IRLF

THE LIBRARY

OF

THE UNIVERSITY
OF CALIFORNIA

PRESENTED BY

PROF. CHARLES A. KOFOID AND
MRS. PRUDENCE W. KOFOID

AIDS TO BACTERIOLOGY

AIDS

TO

BACTERIOLOGY

BY

C. G. MOOR, M.A. (CANTAB.), F.I.C.

CAI'TAIN 1ST LONDON SANITARY COMPANY

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PREFACE TO THE THIRD EDITION

WHILE no notable discovery has been recorded in bac-
teriology since the appearance of the second edition of this
book, much extension and consolidation of previous know-
ledge have been effected. This has necessitated revision
and enlargement of nearly all parts of the book.

A war on highly-cultivated soil, with troops occupying
the same ground for lengthy periods, has very forcibly
obtruded the pathogenic abilities of many faecal bacteria.
Tetanus, typhoid, paratyphoid, and dysentery bacilli, the
bacillus of malignant oedema, and the faecal pyogenic
streptococci, have all been in evidence. Bacillus WdcMi,
under one or other of its many names, has also claimed
much attention in connection with gaseous gangrene.
In the case of these organisms, however, new developments
have been chiefly in the directions of pathology and treat-
ment rather than in bacteriology.

We have considerably extended our article on and
devoted an appendix to the meningococcus — an organism
whose life outside the human body remains largely a
matter for conjecture. We have collected in one chapter
a summary of the little that is known about the filterable
viruses, and have also dealt with anaphylaxis and the
preparation of vaccines. In accordance with the advice
of Professor David Ellis, we have substituted the classifi-
cation of Migula for that of Hueppe.

Few bacteriological investigations are free from possible
fallacy or insusceptible of wrong deduction. The influ-
ence of anti-typhoid inoculation on the Widal test, the
absence of tubercle bacilli in milk from tuberculous udders,
the absence of colon bacilli from polluted water, the
incorrect selection of organisms in the preparation of bac-
terial vaccines, and the fact that the popular Wassermann

M3G0094

vi PREFA CE

reaction is not a true antigen test, are but a few instances
of many which occasionally intrude to mislead the unwary
bacteriologist and to confuse those who rely on his findings.
So far as space allows, we have briefly indicated where,
why, and how, errors may be expected and avoided.

Surgeons on military service, when first called on to deal
with the complicated wounds caused by the pointed-nose
bullet and contaminated with resistant faecal organisms
carried in from soil and clothing, found methods then current
for antisepsis quite inadequate. Discussion of the relative
values of antiseptics for surgical use continues, the ten-
dency at present being to use those dependent for action on
the oxidising properties of chlorine. Sir Almroth Wright
tackles the problem of cleansing wounds by the applica-
tion of very strong saline solution, which results in a con-
siderable exudation of serum. Though it has no gcrmicidal
action for the streptococci, this process is much used, in-
voking both praise and criticism. Magnesium sulphate
and glycerin are also used, and, it is said, kill the strepto-
cocci (A. E. Morison's process).

Bacteriology promises to modify agricultural methods,
and in Chapter XX. we give resumes of what has been
accomplished in soil-sterilisation and nitrogen-fixation.

While most bacteriologists have had some personal ex-
perience of bacterial mutability, to which we make fre-
quent reference, this remains largely of academic interest.
In practice, species tend to crop up fairly true to type, and
exceptional conditions are usually required to produce
important change in attributes.

C. G. M.
W. P.

LONDON,

May, 1916.

CONTENTS

CHAPTER PAGE

I. INTRODUCTION ...... 1

II. BACTERIOLOGICAL APPARATUS .... 29

III. THE PREPARATION AND USE OF NUTRIENT MEDIA 33

IV. THE MICROSCOPIC EXAMINATION OF BACTERIA . 46
V. THE -ACID-FAST ORGANISMS .... 60

VI. SPORE-FORMING PATHOGENIC ORGANISMS . . 75

VII. THE COLON-TYPHOID GROUP .... 88

VIII. THE DIPHTHERIA BACILLUS . . . .110

IX. THE BACILLI OF H^MORRHAGIC SEPTICAEMIA . 121

X. MICRO-ORGANISMS OF SUPPURATION AND SEPTIC

DISEASES ....... 127

XI. PNEUMONIA MALTA FEVER .... 142

XII. THE INFLUENZA AND GLANDERS BACILLI . .145

XIII. THE SPIRILLA 150

XIV. THE TRICHOMYCETES . . . . . 156
XV. THE BLASTOMYCETES 161

XVI. THE HYPHOMYCETES . . . . .167

XVII. THE PATHOGENIC PROTOZOA . . . .174,

XVIII. FERMENTATION ENZYMES SULPHUR AND IRON

BACTERIA BACTERIAL DISEASES OF PLANTS . 188

XIX. DISEASES OF QUESTIONABLE ORIGIN THE FILTER-
ABLE VIRUSES 195

vii

viii CONTENTS

CHAPTER PAGE

XX. THE BACTERIOLOGY OF SEWAGE, SHELLFISH, MEAT,

SOIL, AIR, AND MILK 202

XXI. BACTERIOLOGY OF WATER .... 224
XXII. DISINFECTION AND DISINFECTANTS . . . 244

APPENDIX ....... 265

INDEX 267

AIDS TO BACTERIOLOGY

CHAPTER I
INTRODUCTION

THE Thallophyta, which form the lowest group of cryp-
togainic plants, show no division into root and stem, and
have no fibre- vascular system. They are divided into
algae, which contain chlorophyll, and fungi, which contain
none. Excluding the Hymenomycetes (mushrooms, etc.),
the fungi are microscopic, and most genera and species
come within the purview of bacteriology. The Hypho-
mycetes (moulds) and the Blastomycetes (yeasts and
torula) receive frequent attention from the bacteriolo-
gist, and Chapters XV. and XVI. are devoted to them.
While the study of bacteria, or Schizomycetes, is the
primary object of bacteriology, its scope has gradually
extended beyond the fungi to include organisms too
small to be seen with the microscope (ultra-microscopic
organisms) and such unicellular animals (Protozoa) as
cause disease in man, beast, or plant. Of vital impor-
tance is a knowledge of the circumstances under which
organisms die, so disinfection occupies a prominent place
in the science.

Anatomy. — The cell wall is a true membrane (Ellis and
Meyer), and is generally composed of a nitrogenous sub-
stance very similar to chitin, a skeletin not found in
vertebrates, but of frequent occurrence in the cuticles of
invertebrates.

The cell membrane in many, perhaps all, forms some-
times swells to form a capsule. Large numbers of bac-
teria may cluster together in a jelly-like mass known as a
zooglosa.

The cell contents, or cytoplasm, consists of translucent
protoplasm. Scattered through the cell or massed at the
poles of certain bacteria are granules that stain differently

1

2 AIDS TO BACTERIOLOGY

to the remainder of the cell (metachromatic granules).
These are composed of glycogen, fat, or, according to
Dobell, nucleic acid combined with an organic base. They
are probably in large part reserve food substance (Jordan).

The presence of a nucleus in all species is regarded as
proved by the work of Clifford Dobell.

Movement. — Many bacteria, particularly bacilli and
spirilla, are capable of motion, produced by the little
protoplasmic threads (flagella). Ellis has shown that
flagella arise from the cell substance and not from the
membrane.

Some organisms, such as the cholera spirillum, have a
single flagellum at one end (monotricha), in others there is
one at each pole (amphitricha) ; the flagella may assume
the form of a tuft at one pole (lophotricha), or they may
be scattered round the cell (peritricha). The position of
the flagella is always the same in each species; but in
those species having more than one flagellum the number
is not always constant, and depends on the health of the
culture (Ellis). Even these organisms are not always
motile, but go through a resting stage. Motility is best
seen in young cultures when conditions are favourable
for growth. Flagella are not seen when the organism is
examined under the microscope in the usual way, but they
can be observed in specimens specially stained. This
independent movement must not be confused with the
motion that solid particles, whether bacteria or not, exhibit
when suspended in a fluid medium, known as the ' Brownian
movement,' which is variously attributed to electrical
disturbances and to surface tension.

Size. — The unit of measurement adopted is the ' micron '
(often erroneously called a micro-millimetre), which is
equal to 0-001 millimetre, and is represented by the letter//.
The influenza bacillus measures about 0-5 x 0-2 IJL, while
the spirochsete of relapsing fever may attain 40^ in length.
Great differences in length are not observed among the
majority of species, most of the bacilli, for instance, measur-
ing about 2 //. Even smaller than the influenza bacillus
is the organism causing pleuro-pneumonia in cattle, which
is just visible under the highest powers. ' Ultra-micro-
scopic ' organisms capable of passing through the filter mass
of a porcelain filter cause many diseases (see Chapter
XIX.).

REPRODUCTION 3

Reproduction. — Bacteria are asexual, and propagate
by fission. When a cell has attained the maximum size
for its species, it elongates, with constriction round its
middle, followed by a simple partition. Hence the family
name of Schizomycetes. Two young cells are thus formed
from the mother cell. This process may be repeated as
often as once in twenty minutes if conditions are favourable.
An increase in geometric progression is not consistently
maintained, however, owing to various checks on the
growth. While insufficient food, lack of moisture, and
other conditions, disallow unhampered multiplication,
the chief hindrance is the production of inhibitory sub-
stances by the vital activity of the bacteria themselves.

When bacteria have occupied the same site in the body
or lived in the same culture for a long period, or conditions
for growth are otherwise unfavourable, abnormal shapes
and sizes are produced (involution forms). These gener-
ally take stain less readily, or else do not stain at all.
Other characters, such as pathogenicity or fermentation
power, are likely to diminish, and there is general evidence
of degeneracy.

