

Cultural characters, macroscopical examination, 261

Culture flask, Guy's, 5
    Kolle, 4
    Roux, 5

Cuneate bacilli, 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, 178

Diaphragm, iris, 53

Diastatic enzymes, tests for, 278

Differential atmosphere cultivation, 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 pneumoniæ, immunisation 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, characters of, 267

Egg albumin agar, 213
    broth, (Lipschuetz), 213
  media (Dorset), preparation of, 174
    inspissated, 212
    (Lubenau), 209
    (Tarchanoff and Kolesnikoff), 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: typhoid group, 438

Endogenous spores, 138
  varieties of, 139

Endo-germination, 139

English proof agar, Blaxall, 193

Enumerating colonies on plates, 423
  discs, Jeffer's, 424
    Pakes', 424

Enrichment method in water analysis, 427

Enumeration of micro-organisms, 423

Environmental conditions, 142

Enzyme production, investigation 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, 160

Film preparations, 81
  fixing, 81
  making, 81
  mounting, 85
  staining, 83

Filter candle, closed, 47
    open, 43
    testing efficiency of, 478
    to disinfect, 28
    to sterilise, 29
  flask, 6
  papers, to fold, 156

Filters, 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, 114

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, 18
  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 of 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 (Sabouraud), 193
  proof agar (Sabouraud), 193

Fresh preparations of bacteria, 74

Friedländer'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 anæsthetics, 345

Gentian violet, 91

German lined paper, 69

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-Weigert for sections, 121, 122

Gram-Weigert's differential stain, 109
  modified, 110

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 Hansen), 192


Hæmatin, 95

Hæmatocytometer, 248

Hæmatoxilin, 95

Hæmolysin, definition of, 326
  preparation of, 327
  storage of, 331

Hæmolytic 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

Inosite-free media--bouillon (Durham), 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, preparation 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 hæmatocytology of, 374
    normal temperature, 372
  regulations, 1

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, 125

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-Conradi), 205
  whey, 195
    agar, 196
    gelatine, 196
    (Petruschky), 195

Local anæsthetics, 345
  reaction to infection, 372

Locomotive movement, 80

Loeffler'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 (Loeffler), 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 bacteria, 249
  stage, 52

Media, filtration of, 156
  preparation of, 163
    aerobic culture, 222
    aesculin agar, 204
    agar-agar, 167
    agar gelatine (Guarniari), 194

Media, preparation of anaerobic culture, 180
  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 Mallory), 208
    (Loeffler), 208
    (Lorrain Smith), 208
  bouillon, 163
  bread paste, 193
  brilliant green agar (Conradi), 206
    bile salt agar (Fawcus), 206
  Capaldi-Proskauer, No. I, 186
    No. II, 187
  carbohydrate, 177
  carbolised agar, 202
    bouillon, 202
    gelatine, 202
  carrot, 200
  China green agar (Werbitski), 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 Kolesnikoff), 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 (Sabouraud), 193

Media, preparation of French proof agar (Sabouraud), 193
  Fuchsin agar (Braun), 205
    sulphite agar (Endo), 206
  gelatine, 193
    agar, 193
  glucose formate agar (Kitasato), 180
    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 (Durham), 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 (Loeffler), 207
  malt extract solution (Herschell), 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

Media, preparation of nutrient bouillon, 163
    gelatine, 164
  nutrose agar (Eyre), 172
  oleic acid agar (Fleming), 201
  Omeliansky's nutrient fluid, 189
  Parietti's bouillon, 202
  parsnip, 200
  Pasteur's solution, 191
  peptone rosolic acid water, 186
    water (Dunham), 177
  plaster-of-Paris discs, 192
  potato, 174
    gelatine (Elsner), 204
      (Goadby), 214
  proteid free broth (Uschinsky), 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
    (Libman), 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