The cbcci do not always separate after fission; division
may occur in one plane with the formation of a chain
(streptococci}; in two planes, producing a cluster (staphy-
lococci); or in three planes, forming cubical bales (sarcina).

Spore-formation is not common; it occurs most fre-
quently in bacilli and spirilla, and rarely in micrococci.

Endogenous Spores. — The protoplasm becomes granular,
and some of the granules coalesce to form a highly re-
fractile round or ovoid body enclosed in a tough membrane.
Spores exhibit very great resistance to heat, desiccation,
and chemical agents. They are thus able to preserve their
species through most disadvantageous circumstances.
When favourable conditions recur, the spore loses its
refractile appearance, elongates, and bursts its membrane
to extrude an organism which divides in the usual manner.
Only one spore is formed in a cell, so that this cannot be
regarded as a reproductive process. While in most cases
the diameter of the spore does not exceed that of the parent
organism, it may be greater, and will give a ' drumstick '
appearance if terminal and round; or it may be swollen
and club-shaped (clostridium). The conditions determin-
ing the formation of spores are variable for different

4 AIDS TO BACTERIOLOGY

organisms. The anthrax bacillus spores when in contact
with free oxygen, while the anaerobes generally require
absence of oxygen. An organism containing no spore,
but ready to divide by fission at maturity, is said to be a
vegetative form.

Arthrospores. — Some of the cells formed by fission were
formerly thought to possess the characters of spores. The
formation was known as ' arthrogenous,' and has only
been noticed in the micrococci. The suggestion is based
on a misconception. Eyre states that these so-called
arthrospores have never been observed to germinate, and
they cannot survive a temperature of 80° C. for ten minutes.
It is now universally accepted that these individuals are
not spores.

The formation of spores in imperfectly divided sister
cells has been interpreted, wrongly, according to most
authorities, as ' autogamy.' There is no evidence that
either this or other sexual process occurs among bacteria.

Bacteria cannot arise ab initio, since a living organism
can only be derived from a living organism (biogenesis).
The supporters of the theory of ' spontaneous generation '
(abiogenesis or archebiosis) believed that organisms could
be produced in a sterile medium without the introduction
of living organisms.

Classification. — The bacteria possess so few morpho-
logical attributes, and so many forms are pleomorphic,
that the ingenuity of bacteriologists has hitherto been
incapable of formulating either a scientific or a convenient
classification. The present nomenclature is more of a
hindrance than a help, for so many organisms are crowded
together in a single genus, many of them possessing names
either unwieldy or unsuitable, that but little assistance
can be expected from the classification. We append one
scheme, that of Migula, and comment on it where necessary:

The bacteria are divided into five families: Coccacese,
Bacteriacese, Spirillacese, Chlamydo - Bacteriacea3, and
Beggiatoacese. These again are subdivided into genera,
based partly on the mode of division and partly on the
number and arrangement of the flagella.

I. COCCACE^. — Round or oval cells. The division into
genera is based on the arrangement of the cocci on division.

(1) Streptococcus. — The cocci form chains. Includes
cocci forming pairs (Diplococci).

CLASSIFICATION 5

(2) MicroGoccus.— Irregular grouping, Micrococci form-
ing clusters in an arrangement suggestive of that of grapes
on a bunch are still known as Staphylococci, though
there is a movement among the precise to eradicate the
word.

(3) Sarcina, or Packet Cocci.— Division in three direc-
tions, forming packets of eight or more elements, which
remain associated in more or less cubical masses.
Non-motile.

(5)

II. BACTERIACEJE. — Straight rod forms.

(1) Bacterium. — Non-motile organisms. The name has
been variously applied in other sj^stems of classification.
In one it signified a very short rod, and in another a rod of
a non-sporing species. In its plural form it is used as a
general term for all the organisms included in this table;
otherwise it has fallen into disuse.

(2) Bacillus. — Migula applied the term to rods possessing
both polar and lateral flagella. It is now customary to
apply the name to all rod-shaped organisms — i.e., all of the
Bacteriacece.

(3) Pseudomonas. — Rods with polar flagella only. The
title is now seldom used except for some bacilli concerned
in the nitrogen cycle, and for others concerned in plant
diseases.

III. SPIRILLACE/E. — Curved or spiral rod forms.
Migula divides this family into three genera: non-motile

organisms (Spirosoma); motile forms with a single polar
flagellum (Microspira) ; and motile forms with more than
one polar flagellum (Spirillum). Other classifications
apply the name Spirillum to a larger class, causing much
confusion in nomenclature. The result is that any member
of the family may now be properly referred to as a spirillum,
though the term is often restricted to those organisms
having three or more definite corkscrew turns. Short
curved rods are commonly termed ' vibrios,' or ' comma '
bacilli, but the latter name is frequently used for, and
should be restricted to, the cholera vibrio.

IV. CHLAMYDO-BACTERIACE.E. — Thread forms not con-
taining sulphur granules.

(1) Streptothrix. — Forms showing true but not dichoto-
mous branching.

6 AIDS TO BACTERIOLOGY

(2) Crenothrix. — Threads thicker at apex than at base.
Each thread is tubular, and inside each a linear series of
cells is arranged, each cell possessing a membrane of its
own. The cells are thrown out at the top, and elongate
to form new threads like those of the parent plant.

(3) Leptothrix. — Threads with or without sheaths,
showing no branching.

(4) Cladothrix. — The threads possess pseudo-branches.
Strings of rod-like cells are enclosed in a sheath.

V. BEaaiATOACE^. — Thread forms containing sulphur
granules.

(1) Beggiatoa. — Long motile free-swimming threads of
colourless cells containing strongly refracting granules of
sulphur.

(2) Thiothrix. — Threads that differ from beggiatoa by
having the filaments attached at one end.

Organisms other than the true bacteria are dealt with
later, and descriptions of them are left to the special mono-
graphs and chapters.

Growth of the Bacteria. — Few bacteria can derive their
nourishment from inorganic sources, and an absence of
chlorophyll prevents all species from utilising atmospheric
carbon dioxide. The large majority require complex
organic compounds such as proteins and carbohydrates as
well as mineral salts for food. Suitable mixtures are pre-
pared for laboratory purposes (culture media — v. Chap-
ter III.). While some simple media allow growth of most
species, by the introduction of other constituents certain
bacteria are favoured and nourish, perhaps with specific
indications of their presence, while other species are killed
or suppressed (selective media). By studies of the pref-
erences and conditions of vigorous growth in this and other
respects, the extraction of a required organism from ad-
mixture with others is facilitated.

Bacteria derive their oxygen either from the air (aerobes)
or from compounds containing oxygen (anaerobes). The
' facultative anaerobes ' grow either in the presence or
absence of oxygen. There are gradations in this respect,
from the strictly aerobic species, which require abundance
of oxygen, and will not grow in its absence, to the anaerobic,
which grow in the absence of free oxygen. Strict anaerobes
do not exist. Organisms that can live without oxvgen thrive
better when oxygen is present, but in very small quantity.

EXTERNAL INFLUENCES 7

Moore and Stenhouse Williams killed tubercle bacilli by
three weeks' exposure to an atmosphere enriched with
oxygen, and found the plague bacillus and stapliylococcus
growths were adversely affected (oxyphobia).

As a rule, bacterial growth ceases at temperatures below
12° C. and above 42° C. The range of temperature within
which bacteria will grow is practically constant for each
species, but there is a more narrow margin (the ' optimum '
temperature) in which each does best. The optimum
temperature differs more or less according to the species.
Normal inhabitants of the human body and organisms
pathogenic for man thrive best at blood-heat (37° C.),
the colon bacillus, which grows luxuriantly at 42° C., being
an exception. Bacteria obtained from the lower animals
generally have the normal temperature of the host as an
optimum. The body- temperature of a fowl is 42° C.,
and avian tubercle bacilli thrive at 43° C., a temperature
at which human tubercle bacilli refuse to grow.

Some bacteria isolated from dung and from heated hay
grow best at temperatures between 60° and 70° C. (ther-
mophilic organisms), while others can grow at 0° C.
Growth at unnatural temperature may result in loss of some
characters.

When obtaining their nourishment from some living
body or ' host,' organisms are known as ' parasites.' The
adjective ' obligate ' is prefixed if they can live only on
this ' host.' If the bacteria grow on dead organic matter,
they are called ' saprophytes.' These are also divided into
' obligate ' and ' facultative ' saprophytes. The term
* obligate parasite ' requires to be used with some reserva-
tion. It merely indicates that hitherto attempts to cul-
tivate an organism on culture media have failed, and a
successful attempt thereat automatically transfers it to the
list of facultative parasites.

Resistance to External Influences — Cold. — The germi-
cidal effect of low temperatures is small. Growth usually
ceases below 10° C., but even after exposure to- 252° C.
(the temperature of liquid hydrogen) for ten hours, or to
— 170° C. for several weeks, bacteria are not killed, and
when placed in favourable conditions show that no serious
impairment has taken place. When water freezes naturally
there is a high death-rate among the bacteria therein,
and repeated freezing and thawing has been found more

8 AIDS TO BACTERIOLOGY

destructive to the typhoid bacillus than continuous freez-
ing. S. C. Keith states that living bacteria are hardly
ever found in clear ice, though they are comparatively
abundant in snow ice and bubbly ice. Cooling is valuable
in the preservation of putrescible material, because it
inhibits bacterial growth.