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

Metachromatic 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

Mounting film preparations, 85
  paraffin sections, 119

Mouse cages, 342
  holder, 351
  scales, 341

Mucor mucedo, 126

Mucorinæ, 126

Mueller's desiccator, 307

Muffle furnace, 28

Muirs's capsule stain, 100
  flagella stain, 101

Museum preparations of bacteria, 407
    of tissues, 404
    sealing of, 406

Mycelium, 126

Mycoprotein, 135


Naegeli's solution, 191

Naehrstoff agar (Hesse and Niedner), 199

Naked flame, 28

Neisser's stain modified, 111

Net micrometer, 63

Neutral litmus solution, preparation of, 179
  red, 94

Nitrate bouillon, 185
  peptone solution (Pakes), 186

Nitric organisms in soil, 478

Nitrosoindol reaction, 287

Nitrous organisms in soil, 477

Normal averages (_t.p.r._), 372
  serum, 375

Nosepiece, 57
  double, 58
  triple, 58

Navy's anaerobic method, 244
  jars, 245

Nuclei, to stain, 105

Nucleus of bacteria, 135

Numerical aperture, 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

Oïdium, 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

Opsonin, 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


Pakes' counting disc, 424
  filter reservoir, 45

Papier chardin, 158

Pappenheim's stain, 111

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-typhoid 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, 186
  water (Dunham), preparation of, 177

Percentage formula, 496

Perchloride of mercury, 27

Perisporaceæ, 127

Peritrichous bacilli, 136

Permanent preparations of bacteria, 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

Pipettes, automatic, 13
  blood, 11
  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, 18
  to sterilise, 31

Piridin method of staining spirochætes, 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, 18
  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 experimental animals, 396

Potato gelatine (Eisner), 204
    (Goadby), 214
  medium, preparation of, 174

Potted meat, analysis of, 460

Pouring plates, 227

Preparation of experimental animals, 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, measurement of, 305
  of temperature, measurement of, 298

Rat cages, 342

Raw milk, Saul's test for, 442

Reaction of medium, 305
    optimum, 305
    range of, 305
  scale, 153

Reduced pressure and temperature table, 501

Reducing agents, production, 389
    tests for, 289

Reduction of nitrates, 389

Reichert's thermo-regulator, 218

Relation of bacteria to environment, 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

Rosolic 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

Sarcinæ, morphology of, 132

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
    (Libman), 212
    plates, 250
    (Wertheimer), 211
  bouillon, 210
  collection of, 379
  dextrose water (Hiss), 188
  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, 154

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

Spirochæta, morphology of, 133

Spirochætes 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 (Burri), 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

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 cultures, test for, 290

Sunlight, 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 inoculation 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 filters, 478

Test-tubes, 3
  to clean infected, 19
    new, 18
  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
  Reichert'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

Torulæ, differentiation from saccharomyces, 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, 110, 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 choleræ 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

Washing red blood cells, 388
  tissues, 115

Water, analysis of, 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, 31

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, 110

Zoogloea, 134

Zymogenic bacteria, 131




SAUNDERS' BOOKS

on

Pathology, Physiology

Histology, Embryology

Bacteriology, Biology

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Mallory's

Pathologic Histology

~Pathologic Histology.~ By FRANK B. MALLORY, M. D., Associate Professor of
Pathology, Harvard University Medical School. Octavo of 677 pages, with
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     Dr. Mallory here presents _pathology_ from the morphologic
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Howell's Physiology

~A Text-Book of Physiology.~ By WILLIAM H. HOWELL, PH.D., M. D., Professor
of Physiology in the Johns Hopkins University, Baltimore, Md. Octavo of
1020 pages, 306 illustrations. Cloth, $4.00 net.

~THE NEW (5th) EDITION~

     Dr. Howell has had many years of experience as a teacher of
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Mallory _and_ Wright's Pathologic Technique

~Fifth Edition~

~Pathologic Technique.~ A Practical Manual for Workers in Pathologic
Histology, including Directions for the Performance of Autopsies and for
Clinical Diagnosis by Laboratory Methods. By FRANK B. MALLORY, M. D.,
Associate Professor of Pathology, Harvard University; and JAMES H.
WRIGHT, M. D., Director of the Pathologic Laboratory, Massachusetts
General Hospital. Octavo of 500 pages, with 152 illustrations. Cloth,
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     In revising the book for the new edition the authors have
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Eyre's Bacteriologic Technic

~Bacteriologic Technic.~ A Laboratory Guide for the Medical, Dental, and
Technical Student. By J. W. H. EYRE, M. D., F. R. S. Edin., Director of
the Bacteriologic Department of Guy's Hospital, London. Octavo of 520
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~JUST READY--NEW (2d) EDITION, REWRITTEN~

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     the new edition of Dr. Eyre's work is indispensable. The
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McFarland's Pathology

~A Text-Book of Pathology.~ By JOSEPH MCFARLAND, M. D., Professor of
Pathology and Bacteriology in the Medico-Chirurgical College of
Philadelphia. Octavo of 856 pages, with 437 illustrations, many in
colors. Cloth, $5.00 net; Half Morocco, $6.50 net.

~THE NEW (2d) EDITION~

     You cannot successfully treat disease unless you have a
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McFarland's Biology: Medical and General

~Biology: Medical and General~--By JOSEPH MCFARLAND, M. D., Professor of
Pathology and Bacteriology in the Medico-Chirurgical College of Phila.
12mo, 457 pages, 160 illustrations. Cloth, $1.75 net.

~JUST READY--NEW (2d) EDITION~

     This work is both a _general_ and _medical_ biology. The
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~Frederic P. Gorham, A. M.~, _Brown University_.

     "I am greatly pleased with it. Perhaps the highest praise
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McFarland's Pathogenic Bacteria and Protozoa

~Pathogenic Bacteria and Protozoa.~ By JOSEPH MCFARLAND, M. D., Professor
of Pathology and Bacteriology in the Medico-Chirurgical College of
Philadelphia. Octavo of 878 pages, finely illustrated. Cloth, $3.50 net.

~NEW (7th) EDITION, ENLARGED~

     Dr. McFarland has subjected his book to a most vigorous
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Hill's Histology and Organography

~A Manual of Histology and Organography.~ By CHARLES HILL, M. D., formerly
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Stengel's Text-Book of Pathology

~Fifth Edition~

~A Text-Book of Pathology.~ By ALFRED STENGEL, M. D., Professor of
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~WITH 400 TEXT-CUTS, MANY IN COLORS, AND 7 COLORED PLATES~

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     "This volume is intended to present the subject of pathology
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       *       *       *       *       *

Stiles' Nutritional Physiology

~Nutritional Physiology.~ By PERCY GOLDTHWAIT STILES, Assistant Professor
of Physiology at Simmons College, Boston. 12mo of 295 pages,
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~ILLUSTRATED~

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       *       *       *       *       *

Jordan's General Bacteriology

~A Text-Book of General Bacteriology.~ By EDWIN O. JORDAN, PH.D.,
Professor of Bacteriology in the University of Chicago and in Rush
Medical College. Octavo of 623 pages, illustrated. Cloth, $3.00 net.

~NEW (3d) EDITION~

     Professor Jordan's work embraces the entire field of
     bacteriology, the non-pathogenic as well as the pathogenic
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     engineering, etc.

~Prof. Severance Burrage~, _Associate Professor of Sanitary Science,
Purdue University._

     "I am much impressed with the completeness and accuracy of
     the book. It certainly covers the ground more completely
     than any other American book that I have seen."

       *       *       *       *       *

Buchanan's Veterinary Bacteriology

~Veterinary Bacteriology.~ By ROBERT E. BUCHANAN, PH.D., Professor of
Bacteriology in the Iowa State College of Agriculture and Mechanic Arts.
Octavo, 516 pages, 214 illustrations. Cloth, $3.00 net.

~THE BEST PUBLISHED~

     Professor Buchanan discusses thoroughly all bacteria causing
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     consideration of immunity, opsonic index, reproduction,
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     toxins, antitoxins, tuberculins, and vaccines that have
     proved of diagnostic or therapeutic value. Then, in addition
     to bacteria and protozoa proper, he considers molds,
     mildews, smuts, rusts, toadstools, puff-balls, and the other
     fungi pathogenic for animals.

~B. F. Kaupp, D. V. S.~, _State Agricultural College, Fort Collins._

     "It is the best in print on the subject. What pleases me
     most is that it contains all the late results of research.
     It fills a long felt want."


Heisler's Embryology

~A Text-Book of Embryology.~ By JOHN C. HEISLER, M.D., Professor of
Anatomy in the Medico-Chirurgical College, Philadelphia. Octavo volume
of 435 pages, with 212 illustrations, 32 of them in colors. Cloth, $3.00
net.

~THIRD EDITION--WITH 212 ILLUSTRATIONS, 32 IN COLORS~

This edition represents all the advances recently made in the science of
embryology. Many portions have been entirely rewritten, and a great deal
of new and important matter added. A number of new illustrations have
also been introduced and these will prove very valuable. Heisler's
Embryology has become a standard work.