Desiccation. — Moisture is absolutely necessary for the
growth of bacteria. Ordinary drying in the air has a
detrimental effect on the vegetative forms of bacteria,
spores suffering less or not at all. Resistance varies with
the species. The tubercle bacillus, which retains its
virulence after five months' desiccation, is much more
resistant than the cholera spirillum, which is incapable of
development after three hours' drying in the form of a very
thin film. In spite of the large numbers of colon bacilli
continually being deposited in our streets, Gordon failed
to find the organism in 500 litres of air of the East Central
district of London.

Heat. — The Thermal Death-point (Eyre) for vegetative
forms is ' that temperature which with certainty kills a
watery suspension of the organisms in question after an
exposure of ten minutes.' Eyre defines the moist t.d.p.
for spores as ' that time exposure to a fixed temperature
of 100° C. necessary to effect the death of all the spores
present in a suspension.' Eyre's dry thermal death-
point for both vegetative forms and spores is the tem-
perature that kills each form in a thin film after a time
exposure of ten minutes. The thermal death-point can be
determined with a fair degree of accuracy for each species,
and is much higher for spores than for the vegetative
forms. Heat may be applied either in the absence or
presence of moisture, known respectively as ' dry heat '
and ' moist heat.'

Dry heat is much less efficient than moist heat, the
death of the protoplasm taking place more readily in the
presence of moisture. In practical disinfection moist heat
is therefore used where possible, especially where penetra-
tion of fabrics is required (see p. 245). Steam may be
applied under pressure, as in an autoclave, when a fifteen
minutes' exposure to 125° C. suffices to destroy all
known organisms. When moisture is present, most vege-
tative forms are killed by an exposure to 65° C. for ten
minutes, while an exposure for an hour and a half at

STERILISATION 9

120° C. to 128° C. is necessary to attain the same result
with dry heat. While the spores of most pathogenic
bacteria are destroyed by boiling for a short time, those of
non-pathogenic species require a much longer exposure.
A temperature of 140° for three hours is necessary to
secure destruction of the spores of some organisms by dry
heat. B. subtilis spores are particularly resistant, and
Ellis found they resisted boiling at 100° C. for six hours
without injury to their germinating power.

Fractional Sterilisation. — Instead of performing sterili-
sation in one operation, ' discontinuous ' or ' fractional '
sterilisation may be employed. For this the medium or
other material is exposed to heat for a few minutes on two
or three successive days. Any spores surviving the first
heating germinate, and the resulting organisms succumb to
the second and third sterilisations. Hewlett ascribes some
of the sterilising effect simply to the injurious action of
alternate heating and cooling. As spores may take days
or even weeks to germinate, the procedure is not always
certain in its action, and it is thus possible for spores to
germinate in a medium believed to be sterile.

Light. — Sunlight and, to a less degree, the electric arc
are very injurious to certain forms of bacteria. The red
and yellow rays of the spectrum have little effect, germi-
cidal action being exerted by the blue and violet portions.
The violet rays are therapeutically applied in the Finsen
light treatment of lupus vulgaris. Water and the ordinary
milk of commerce can be sterilised without appreciable
rise of temperature by exposure to the ultra-violet rays of
a quartz-mercury lamp. Hewlett and Barnard find that
these rays have practically no power of penetration, and
are stopped even by thin glass. The action of light is
superficial, even a short depth of water stopping the action.
Probably sunlight does not materially assist the purifica-
tion of rivers. Definite germicidal action cannot yet be
attributed to the Rontgen rays, while radium emanations
require long exposure and close contact to exert appreciable
action.

Pressure. — If suddenly applied or released, pressure
may rupture bacterial cells; otherwise no appreciable effect
has been observed.

Electricity. — The products of electrolysis may destroy
bacteria, and currents of high potential may inhibit

io AIDS TO BACTERIOLOGY

growth, C. Russ (Proc. Roy. Sor,., 1909, p. 314) shows
that under the influence of a suitable current certain
bacteria aggregate at one or other electrode, and uses this
action for the collection of bacteria from a fluid medium.
Thus, if sodium chloride be used, nearly all go to the
positive electrode, a large number being killed. It is
suggested that successful ionisation of suppurating wounds
is effected by drawing the bacteria out of the tissues and
then exposing them directly to the destructive effect of the
electric current.

Products of Metabolism in Bacteria.— The action of
bacteria on the surrounding medium is generally of an
analytical nature, complex nitrogenous substances and
carbohydrates being decomposed into simpler substances
Jordan attempts to classify the chemical products of
bacteria under the following heads: (i.) The secretions or
substances which subserve some purposeful end in the cell
economy; (ii.) the excretions or substances ejected because
useless; (iii.) the disintegration products formed by the
breaking down of food substances; and (iv.) the true cell
substance. The word ' aporrhegma ' has been applied
to any substance split off by biological actions, but its
employment is not encouraged by the precise.

Production of Heat. — Bacteria of thermophilic character
are responsible for self -heating of hay and some forms of
spontaneous combustion, especially the firing of moist
cotton.

Photogenesis. — Phosphorescence, especially that seen on
fish, is often due to bacteria. The photogenic bacteria
are generally, but not always, of marine origin.

Some bacteria produce a fluorescence in the culture
medium.

Chrom,ogenesis. — A number of bacteria produce pigments,
often lipochromes. The raison d'etre of these pigments is
uncertain, but probably they are excretory products of no
service to the organism. B. prodigiosus produces a red
pigment. On one occasion it contaminated a water-supply
and infected the bread made with it. The same organism
is the causative agent of the ' bleeding host.' Chromidrosis
(coloured sweat) is attributed to bacteria. While the term
' chromogen ' is restricted to bacteria that produce a pig-
ment either for retention in their cells or for excretion,
many others produce a pigment or change of colour in

PRODUCTS OF METABOLISM

culture media. Beycrinck gives some instances that result
from oxidation by bacterial action: The oxidation of
quinic acid to protocatechuic acid is brought about by
Micrococcus calco-aceticus andB.fluorescens non-liquefaciens.
Quercitol is oxidised to pyrogallol by Pseudomonas aro-
matica. Melanine can be formed from tyrosine by Vibrio
tyrosinatica, isolated from sea-water and sewage. Aceto-
bacter melanogenum, occurring in vinegar, converts peptone
into a caramel-like substance.

Fermentation. — By bacteria (Chapter XVIII.); by
moulds (Chapter XVI.); by yeasts (Chapter XV.); and by
enzymes, or ' zymolysis ' (Chapter XVIII.).

Putrefaction. — Nitrogenous substances, such as the
proteins, are decomposed by bacteria, particularly by those
of the Proteus group. The insoluble albumins, etc., are
first converted into albumoses and peptones; then ammo-
acids are produced, and a variety of other substances, such
as fatty acids, basic bodies, and gases.

Indole. — Indole is one of the final products of the
decomposition of proteins, and is of importance in bacterial
diagnosis, as organisms, otherwise very similar, may
differ in regard to the production of this substance. To
ascertain if an organism produces indole, it is inoculated
into peptone water (2 per cent.) or into a glucose-free
broth. The culture is incubated for twenty-four hours or
longer. Two c.c. of a stock solution of sodium nitrite
(5 per cent.) are diluted to 100 c.c. One c.c. of this weak
solution is added to the culture (the volume of which
should be about 10 c.c.). A little concentrated hydrochloric
acid is then allowed to trickle down the wall of the in-
clined test-tube to liberate nitrous acid, which gives a
pink colour with the indole. If a definite reaction is not
obtained, the tube is placed in the blood-heat incubator for
half an hour to intensify the colour. At the same time as
the peptone water is inoculated, another tube should be
infected with an organism known to produce indole, and
an uninoculated control- tube placed with the others in the
incubator. The purity of the reagents and the power of
the peptone to allow production of indole is thus secured.
As a more delicate reaction for indole the following reagent
may be used: 4 grammes of paradimethylamidobenzalde-
hyde are dissolved in 380 c.c. of absolute alcohol and 80 c.c.
of concentrated hydrochloric acid. To 2 c.c. of the culture

12 ' AIDS TO BACTERIOLOGY

similar quantities of the reagent and of a saturated aqueous
solution of potassium persulphate are added, when indole
produces a rose-pink colour.

The cholera spirillum reduces peptone with formation of
nitrites, and gives the reaction on the addition of acid alone.

The production of indole depends on the presence of the
tryptophan group in the culture medium,and Zipf el has pro-
posed the following medium as most suitable : ammonium
lactate (0-5 per cent.), dicalcium phosphate (0-2 per cent.),
magnesium sulphate (0-02 per cent.), and tryptophan
(0-03 per cent.).

Production of Acid and Alkali. — Many bacteria produce
ammonia in a sugar-free broth, while the power of some
to ferment sugars, with the production of acid, and some-
times of gas as well, is used for diagnostic purposes.

Nitrification, Denitrification, Nitrogen Fixation. — The
bacteria concerned in the nitrogen cycles are dealt with
in the chapter on ' The Bacteriology of Soil.'

The Ptomines (Cadaveric Alkaloids). — During the de-
composition of proteins, bodies similar in constitution
to the vegetable alkaloids may be formed. Some, like
methylamine, dimethylamine, and trimethylamine, are
non-poisonous; but others, such as muscarin (found in
poisonous mushrooms), tyrotoxicon (found in poisonous
cheese, milk, and ice-cream), and mytilotoxin (found in
mussels), have very toxic properties. It is doubtful
whether these products are responsible for so-called pto-
mine poisoning. Many outbreaks are due to infection with
an organism (such as B. enteritidis). See ' Bacteriology of
meat,' Chapter XX.

Toxins. — Bacteria may give rise to disease in various
ways: by appropriation of nutriment, by abstraction of
oxygen from the tissues, and obstruction of capillary
vessels through their excessive multiplication. Their most
important method of aggress, however, is in the production
of poisonous bodies, known as ' toxins.' Toxins are
supposed to be protein in nature, and in many cases re-
semble enzymes. They are probably not products of
disintegration, like the ptomines, but are specific meta-
bolic products of the bacterial cell.

Toxins may be retained in the bacterial cell as an
integral part thereof (endotoxins or intracellular toxins),
or, after their formation in the cell, they may be excreted

PATHOGENESIS 13

into the surrounding medium (extracellular toxins). The
diphtheria and tetanus bacilli produce extracellular
toxins, and, if a fluid culture be passed through a Pasteur
filter, the toxins pass through, giving a toxic filtrate. The
bacilli of typhoid, anthrax, plague, and cholera, produce
endotoxins, which are retained in the organisms in the
filter, and the filtrate has but slight toxic properties.

Pathogenesis. — The application of the adjective ' patho-
genic ' to an organism does not signifiy more than that,
given a certain set of conditions in a susceptible host, it is
able to produce disease. No sharp line of demarcation
can be drawn between the pathogenic and non-pathogenic
organisms, for even those regarded as harmless may on
occasion produce ill-effects, as when the B. subtilis is
introduced into the human eye.

Koctis Postulates. — Before an organism can be regarded
as specific for a certain disease, it must be shown to con-
form to certain requirements which have been formulated
by Koch:

1. The organism must be present in the tissues, fluids, or
organs, of the animal affected with, or deadf rom, the disease.

2. The organism must be isolated and cultivated outside
the body on suitable media for successive generations.

3. The isolated and cultivated organism, on inoculation
into a suitable animal, should reproduce the disease.

4. In the inoculated animal the same organism must be
found.

To these Hewlett adds —

5. Chemical products with a similar physiological action
may be obtainable from the artificial cultures of the
micro-organism and from the tissues of man or animals
dead of the disease.

6. Specific serum and other reactions, agglutinative,
bacteriolytic, complement fixative, etc., are generally
obtainable, under certain conditions, if the blood of the
infected person or animal be allowed to act on the specific
organism producing the infection.

Although an organism fail to conform to all these con-
ditions, other considerations may justify its association
with the affection.

Methods of Spread of Infection. — Contagion was the
term applied to infective matter when contact with a
diseased person was supposed to be necessary for the

H AIDS TO BACTERIOLOGY

acquirement of the disease, while if the contaminating
matter was conveyed aerially it was known as infection.
At the present time distal aerial convection is seriously
entertained only in regard to smallpox, and even over
this disease opinions differ. Now, no distinction is made
between infection and contagion, and it is usual to class
diseases associated with micro-organisms as ' infective.'
The principal methods of infection are —

1. Pulmonary infection, the bacteria or spores being
inspired. Except where the bacteria are protected, as when
in moist droplets, infection through the air is not common.

2. Intestinal infection, the organisms being swallowed
with food, water, or dust.

3. Inoculation through a wounded or unwounded
surface of the skin or mucous membrane.

4. Inoculation through the agency of some biting insect
or other intermediate host, a developmental cycle usually,
if not always, being passed in the host.

Infection by contagion, fomites, etc., may also occur, in
which the manner of entrance of the virus into the body
is not precisely understood.

Infection may be restricted to a particular portion of
the body (local infection), or may be distributed more or
less consistently thereover (general infection). Septi-
ccpmia is the term applied to the infection when the
organism is carried over the body in the blood-stream,
saprcemia when the organism is localised and the bacterial
products alone enter the system, and pycemia to the
development of metastatic abscesses in the liver, joints,
lymphatic glands, etc.

Epidemics. — Many diseases are more or less persistent
in a restricted locality (endemic). Thus leprosy is endemic
in the Sandwich Islands, cholera in the Ganges Delta, and
smallpox in the Soudan. On occasion a disease may spread
over wide areas, when it is said to be ' epidemic,' or if
spreading over the globe, more or less, it is ' pandemic.'

Susceptibility. — The power of an organism to infect is
determined by many conditions. Hunger, thirst, ex-
cessive fatigue, exposures to extremes of temperature,
debility, and immaturity, all predispose an individual to
infection. On the part of the organisms many considera-
tions are involved. If subcultured through many genera-
tions on artificial media, most become ' attenuated ' —

SUSCEPTIBILITY 15

i.e., much of the virulence is lost. A much larger dose
will then be required to produce an effect on a susceptible
animal, but the virulence may be enhanced by passage
through a suitable animal.

The virulence of an organism may be ' attenuated '
artificially; for example, by exposing cultures of anthrax
to a temperature of 40° C. for some time, they become
attenuated to such a degree that they will kill nothing
larger than mice.

While many animals exhibit more or less susceptibility
to an organism, some may absolutely resist infection
therewith (natural immunity).

In the case of several diseases, notably in smallpox, to
a less degree in measles, mumps, whooping-cough, and
scarlet fever, it is not often that the same person is at-
tacked twice by the same disease. That is to say, one
attack is ' protective,' and in the above-mentioned
diseases the ' protection ' may last a lifetime, but extends
only to that particular disease, and does not in any way
protect against other diseases (acquired immunity}. On
the other hand, an attack from certain other diseases may
even predispose the patient to a second attack of the same
disease. This is true of influenza, diphtheria, pneumonia,
and erysipelas. A distinct predisposition may be caused
to attack by other diseases — thus, diphtheria and scarle.t
fever mutually predispose to one another.

The number of bacteria introduced is important, as a
cell or tissue may successfully repel a limited number,
but succumb to a greater.

Combinations of infectious diseases are sometimes met
with, such as syphilis with gonorrhoea, diphtheria with
scarlet fever, and pneumonia with typhoid fever. These
are known as ' mixed infections,' but if, as is generally the
case, one disease has lessened the immunity to the one
acquired last, the latter is known as a ' secondary infec-
tion.'

Symbiosis. — Symbiosis is the cohabitation of two different
organisms for mutual benefit, or their co-operation to
produce certain reactions. The presence of streptococci
appears to enhance the virulence of the diphtheria bacillus.
The effect on animals of an organism may be greatly en-
hanced by the injection along with it of some other
organism that has not pathogenic properties, but which,

16 AIDS TO BACTERIOLOGY

in some way that we do not yet understand, adds greatly
to the virulence of the pathogenic organism which it
accompanies. Attenuated cultures of B. anthracis may
reacquire virulence if injected simultaneously with a
culture of B. prodigiosus, and attenuated tetanus bacilli
become greatly exalted in virulence when cultivated with
the Proteus vulgaris.

Streptococci and colon bacilli are not uncommonly con-
cerned in cystitis. When the urine is alkaline, the strep-
tococci attain predominance, while an acid urine sup-
presses them and allows the colon bacilli to get the upper
hand.

Antagonism of Species. — One species suppresses another
by exhausting the food material or by excreting metabolic
products detrimental to the growth of the other.

Immunity. — Insusceptibility to the attack of a patho-
genic organism may be natural to a tribe (racial immunity),
or to a person (individual immunity).

The animal economy stubbornly resists attack by
pathogenic bacteria. If any be swallowed, the acidity of
the gastric juice will probably destroy them, and the normal
flora of the intestine tend to suppress harmful organisms.
The nasal secretion entangles and destroys organisms that
have been inspired. The tonsillar epithelium acts as a
bacterial filter, preventing passage of bacteria at times,
but allows free entry into the lymph-channels at
others. Wright regards this as a physico-chemical pro-
cess 'affecting the surface tension of colloids, of which the
cells and bacteria are composed. Even milk, when first
drawn, has a germicidal action. When infection takes place
through skin or mucous membrane, the phagocytes can
dispose of many alien bacteria (see below). Resistance
is also offered to toxins, which are destroyed or eliminated
by various processes, such work being a notable function
of the liver. All these processes are non-specific, with more
or less restricted capacity for eliminating bacteria and their
toxic products. When, owing to the number of bacteria
or amount of their toxins offered, they are overcome, the
infection will obtain a hold unless a degree of immunity
specific for the particular organism or toxin is available.
Artificial immunity may be active or passive.

Active immunity may be produced by one of the follow-
ing methods:

IMMUNITY 17

1. By injection of the virulent organisms in non- lethal
doses.

2. By injection of the dead organisms.

3. By injection of the toxic products prepared from
filtered broth cultures of the organism.

4. By the injection of the living, but attenuated, organ-
ism prepared by one of the undermentioned methods:

(a) By passing through a naturally resistant animal.
(6) By growth at unsuitable temperatures or in unsuit-
able atmosphere.

(c) By frequent and prolonged subculturing.

(d) By growth in the presence of very weak antiseptics.

(e) Growth in a medium of unfavourable composition
or reaction.

5. By feeding dead cultures of an organism. Achieve-
ment of a degree of immunity by this means is slower and
less certain than by others. It has been more successful
with glucosides such as ricin and abrin, and with snake
venom.

An immunised animal is said to be ' protected ' against
the specific disease. Protection against one disease some-
times also carries an immunity, though to a less degree,
against another.

Passive Immunity. — The serum of a protected animal
has an antagonistic effect on the virulent bacteria if in-
jected into a second animal at the same time as, or shortly
after, infection. Such immunity is transient. The serum
of an animal highly immunised against a particular toxin
is properly known as ' antitoxic serum ' ; that of an animal
highly protected against a particular organism in a virulent
condition is known as ' antimicrobic,' or ' antiserum.'