~G. Carl Huber, M.D.~, _Professor of Embryology at the Wistar Institute,
University of Pennsylania._

     "I find this edition of 'A Text-Book of Embryology,' by Dr.
     Heisler, an improvement on the former one. The figures added
     increase greatly the value of the work. I am again
     recommending it to our students."

       *       *       *       *       *

Böhm, Davidoff, _and_ Huber's Histology

~A Text-Book of Human Histology.~ Including Microscopic Technic. By DR. A.
A. BÖHM and DR. M. VON DAVIDOFF, of Munich, and G. CARL HUBER, M.D.,
Professor of Embryology at the Wistar Institute, University of
Pennsylvania. Handsome octavo of 528 pages, with 361 beautiful original
illustrations. Flexible cloth, $3.50 net.

~SECOND EDITION, ENLARGED~

     The work of Drs. Böhm and Davidoff is well known in the
     German edition, and has been considered one of the most
     practically useful books on the subject of Human Histology.
     This second edition has been in great part rewritten and
     very much enlarged by Dr. Huber, who has also added over one
     hundred original illustrations. Dr. Huber's extensive
     additions have rendered the work the most complete students'
     text-book on Histology in existence.

~Boston Medical and Surgical Journal~

     "Is unquestionably a text-book of the first rank, having
     been carefully written by thorough masters of the subject,
     and in certain directions it is much superior to any other
     histological manual."

       *       *       *       *       *

Wells' Chemical Pathology

~Chemical Pathology.~--Being a Discussion of General Pathology from the
Standpoint of the Chemical Processes Involved. By H. GIDEON WELLS,
PH.D., M.D., Assistant Professor of Pathology in the University of
Chicago. Octavo of 616 pages. Cloth, $3.25 net.

~JUST READY--NEW (2d) EDITION~

     Dr. Wells' work is written for the physician, for those
     engaged in research in pathology and physiologic chemistry,
     and for the medical student. In the introductory chapter are
     discussed the chemistry and physics of the animal cell,
     giving the essential facts of ionization, diffusion, osmotic
     pressure, etc., and the relation of these facts to cellular
     activities. Special chapters are devoted to _Diabetes_ and
     to _Uric-acid Metabolism and Gout_.

~Wm. H. Welch, M.D.~ _Professor of Pathology, Johns Hopkins University._

     "The work fills a real need in the English literature of a
     very important subject, and I shall be glad to recommend it
     to my students."

       *       *       *       *       *

Lusk's Elements of Nutrition

~Elements of the Science of Nutrition.~ By GRAHAM LUSK, PH.D., Professor
of Physiology at Cornell Medical School. Octavo volume of 302 pages.
Cloth, $3.00 net.

~THE NEW (2d) EDITION--TRANSLATED INTO GERMAN~

     Prof. Lusk presents the scientific foundations upon which
     rests our knowledge of nutrition and metabolism, both in
     health and in disease. There are special chapters on the
     metabolism of diabetes and fever, and on purin metabolism.
     The work will also prove valuable to students of _animal
     dietetics_ at agricultural stations.

~Lewellys F. Barker, M. D.~ _Professor of the Principles and Practice of
Medicine, Johns Hopkins University._

     "I shall recommend it highly to my students. It is a comfort
     to have such a discussion of the subject in English."


Daugherty's Economic Zoölogy

~Economic Zoölogy.~ By L. S. DAUGHERTY, M. S., PH. D., Professor of
Zoölogy, State Normal School, Kirksville, Mo., and M. C. DAUGHERTY,
author with Jackson of "Agriculture Through the Laboratory and School
Garden." Part I: _Field and Laboratory Guide_. 12mo of 237 pages,
interleaved. Cloth, $1.25 net. Part II: _Principles._ 12mo of 406 pages,
illustrated. Cloth, $2.00 net.

~ILLUSTRATED~

     There is no other book just like this. Not only does it give
     the salient facts of structural zoölogy and the development
     of the various branches of animals, but also the natural
     history--the _life and habits_--thus showing the
     interrelations of structure, habit, and environment. In a
     word, it gives the principles of zoölogy and _their actual
     application_. The economic phase is emphasized.