To account for acquired immunity the following theories
have been adduced:

Exhaustion or Pabulum Hypothesis. — The bacteria are
assumed to abstract from the blood some compound
necessary for their growth, so that, once this pabulum is
exhausted, a second attack cannot take place until it has
been re-formed. As an organism will grow in the blood
or tissues removed from an animal immune against it,
this theory is untenable. Ehrlich, in his Atrepsy Theory
modifies the hypothesis by assuming the presence of
' chemo-receptors ' for binding the poison before it can act.

The Antidote or Retention Hypothesis. — After the first

1 8 AIDS TO BACTERIOLOGY

attack the organisms are presumed to leave behind them
some product of metabolism that is inimical to their
existence. A later form of this theory was that antitoxin
was a modified toxin, which supposition has been dis-
proved.

The Phagocytosis Theory (Metchnikoff). — The large
mononuclear leucocytes and the polymorphonuclear
leucocytes ingest and destroy such bacteria as obtain
access to the blood-stream. If infection occurs in one
locality, there is a simultaneous movement of leucocytes
to that spot to cope with the bacteria (positive chemiotaxis).
When no such attraction takes place, negative chemiotaxis is
said to occur. Metchnikoff ascribes the process of im-
munisation to the ' education ' of the phagocytes. It is
supposed that phagocytes contain digestive ferments
(cytases) which effect bacteriolysis (solution of bacteria)
intracellularly. Or, on the breaking up of the phagocyte
(phagolysis), bacteriolysis may be effected extracellularly,
and the cytases are then the alexines or complements of
Ehrlich's theory. While phagocytosis constitutes a most
important factor in the production of immunity, it requires
further development to explain passive immunity.

Phagocytosis depends on the presence in the serum of
' opsonins,' which act on the bacteria, and in some way
render them suitable for ingestion by phagocytes.

Opinions are divided on the question whether normal
blood contains specific opsonins for different diseases, or
whether the opsonins are ' common ' and act irrespective
of the bacteria proffered. However, in the process of
immunisation, opsonins of specific character are developed.

EhrlicJi's Side-Chain Theory. — Ehrlich assumes that
protoplasm is composed of complex molecules possessing
very unstable ' side-chains,' ready to combine with other
atomic groups if suitable ones come within the sphere of
action. In the production of antitoxin the following is
assumed to take place: A group on the toxic body (or
toxoid), called the haptophore group, combines with a
group on the cell called the receptor group. If the ceil is
not too much damaged, it produces an excess of receptor
groups more than sufficient to combine with the toxin,
thus following Weigert's law that continued stimulation
is attended by overproduction — hypertrophy. After re-
peated injections of toxin, so many receptor groups are

PRODUCTION OF ANTIBODIES IQ

formed that the cell cannot hold them, and they become
detached, and float about, ready to unite with any fresh
toxin introduced. These free receptor cells constitute
the antitoxins, agglutinins, etc. Besides the haptophore
group, the toxin molecule contains another group called a
toxophore group, which, if it unites with another group
on the cell known as the toxophile group, after the union
of the haptophore and receptor groups, sets up poisoning.
If, however, the toxin come in contact with free receptor
cells (antitoxin, etc.), with consequent union of its hapto-
phore groups thereto, it cannot produce poisoning, al-
though its toxophore groups are free, because the first
essential necessary, the fixation of the toxin to the cells by
the combination of the haptophore and receptor groups,
is no longer possible. The toxophore group is more readily
destroyed than the haptophore group. By heating a
toxin for some time from 140° to 158° F. its toxicity is
destroyed, although it still possesses an affinity for anti-
toxin, because the toxophore group, which conditions
poisoning, has been destroyed, but the haptophore group,
which unites with antitoxin, is intact.

Production of Antibodies — Antitoxin. — When an animal
is rendered immune to a toxin a substance known as
' antitoxin ' is developed in the blood, which possesses
the power of neutralising the toxin.

Aggressins. — Some believe that before infection can
take place the bacteria must elaborate additional toxins
(aggressins) in the body to overcome the natural resistance.
This is presumed to be effected by paralysing the leucocytes
and so stopping phagocytosis. Perhaps the aggressins
are identical with the true endotoxins of micro-organisms.

Hcemolysins (see p. 20).

Cytotoxins. — The blood-serum of an animal injected
with such cells as leucocytes, spermatozoa, ciliated epi-
thelium, or cells from the liver, kidney, or nerves, possesses
the power of immobilising and destroying cells of the same
origin as those with which the animal was injected. The
phenomenon is known as ' cytolysis.'

Agglutinins. — The serum of an animal suffering from,
or infected with, typhoid or cholera acquires after a few
days the power of causing the aggregation together of
typhoid bacilli or cholera vibrios respectively, when these
are mixed with it. The agglutination is caused by

20 AIDS TO BACTERIOLOGY

agglutinins (see Widal Reaction, p. 104, and Saturation
Test, p. 152).

A serum made from one organism may agglutinate
bacteria of a closely allied species, but in a less marked
manner.

Precipitins. — An antiserum produced by the injection
of an animal with a substance containing proteins, if added
to a solution of the substance with which the inoculation
was performed, causes a cloudiness or precipitate. The
test is very delicate, especially when performed as a
* ring test ' in tiny tubes. It is used for the detection
of horse-flesh in sausages, of castor beans in cattle cake,
and for ascertaining the species of animal from which a
blood- stain came. The sole drawback to the test is the
length of time (up to six weeks) necessary to produce an
antiserum. Feeble reactions are also obtained with
proteins allied to that used in the production of the anti-
serum.

Antigens. — Any substance which, when introduced into
the blood, provokes the formation of antibodies for the
defence of the organism is called an antigen. Blood-
corpuscles, viruses, micro-organisms, etc., are thus all
included in the term ' antigen ' without prejudging their
nature. The antibody formed through the introduction
of an antigen consists of two bodies. One is ' ther-
molabile ' (destroyed by a temperature of 55° C.), and is
termed an alexin, complement, or addiment. The other
body is ' thermostable,' and requires a temperature of
75° C. for its destruction. It may be called by various
names — amboceptor, sensitiser, agglutinin, or precipitin.
While the alexin is present in the serum of every animal,
whether healthy or diseased, the amboceptor is specific,
being peculiar to a particular antigen.

Haemolysis. — If blood-corpuscles be injected into an
animal, the blood of the latter acquires hsemolytic proper-
ties, and dissolves red corpuscles of the same origin as
those which were injected. The solvent agent produced
(' hsemolysin ') contains a complement and an amboceptor
or immune body. For Haemolysis Test see p. 152.

Fixation or Absorption of the Complement. — Cholera-
immune serum, inactivated (i.e., the complement is
destroyed) by heating to 56° C. for half an hour, is mixed
with the cholera vibrio, when after a time the vibrios are

HAEMOLYSIS 21

found to have absorbed the immune body. The test can
be employed for other organisms and for red blood-cor-
puscles.

The same complement will sensitise either haemolytic
or bacteriolytic immune bodies. A mixture of typhoid
bacilli, inactivated typhoid-immune serum, and guinea-
pig serum, is incubated at blood-heat for two hours, then
'sensitised' corpuscles (e.g., a mixture of inactivated
serum hsemolysing sheep corpuscles plus washed sheep
corpuscles) are added and incubation continued for a
further two hours. No haemolysis occurs. The bacillary
amboceptor has absorbed all the guinea-pig complement,
leaving none to activate the amboceptor of the serum
haemolytic for sheep corpuscles. Consequently no haemol-
ysis of the sheep corpuscles is possible (Bordet and Gengou
phenomenon). Should normal serum (inactivated, of
course) be used instead of typhoid-immune serum, there is
no amboceptor to absorb the guinea-pig complement,
and the latter is available for absorption by the inactivated
hsemolytic serum, and haemolysis occurs.

The following test is based on this phenomenon:
Wassermann's Test (Fixation Test, or the Antigen Test). —
A guinea-pig or rabbit is inoculated several times intra-
venously with the washed blood-corpuscles of a rabbit
or sheep, with the consequent production of a hsemolytic
serum specific for the corpuscles of a rabbit or sheep
respectively, and the serum is inactivated. An antigen
is prepared by mincing and triturating the liver of a
syphilitic foetus in physiological salt solution. The serum
from the patient (the test fluid) is inactivated in the same
way as the haemolytic serum— by heating to 56° C. for
thirty minutes, thus destroying the alexin. A complement
is made by diluting guinea-pig serum tenfold. The test
fluid is added to the antigen extract, some complement
is added, and the mixture left for four hours at 20° C.
The faptnolytic, system, (a mixture of inactivated haemolytic
serum and the washed blood- corpuscles for which it is
specific) is added, and if after four hours no haemolysis has
taken place syphilitic taint is present. The antibody
in the patient's blood has attacked the treponem.es which
abound in the liver of the infected foetus, the complement
is absorbed, and there is none left to cause the inacti-
vated haemolytic serum to dissolve the blood-corpuscles.

22 AIDS TO BACTERIOLOGY

Conversely, if no syphilitic antibody exists in the patient's
blood, the complement is left free, and is deviated to the
sensitiser of the haemolytic serum, and allows this to cause
haemolysis of the blood-corpuscles.

Controls with normal and with syphilitic sera with and
without antigen are put through at the same time. Cul-
tures of the treponemes do not prove suitable antigens,
but various other substances act as well as the liver of a
syphilitic foetus — e.g., alcoholic extract of normal heart-
muscle + cholesterin. For this reason the reaction is
not a true antigen test. (See also p. 182.)

Bacteriolysis and Antimicrobic Sera.— If an animal
be treated with gradually increasing doses of an organism,
an immunity against this organism is, to a certain extent,
created. If a mixture of the animal's serum with the
bacteria be injected into an animal, subsequent examina-
tion shows the bacteria in a state of dissolution (bacteri-
olysis orPfeiffer's phenomenon). InPfeiffer^s reaction this
solution of bacteria is applied to determine the species of
an organism, particularly of the cholera vibrio. Two
milligrammes (a loopful) of an eighteen or twenty-four
hour agar culture of the virulent isolated vibrio is sus-
pended in 1 c.c. of broth containing 0-001 c.c. of the serum
of an animal very efficiently immunised to cholera. The
mixture is injected into the peritoneal cavity of a 250
gramme guinea-pig. After intervals of thirty and sixty
minutes, some of the peritoneal fluid is abstracted by a
sterile capillary pipette, and a hanging -drop preparation
made therefrom. If the organism be V. cholerce, the
bacteria are seen broken down into granules. A control
experiment with normal serum is made.

The bodies bringing about this phenomenon are known
as bacteriolysins. Bacteriolysis is brought about by two
substances, an immune body different for each organism,
and only existing subsequent to treatment with the
specific organism, and an addiment, complement, or alexin,
present in normal serum. The complement is highly
unstable, and present in small amount, thus restricting
the curative power of antityphoid and anticholera sera.

Deviation of the Complement. — An excess of immune
body in a serum proves as inefficient for bacteriolysis as
too small a quantity. Some amboceptors unite with the
receptors of the bacterial cells, while others combine with

BACTERIAL VACCINES 23

the complement. No free complement is left to combine
with those amboceptors that are attached to the bacterial
cells. The complex (amboceptor-f complement + organ-
ism) that is necessary for bacteriolysis is therefore not
provided.

Serum-Therapy. — By repeated injection of animals
with gradually increasing doses, sublethal at first, of either
the specific toxin or the living culture, a state of gradually
increasing resistance is acquired by the animals against
the toxin or the microbe. The blood attains an immunis-
ing power that is transferable to a new subject. If in-
jected into a fresh animal, the blood confers on the latter
resistance against the specific infection. The immunising
power of such blood-serum comes into action even after
infection has already taken place — that is to say, the blood-
serum has a curative therapeutic action.

Bacterial Vaccines. — The injection of sterilised cultures
of certain pathogenic organisms serves to protect against
the diseases concerned. Such vaccines may be adminis-
tered as prophylactics in anticipation of exposure to the
specific infection; or they may be used as therapeutic
measures after infection has taken place. Inasmuch
as slightly different organisms of the same species may be
involved in different cases of an infection, a vaccine is
usually made from perhaps ten different strains (poly-
valent vaccine). A vaccine is specific — i.e., only likely to
suppress or cure infection when this is produced by an
organism of a character identical with that used for the
vaccine. By use of a polyvalent vaccine the probability
that the article specific for the infection is used corre-
spondingly increases. A vaccine supplied from organisms
isolated from previous cases of the disease (or of other
diseases due to the same organism) is known as a stock
vaccine. If prepared from cases occurring in the same
institution or neighbourhood, it is produced from strains
of local flora. When infection of a case has already taken
place, and the responsible organism is isolated, a vaccine
may be prepared from it (autogenous or personal vaccine).

A description of the preparation of an autogenous vaccine
will show the principles employed. The responsible
organism is identified by microscopical examination of
the material obtained, this being supplemented if possible
by cultural and agglutination experiments. It is isolated

24 AIDS TO BACTERIOLOGY

on appropriate medium, and three or four ' streak ' or
rather ' slant ' cultures made, the whole of the surface
of the slanted medium being used. About 1 c.c. of sterile
salt solution (O'l per cent.) is poured into one of the tubes,
and by rubbing up with a sterile platinum loop the growth
is detached and forms a milky emulsion. This is poured
into a sterile test-tube or flask and the culture tube is
rinsed out with a few more drops of the salt solution. The
process is repeated with the other slants, and their emul-
sions added to the first, so that a total volume of about
5 c.c. is obtained. The masses present in the emulsion
have to be thoroughly broken up, and the bacterial content
ascertained by Wright's method: A small definite volume
of the emulsion is mixed with an equal volume of blood, and
smears made on slides are stained with one of the blood-
stains. The relative numbers of red cells and bacteria
are determined. Human blood, if from a male, contains
five million red cells per cubic millimetre, or a thousand
times this number per c.c. A calculation therefore gives
the number of bacteria per c.c. The emulsion is diluted to
a strength suitable for administration, with sterile normal
saline containing 0-5 per cent, carbolic acid, and sterilised
for an hour or an hour and a half in a water- bath at 56°
to 60° C. This will not always suffice for sterilisation.
In such cases a further sterilisation at 60° C. for one hour,
twenty-four hours later, is to be preferred to a single
sterilisation at a higher temperature. With some of the
cocci a temperature of 65° or even 75° C. is necessary.
Prolonged heating or the use of too high a temperature
lowers the activity of the vaccine. Before use a subculture
must be made from the vaccine to prove its sterility, and
all through the process of preparation rigorous sterility of
apparatus, diluting solution, etc., must be maintained.

A main cause of failure in vaccine treatment lies in the
selection of the wrong organism. This is liable to occur
with the colon bacillus, of which there are said to be 150
varieties, so that the infecting organism may not be
matched even in a polyvalent vaccine. It is often difficult,
sometimes impossible, to say at a first examination which
of the organisms found is the eetiological agent, and when a
vaccine fails a further examination is necessary. Perhaps
two or three bacteria are responsible, and the vaccine has
only been prepared from one, with the result that the

ANAPHYLAXIS 25

others carry on until a mixed vaccine is discovered to be
necessary. Success in vaccine-therapy also depends on
the correct dosage and the observance of a right interval
between doses.

Anaphylaxis. — Instead of an injection rendering the
subject less sensitive to further injections of the same
substance, the reverse sometimes occurs, certain poisons
creating a peculiar sensibility on the part of the organism
towards themselves under conditions that would lead one
to anticipate a tolerance. Delille defines anaphylaxis as
' a state of acquired vulnerability in an organism to a
second injection of a substance to which, at the time of
its first injection, it was indifferent.' For the production
of anaphylaxis an interval (the latent or incubation period)
must elapse between the first or sensitising dose and the
second or reacting dose. The minimum length of time
is said to be ten days, and should the second dose be
administered within this time anaphylaxis will not develop.
The length of time for which a sensitising dose will remain
effective in increasing sensibility is not known.

Anaphylaxis is in some measure specific — i.e., the
second injection must be of the same nature as the first.
While it has been mainly studied by the injection of horse
serum into guinea-pigs, the phenomenon has been obtained
with proteins, toxins, animal sera, glycogen, peptone,
trypsin, saponin, sodium oleate, in fact it is asserted that
any colloid will induce anaphylaxis. While crystals do
not produce the reaction, quinine, antipyrin, and iodoform
are apparent exceptions, and in some predisposed persons
inevitably lead to urticaria and anaphylactic sickness.

It is necessary that the substance injected be foreign to
the animal used, and that the sani3 or an allied substance
be employed for the reacting as for the sensitising in-
jection. A guinea-pig sensitised with horse serum will
not react if the second injection be sheep serum or goat
serum, but it should react if the second injection be donkey
or mule serum.

Animals injected with a particular organism are
anaphylactised by the corresponding toxin in a strictly
specific manner. Curious to say, it is necessary that the
sensitising and the exciting dose must take place through
the same route.

Anaphylaxis in vitro may be induced by adding horse

26 AIDS TO BACTERIOLOGY

serum to the serum of a rabbit sensitised to horse serum,
in a test-tube. If immediately injected into a rabbit
anaphylactic symptoms, and perhaps death, follow. So
small a dose as a millionth part of a c.c. of horse serum
will sensitise a guinea-pig. If after a period of incubation
a second dose be given, the symptoms of the so-called
anaphylactic shock appear. Death may occur almost
immediately; if not, the animal becomes restless, falls over,
and after diarrhoea, convulsions, and respiratory failure,
paralysis follows. Death may occur in this state or
rapid recovery may follow (Theobald Smith phenomenon).
The hypersensitive state may be transmitted by the
female guinea-pig to her progeny. The blood of anaphy-
lactised animals, if injected into normal animals, confers
anaphylaxis after a large number of injections, sometimes,
indeed, after a single injection (passive anaphylaxis).

No satisfactory explanation of anaphylaxis has been
made. Gay and Southard consider that the serum con-
tains a substance provisionally termed anaphylactiri,
which remains as a constant irritant to the cells of the
body, increasing their reactivity for the other constituents
of the foreign serum. Vaughan and Wheeler believe that
anaphylaxis must be due to a toxic fragment of the pro-
tein molecule. Richet thinks that in the injected animal
there is produced a substance (toxogenin) which is not
toxic in itself, but yields a toxic substance (apotoxin) on
combination with antigen. The toxicity of the apotoxin
(or precipitin) is increased by combination with the
alexin of the blood.

In the serum the precipitin content runs parallel with
the severity of the symptoms, and disappears after the
anaphylactic condition passes off. Halliburton has
suggested that this explains the difference between the
normal and the sensitive animal.

The frequency with which normal horse serum and anti-
diphtheritic serum are used in treatment, proves ana-
phylactic shock to be of rare occurrence among human
beings. Some workers question if death ever occurs,
outside of cases of status lymphaticus.