     Part I--the _Field and Laboratory Guide_--is designed for
     practical instruction in the field and laboratory. To
     enhance its value for this purpose blank pages are inserted
     for notes.

       *       *       *       *       *

Drew's Invertebrate Zoölogy

~A Laboratory Manual of Invertebrate Zoölogy.~ By GILMAN A. DREW, PH. D.,
Assistant Director at Marine Biological Laboratory, Woods Hole, Mass.
With the aid of Former and Present Members of the Zoölogical Staff of
Instructors. 12mo of 213 pages. Cloth, $1.25 net.

~JUST READY--NEW (2d) EDITION~

     The subject is presented in a logical way, and the type
     method of study has been followed, as this method has been
     the prevailing one for many years.

~Prof. Allison A. Smyth, Jr., Virginia Polytechnic Institute~

     "I think it is the best laboratory manual of zoölogy I have
     yet seen. The large number of forms dealt with makes the
     work applicable to almost any locality."

       *       *       *       *       *

Norris' Cardiac Pathology

~Studies in Cardiac Pathology.~ By GEORGE W. NORRIS, M.D., Associate in
Medicine at the University of Pennsylvania. Large octavo of 235 pages,
with 85 superb illustrations. Cloth, $5.00 net.

~SUPERB ILLUSTRATIONS~

     The wide interest being manifested in heart lesions makes
     this book particularly opportune. The illustrations are
     superb and are faithful reproductions of the specimens
     photographed. Each illustration is accompanied by a detailed
     description; besides, there is ample letter press
     supplementing the pictures. Considerable matter of a
     diagnostic and therapeutic nature has been interwoven.

~Boston Medical and Surgical Journal~

     "The illustrations are arranged in such a way as to
     illustrate all the common and many of the rare cardiac
     lesions, and the accompanying descriptive text constitutes a
     fairly continuous didactic treatise."

       *       *       *       *       *

McConnell's Pathology

~A Manual of Pathology.~ By GUTHRIE MCCONNELL, M.D., Professor of
Bacteriology and Pathology at Temple University, Philadelphia. 12mo of
523 pages, with 170 illustrations. Flexible leather, $2.50 net.

~NEW (2d) EDITION~

     Dr. McConnell has discussed his subject with a clearness and
     precision of style that make the work of great assistance to
     both student and practitioner. The illustrations have been
     introduced for their practical value.

~New York State Journal of Medicine~

     "The book treats the subject of pathology with a
     thoroughness lacking in many works of greater pretension.
     The illustrations--many of them original--are profuse and of
     exceptional excellence."

       *       *       *       *       *

Hektoen and Riesman's Pathology

AMERICAN TEXT-BOOK OF PATHOLOGY. Edited by LUDVIG HEKTOEN, M.D.,
Professor of Pathology, Rush Medical College, Chicago; and DAVID
RIESMAN, M.D., Professor of Clinical Medicine, Philadelphia Polyclinic.
Octavo of 1245 Pages, 443 illustrations, 66 in colors. Cloth, $7.50 net;
Half Morocco, $9.00 net.


Dürck _and_ Hektoen's Special Pathologic Histology

~Atlas and Epitome of Special Pathologic Histology.~ By DR. H. DÜRCK, of
Munich. Edited, with additions, by LUDVIG HEKTOEN, M. D., Professor of
Pathology, Rush Medical College, Chicago. In two parts. Part
I.--Circulatory, Respiratory, and Gastro-intestinal Tracts. 120 colored
figures on 62 plates, and 158 pages of text. Part II.--Liver, Urinary
and Sexual Organs, Nervous System, Skin, Muscles, and Bones. 123 colored
figures on 60 plates, and 192 pages of text. Per part: Cloth, $3.00 net.
_In Saunders' Hand-Atlas Series._

     The great value of these plates is that they represent in
     the exact colors the effect of the stains, which is of such
     great importance for the differentiation of tissue. The text
     portion of the book is admirable, and, while brief, it is
     entirely satisfactory in that the leading facts are stated,
     and so stated that the reader feels he has grasped the
     subject extensively.

~William H. Welch, M.D.,~ _Professor of Pathology, Johns Hopkins
University, Baltimore._

     "I consider Dürck's 'Atlas of Special Pathologic Histology,'
     edited by Hektoen, a very useful book for students and
     others. The plates are admirable."