Serum Disease or Serum Sickness. — About one-third
of persons injected with horse serum for the first time are
found to be sensitive to it, an urticaria, more or less cedema,
and sometimes arthritis, appearing between the sixth and

SNAKE VENOM 27

Uventioth days after injection. The only explanation
offered is that part of the serum used as an injection
remains unaltered in the subject, while the remainder
sensitises the serum of the subject. In fact, a sort of
auto-anaphylaxis occurs.

Snake Venom. — The venoms of different species of
poisonous snakes differ greatly in composition. Some
appear to contain proteolytic enzymas which are supposed
to produce the softening of the inuscles in the animals
attacked. Cobra venom contains a hsemolysin, innocuous
by itself, but activated by normal complement in the
blood-serum of the victim. This hsemolysin is also activated
by lecithin.

Rattlesnake venom acts partly by lysis of the endothelial
lining of bloodvessels, the specific toxin from its effects
being called ' haernorrhagin.' Other venoms, such as that
of the krait, produce intravascular thrombosis through an
almost instantaneous coagulation of the blood. Many
venoms are neurotoxic, the neurotoxins of different species
selecting different sites for activity, one acting on the
respiratory centre, another on the nerve endings in muscle.
Serpent venom, unlike true bacterial toxins, is unaffected
in virulence by a considerable degres of heating.

Antivenin or Antivenomous Serum. — Calmstte injects
horses with gradually increasing doses of cobra venom
mixed with diminishing quantities of a 1 in 60 solution of
hypochlorite of lime. When they have acquired sufficient
immunity, the venoms of as many species of reptile as
possible are injected. The process of immunisation lasts
at least fifteen months.

Calmette's serum is active to the extent of 1 to 200,000 —
that is to say, it is sufficient to inject as a prophylactic
dose a quantity of serum into a rabbit equal to o^oooo of
its body-weight; 0-5 c.c. of this serum is sufficiently active
to protect a rabbit against a dose of venom, which other-
wise would be lethal in three or four hours, if it is not
injected later than half an hour after the bite. The dose
of the antivenin for a human being is, according to Calmette,
10 to 20 c.c., but Lamb and Hanna consider that it should
be 30 to 40 c.c., injected as soon after the bite as is prac-
ticable.

It has been asserted that cobra antivenin protects
animals against any snake poisons; but Martin and

28 A IDS TO BACTERIOLOGY

Tidswell have shown that antivenomous serum is just
as specific as other antisera.

Bacterial Mutability. — The production of involution
forms (p. 3) and of attenuated bacteria (p. 14) have been
already dealt with. Appearance in different forms at
different times — e.g., as a bacillus and leptothrix — is called
Isomorphism.

Bacteria that normally are ' acid-fast ' or ' Gram-posi-
tive ' may fail to resist decolorisation by acid in theZiehl-
Neelsen process, or may becoms ' Gram-negative,' when
old. This is most noticeable among the Streptothricice.

Other changes in character occur especially in the colon-
typhoid group. Twort endowed a strain of typhoid bacilli
with lactose-fermenting power. Revis states that the
fermentation of a sugar or polyhydric alcohol takes place
in two stages — a preliminary acid formation and a subse-
quent gas formation. Revis succeeded in causing an
organism to lose its power to produce gas while retaining
its capacity to produce acid, the resulting variety being
of a permanent character.

It should be noted that an organism may ferment a
specific carbohydrate obtained from one dealer, and have
no action on the same substance as supplied by another.

By exposure to ultra-violet rays Mine. Victor Henry
converted anthrax bacilli into cocci and other forms which
are apparently stable. It also appeared to lose its capacity
for secreting proteolytic enzymes.

Simonini found thorium salts to modify morphology,
staining reactions, and physiological characters of Shiga-
Kruse and Flexner dysentery bacilli and B. diphtheria,
B. coli showing less response.

Theile and Embleton showed that a guinea-pig pre-
viously sensitised to B. mycoides died after inoculation
with this organism, and the post-mortem appearances were
indistinguishable from those of anthrax. After passage
through the sensitised animal, the organism was capable
of producing disease in a normal animal. Similarly,
smegma and Timothy- grass bacilli produced in animals
specifically sensitised post-mortem appearances indis-
tinguishable from those due to an intraperitoneal injection
of tubercle bacilli.

Dostal claims to have converted the tubercle bacillus
into a non-acid-fast and non-pathogenic organism.

MICROSCOPE 29

CHAPTER II
BACTERIOLOGICAL APPARATUS

The Microscope. — A microscope for bacteriological work
should be absolutely rigid, and the fine adjustment
should be sensitive and precise. It must be fitted with
a suitable substage condenser, with an arrangement for
focussing, and iris diaphragm. A triple nose-piece avoids
unscrewing the objectives to obtain variations in power,
and saves not only time, but much wear and tear.
The following objectives are required: |-inch, ^-inch,
and a j^-inch oil immersion. These objectives com-
bined with an ' A ' or ' B ' eye-piece will give magnifi-
cations to 1,100 diameters, which is ample for all ordinary
purposes.

A mechanical stage of an easily detachable form is
desirable, especially for the systematic examination of
blood-films, etc.

A brilliant illumination is essential for the examination
of bacteria, particularly when in tissues. A paraffin lamp
with flat flame, the edge of the flame being used, is very
satisfactory; or, if electric current be available, a Nernst
lamp enclosed in a frosted globe, or the Barnard lamp.
Daylight is not always suitable, but it is as well to be able
to use it on occasion.

Micro-organisms in liquids and tissues are only visible
through the shadows caused by the differences in the
refractive power of the various structures. Consequently
the hole in the diaphragm must be diminished. In the
case of stained specimens, however, an open diaphragm
can be used, and the preparation examined with the full
aperture of the condenser.

After using the oil-immersion objective, the cedar oil
should be removed with soft filter-paper and the lens then
wiped with a silk handkerchief. Should the oil be allowed
to dry on at any time, a little fresh oil should be put over
it and allowed to stand a short time; this will soften the
hardened oil, when the whole may be cleaned off together,
or it may be gently cleaned with a rag moistened with
xylol.

30 AIDS TO BACTERIOLOGY

Limits of Microscopical Vision. — There is a limit to
the visibility of microscopical objects. With the very best
optical appliances and the use of monochromatic violet
light it is impossible to see more than about 167,000 lines
to the inch, an object measuring less than about 0-14//, not
being perceptible. By means of transverse illumination,
ultra -microscopic particles may be rendered visible as
diffraction discs, and particles measuring far less than
half a wave-length of light can be made visible (Siedentopf ' s
apparatus).

The Hot-Air Steriliser.— An iron box, with double
walls, fitted with a door in front and supported on four
legs. It is heated by means of. a rose gas-burner from
below, and the temperature of the interior is indicated by
means of a thermometer inserted through a hole in the
top. If necessary, a mercury-gas regulator may be
inserted through a second opening.

The temperature in these ovens is by no means uniform ;
it therefore must be ascertained that the objects exposed
for sterilisation really reach the desired temperature.

Test-tubes, Petri dishes, pipettes, etc., may be thoroughly
sterilised by exposure to a temperature of 150° C. for one
hour. The door must not be opened until the temperature
has dropped to 60° C. Neglect to observe this precaution
courts cracked glass. Inoculating-wires, forceps, etc.,
are best sterilised by passing through the flame of the
Bunsen burner.

The Steam Steriliser. — This is a cylindrical vessel of
copper, about \ metre high by about 30 centimetres wide,
jacketed with non-conducting material, and provided with
a lid. The lid is covered with felt, and is perforated to
receive a thermometer. Inside the vessel is a diaphragm
or grating about two-thirds down which divides the
interior into two portions: the upper, or ' steam- chamber/
and the lower, or ' water-chamber.' A water-gauge
indicates the water-level. The apparatus stands upon
three legs, and is heated by a large Fletcher burner, keep-
ing the water in vigorous ebullition, so that steam issues
freely from the top. A uniform temperature of 100° C.
is thus maintained in the apparatus. The steriliser is
fitted with a wire basket or metal rack for the reception
of test-tubes containing nutrient media.

This apparatus is employed for sterilising media and

STERILISER 31

apparatus which cannot be exposed to temperatures above
100° C. Glass utensils may be steamed for from one to
two hours.

The High-Pressure Steam Steriliser. — High-pressure
steam in an autoclave acts with greater rapidity than
ordinary steam. Although not necessary for ordinary
use, in the sterilisation of soil it must be used. Certain
spores resist ordinary steaming for three hours, but
are destroyed in fifteen minutes by steam at 110° to
120° C.

Sterilisation by Chemical Agents. — For washing instru-
ments, and for disinfecting the hands, solutions of 1 in
1,000 of corrosive sublimate, 1 in 20 solution of phenol, or
1 in 50 solution of lysol, are used. When chemical agents
are used, risk is incurred by traces of the germicide escaping
removal, and destroying the organisms under examination
or introducing other elements of uncertainty into the
work. For ordinary purposes it is best to rely upon the
careful fulfilment of all the details required in the sterilisa-
tion by the usual methods.

Glass pipettes, etc., may be rapidly sterilised by rinsing
with o per cent, phenol, then with absolute alcohol, and
lastly with ether, the ether finally being driven off by
careful heating over the Bunsen.

Steel instruments, etc., are best boiled in water con-
taining a little sodium carbonate.

Sterilisation by Filtration. — Air and other gases are
readily freed from micro-organisms by drawing them
through a tube containing a plug of dry sterile cotton-
wool or packed with sugar or sand.

Water or other liquid which is not too viscid is sterilised
by passage through unglazed porcelain cylinders (Pasteur-
Chamberland filter). These filters are used for the pur-
poses of concentration of bacteria in a liquid, or the separa-
tion of bacteria from their products.