       *       *       *       *       *

Sobotta _and_ Huber's Human Histology

~Atlas and Epitome of Human Histology.~ By PRIVATDOCENT DR. J. SOBOTTA, of
Würzburg. Edited, with additions, by G. CARL HUBER, M. D., Professor of
Histology and Embryology in the University of Michigan, Ann Arbor. With
214 colored figures on 80 plates, 68 text-illustrations, and 248 pages
of text. Cloth, $4.50 net. _In Saunders' Hand-Atlas Series._

~INCLUDING MICROSCOPIC ANATOMY~

     The work combines an abundance of well-chosen and most
     accurate illustrations, with a concise text, and in such a
     manner as to make it both atlas and text-book. The great
     majority of the illustrations were made from sections
     prepared from human tissues, and always from fresh and in
     every respect normal specimens. The colored lithographic
     plates have been produced with the aid of over thirty
     colors.

~Boston Medical and Surgical Journal~

     "In color and proportion they are characterized by
     gratifying accuracy and lithographic beauty."


Bosanquet on Spirochætes

~Spirochætes~: A Review of Recent Work, with Some Original Observations.
By W. CECIL BOSANQUET, M.D., Fellow of the Royal College of Physicians,
London. Octavo of 152 pages, illustrated. $2.50 net.

~ILLUSTRATED~

     This is a complete and authoritative monograph on the
     spirochætes, giving morphology, pathogenesis,
     classification, staining, etc. Pseudospirochætes are also
     considered, and the entire text well illustrated. The high
     standing of Dr. Bosanquet in this field of study makes this
     new work particularly valuable.

       *       *       *       *       *

Levy _and_ Klemperer's Clinical Bacteriology

~The Elements of Clinical Bacteriology.~ By DRS. ERNST LEVY and FELIX
KLEMPERER, of the University of Strasburg. Translated and edited by
AUGUSTUS A. ESHNER, M. D., Professor of Clinical Medicine, Philadelphia
Polyclinic. Octavo volume of 440 pages, fully illustrated. Cloth, $2.50
net.

~S. Solis-Cohen, M.D.~, _Professor of Clinical Medicine, Jefferson Medical
College_, Philadelphia.

     "I consider it an excellent book. I have recommended it in
     speaking to my students."

       *       *       *       *       *

Lehmann, Neumann, _and_ Weaver's Bacteriology

~Atlas and Epitome of Bacteriology~: INCLUDING A TEXT-BOOK OF SPECIAL
BACTERIOLOGIC DIAGNOSIS. By PROF. DR. K. B. LEHMANN and DR. R. O.
NEUMANN, of Würzburg. _From the Second Revised and Enlarged German
Edition._ Edited, with additions, by G. H. WEAVER, M. D., Assistant
Professor of Pathology and Bacteriology, Rush Medical College, Chicago.
In two parts. Part I.--632 colored figures on 69 lithographic plates.
Part II.--511 pages of text, illustrated. Per part: Cloth, $2.50 net.
_In Saunders' Hand-Atlas Series._

       *       *       *       *       *

Dürck and Hektoen's General Pathologic Histology

ATLAS AND EPITOME OF GENERAL PATHOLOGIC HISTOLOGY. By PR. DR. H. DÜRCK,
of Munich. Edited, with additions, by LUDVIG HEKTOEN, M. D., Professor
of Pathology in Rush Medical College, Chicago. 172 colored figures on 77
lithographic plates, 36 text-cuts, many in colors, and 353 pages. Cloth,
$5.00 net. _In Saunders' Hand Atlas Series._


    American Text-Book of Physiology      Second Edition

AMERICAN TEXT-BOOK OF PHYSIOLOGY. In two volumes. Edited by WILLIAM H.
HOWELL, PH. D., M.D., Professor of Physiology in the Johns Hopkins
University, Baltimore, Md. Two royal octavos of about 600 pages each,
illustrated. Per volume: Cloth, $3.00 net; Half Morocco, $4.25 net.

     "The work will stand as a work of reference on physiology.
     To him who desires to know the status of modern physiology,
     who expects to obtain suggestions as to further physiologic
     inquiry, we know of none in English which so eminently meets
     such a demand."--_The Medical News._


    Warren's Pathology and Therapeutics      Second Edition

SURGICAL PATHOLOGY AND THERAPEUTICS. By JOHN COLLINS WARREN, M. D., LL.
D., F. R. C. S. (Hon.), Professor of Surgery, Harvard Medical School.
Octavo, 873 pages, 136 relief and lithographic illustrations, 33 in
colors. With an Appendix on Scientific Aids to Surgical Diagnosis and a
series of articles on Regional Bacteriology. Cloth, $5.00 net; Half
Morocco, $6.50 net.


Gorham's Bacteriology

A LABORATORY COURSE IN BACTERIOLOGY. For the Use of Medical,
Agricultural, and Industrial Students. By FREDERIC P. GORHAM, A. M.,
Associate Professor of Biology in Brown University, Providence, R. I.,
etc. 12mo of 192 pages, with 97 illustrations. Cloth, $1.25 net.

     "One of the best students' laboratory guides to the study of
     bacteriology on the market.... The technic is thoroughly
     modern and amply sufficient for all practical
     purposes."--_American Journal of the Medical Sciences._


    Raymond's Physiology      New (3d) Edition

HUMAN PHYSIOLOGY. By JOSEPH H. RAYMOND, A. M., M. D., Professor of
Physiology and Hygiene, Long Island College Hospital, New York. Octavo
of 685 pages, with 444 illustrations. Cloth, $3.50 net.

     "The book is well gotten up and well printed, and may be
     regarded as a trustworthy guide for the student and a useful
     work of reference for the general practitioner. The
     illustrations are numerous and are well executed."--_The
     Lancet_, London.


    Ball's Bacteriology      Seventh Edition, Revised

ESSENTIALS OF BACTERIOLOGY: being a concise and systematic introduction
to the Study of Micro-organisms. By M. V. BALL, M. D., Late
Bacteriologist to St. Agnes' Hospital, Philadelphia. 12mo of 289 pages,
with 135 illustrations, some in colors. Cloth, $1.00 net. _In Saunders'
Question-Compend Series._

     "The technic with regard to media, staining, mounting, and
     the like is culled from the latest authoritative
     works."--_The Medical Times_, New York.


    Budgett's Physiology      New (3d) Edition

ESSENTIALS OF PHYSIOLOGY. Prepared especially for Students of Medicine,
and arranged with questions following each chapter. By SIDNEY P.
BUDGETT, M. D., formerly Professor of Physiology, Washington University,
St. Louis. Revised by HAVAN EMERSON, M. D., Demonstrator of Physiology,
Columbia University. 12mo volume of 250 pages, illustrated. Cloth, $1.00
net. _Saunders' Question-Compend Series._

     "He has an excellent conception of his subject.... It is one
     of the most satisfactory books of this class"--_University
     of Pennsylvania Medical Bulletin._


    Leroy's Histology      New (4th) Edition

ESSENTIALS OF HISTOLOGY. By LOUIS LEROY, M. D., Professor of Histology
and Pathology, Vanderbilt University, Nashville, Tennessee. 12mo, 263
pages, with 92 original illustrations. Cloth, $1.00 net. _In Saunders'
Question-Compend Series._

     "The work in its present form stands as a model of what a
     student's aid should be; and we unhesitatingly say that the
     practitioner as well would find a glance through the book of
     lasting benefit."--_The Medical World_, Philadelphia.


Barton and Wells' Medical Thesaurus

A THESAURUS OF MEDICAL WORDS AND PHRASES. By WILFRED M. BARTON, M. D.,
Assistant Professor of Materia Medica and Therapeutics, and WALTER A.
WELLS, M. D., Demonstrator of Laryngology, Georgetown University,
Washington, D.C. 12mo, 534 pages. Flexible leather, $2.50 net; thumb
indexed, $3.00 net.


    American Pocket Dictionary      New (8th) Edition

DORLAND'S POCKET MEDICAL DICTIONARY. Edited by W. A. NEWMAN DORLAND, M.
D., Editor "American Illustrated Medical Dictionary." Containing the
pronunciation and definition of the principal words used in medicine and
kindred sciences, with 64 extensive tables. 677 pages. Flexible leather,
with gold edges, $1.00 net; with patent thumb index, $1.25 net.

     "I can recommend it to our students without reserve."--J. H.
     HOLLAND, M.D., _of the Jefferson Medical College_,
     Philadelphia.





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