For experimental purposes these filters must be cleaned
and sterilised before being used. This operation does
not affect the Pasteur filter, but tends to disintegrate the
Berkefeld (see p. 239).

The Microtome. — A large number of machines for the
cutting of sections of tissues have been introduced. For
some the tissue is frozen before cutting, for others it is
first impregnated with paraffin or celloidin.

32 AIDS TO BACTERIOLOGY

The Incubator. — The pathogenic bacteria grow best at
the temperature of the body of the host, and for their
culture an incubator with a temperature of 37° C. is
employed. Many of the saprophy tic forms will not develop
at so high a temperature, and they are cultivated either in
a warm room or in a ' cool ' incubator, at about 22° C.
The incubator consists essentially of a double-walled
chamber, the space between the walls being filled with
water warmed by a gas-burner. The outer wall is covered
with some non-conducting material. In the Hearson
incubator a regular temperature is secured by an Excelsior
gas-valve, in which the pressure of ether and other vapours
is employed in a flexible envelope; this, acting upon a
lever, controls the gas-supply. Page and Reichert thermo-
stats are also used, where a fall in temperature occasions
the contraction of mercury and allows more gas to pass,
while if too hot, mercurial expansion ensues, and by par-
tially cutting off the gas, diminishes the flame. In case
the main gas opening should become closed by the expan-
sion of the mercury, a by-pass allows the maintenance of
a pilot light.

The ' cool ' incubator is similar in principle, but is sur-
mounted by a vessel containing ice. The regulation of
temperature within the chamber is effected by a small
stream of water, which runs continuously through the
apparatus in one of three directions, the choice being
automatically determined by a thermostatic capsule. A
third incubator, giving a temperature of 42° C., is useful
for the culture of typhoid and colon bacilli in water, etc.,
examinations.

Centrifuges. — For the removal of fine particles from
suspension, centrifugal force is employed. The Gerber
machine, as used in milk analysis, when fitted with centri-
fuge tubes, answers well, and special machines are made
for the purpose. For small quantities of material, haema-
tocrites are used.

Inoculating Needles. — For the transference and spread-
ing, etc., of material, pieces of platinum wire fused into
glass, or fixed into aluminium rods, are used. For ordinary
purposes O4 millimetre (27-28 B.W.G.) wire is suitable, but
the greater stiffness of a 0*7 millimetre wire is sometimes
necessary. Needles, both straight and with loops (of vary-
ing sizes up to 4 millimetres in diameter or bigger) are used.

NUTRIENT MEDIA 33

Test-Tubes. — 6 x £ inches is the most useful size. For
special purposes smaller and larger ones are required.

Cornet forceps for cover-glasses, dissecting forceps of
various patterns, and other instruments, are needed,
together with flasks for holding media.

CHAPTER III

THE PREPARATION AND USE OF NUTRIENT
MEDIA

IT is necessary, in order to obtain a satisfactory know-
ledge of the biological characters of a micro-organism,
to obtain a pure culture — that is, a culture containing
one species only.

When, by exposure to air or by other means, isolated
bacteria lodge on the surface of a nutrient medium,
they are fixed in situ and commence to grow, each organism
producing a colony which eventually becomes visible
macroscopically. This character is used for the isolation
of organisms. Pure cultures of bacteria are hardly ever
met in practice, and a very common method of separating
individual bacteria is to disperse the liquid containing
them over or through some solid medium in such a dilution
that individual bacteria can form sufficiently large colonies
without their meeting. This is generally effected in
Petri dishes.

By the introduction of a part of a colony into a tube of
sterile medium, a pure culture is obtained after incubation,
and the larger quantity of growth provides more material
for examination. The appearances of the growths on
various media constitute important, and often positive,
means of identification.

In culture, the store of nutrient material becomes
gradually used up, and reproduction stops. It is necessary,
therefore, to reinoculate them from time to time into fresh
media. Bacteria are artificially cultivated in both liquid
and solid culture media.

Nutrient media are employed in test-tubes, small conical
(Erlenmeyer's) or other flasks, or Petri dishes. All test-
tubes, flasks, etc., are thoroughly cleansed with 25 per cent.

3

34 AIDS TO BACTERIOLOGY

hydrochloric acid, and well rinsed with water to remove
all traces of acid. The tubes are then allowed to drain
until nearly dry, when they are finally rinsed out with a
little strong alcohol, drained, and allowed to dry. Or the
tubes may be cleaned by boiling in water containing soap
powder for thirty minutes, followed by cleansing with
brush and thorough rinsing with water. They are then
plugged with sterilised cotton-wool, and sterilised for an
hour at 140° to 150° C. in the hot-air steriliser. Cotton-
wool is sterilised by pulling loosely apart and heating for
an hour at 145° C. in the hot-air steriliser. Though
sometimes desirable, it is not always necessary to sterilise
tubes and cotton-wool before filling with media. The
sterilisation after tubing is generally sufficient.

Reaction of Media. — A reaction slightly acid to phenol-
phthalein (equivalent to a faintly alkaline reaction to
litmus) is generally most suitable.

Standardisation of Media. — Variations in reaction of
media influence the character of the growths. For the
enumeration of organisms and for descriptive work
standard media are necessary. The signs + and— indicate
acidity and alkalinity to phenolphthalein respectively.
The American Committee describe +1-5 per cent, reaction,
when to every 100 c.c. of medium neutral to phenolphtha-
lein 1 '5 c.c. of normal hydrochloric acid are added. English
workers, following Eyre, use an acidity of +1-0 per cent.
(+ 10 on Eyre's scale).

The procedure is briefly as follows: An aliquot portion
of the nutrient medium is taken, say 20 c.c. ; this is diluted
with warm distilled water, boiled for a minute, a few
drops of a solution of phenolphthalein are added, and
decinormal solution of sodium hydrate is run in drop by
drop from a burette to the hot solution until a pink colora-
tion is obtained. The correct volume in c.c. of normal, or,
better, dekanormal, soda solution to be added to the bulk
is calculated and added; the reaction of the medium will
then be neutral to phenolphthalein, but strongly alkaline
to litmus. The alkalinity is too great for the optimum
growth of most organisms, and it is reduced by the addition
of normal hydrochloric acid to the extent of 1 c.c. per 100
c.c. of medium. The reaction is then said to be + 10
(Eyre's scale) or 4- 1*0 per cent. (American scale).

Instead of first neutralising and then adding normal

NUTRIENT MEDIA 35

acid or alkali, sufficient alkali may be added to reduce the
reaction to the required point. After standardisation, the
medium is heated on a water bath for half an hour to bring
down the precipitate caused by the change in reaction (mag-
nesium ammonium phosphate, Jordan), and then filtered.

Preparation of Beef Broth. — One pound of beefsteak,
freed from fat and connective tissue, is cut up and passed
through a mincing-machine. The finely-minced meat
is digested with 1,000 c.c. of water. It is then boiled,
with constant stirring, for thirty minutes in a tinned or
enamelled saucepan, which is kept well covered. The
broth is strained through muslin, and then made up with
distilled water to 1,000 c.c., to replace that evaporated
during the boiling. This is the ' acid-beef broth.' To the
broth is then added 5 grammes of sodium chloride and 10
grammes of peptone. The latter is first rubbed up with a
little of the broth in a glass mortar, after which it is added
to the bulk.

The mixture is now boiled for five minutes, and then
very carefully neutralized with a solution of sodium car-
bonate or hydrate, making the solution very faintly
alkaline to litmus-paper. The alkaline solution is added
carefully, drop by drop, shaking the flask well between
each addition. The solution is again boiled for ten
minutes, with constant agitation. The reaction is again
tested with litmus -paper, and if still faintly alkaline, the
solution is filtered into a flask through a double-pleated
filter-paper. Or the broth may be standardized to + 10
(vide supra). The filtered product, which should be
absolutely clear and bright, is then run into flasks (which
are plugged with sterile cotton- wool) and sterilised on three
successive days in the steamer for fifteen to twenty minutes
on each occasion. Bullock's heart or sheep's heart may
be used instead of steak.

Lemco Broth. — Although some workers prefer beef broth,
one in which extract of meat is used serves equally for
most purposes, and is now generally used. Lemco, 20
grammes; peptone, 20 grammes; salt, 10 grammes;
distilled water, 1 litre. Boil for thirty minutes, standardise,
and filter. This is more easy to prepare, and varies less
in composition, than beef broth.

If, in spite of filtration, the broth remains turbid, the
white of an egg is added to the cooled broth (50° C.),

36 AIDS TO BACTERIOLOGY

well mixed, and raised and maintained at the boiling-
point for ten minutes. The precipitated albumin is then
filtered off, and the filtrate sterilised as above directed.

Stitt uses 3 grammes of Lemco to the litre, and, as the
msdium has almost invariably a reaction of +0'75 (Ameri-
can scale), considers it is usually unnecessary to titrate
and adjust the reaction unless precision is demanded.

Glycerin Broth. — Five c.c. of glycerin to every 100 c.c.
of beef broth.

Glucose and Lactose Broth. — To each 100 c.c. of broth
is added 1 to 2 grammes of pure glucose or lactose. Used
in the cultivation of anaerobic bacteria.

Nutrient Gelatin. — To 1 litre of acid beef broth are
added 100 grammas of ' gold label ' gelatin, 10 grammes
of peptone, and 5 grammes of salt. The mixture is
placed on a water-bath until solution is complete, and
then rendered faintly alkaline to litmus-paper with
sodium hydrate solution. -After cooling to 50° C., the
white of an egg is added, and after stirring it is steamed
for one hour. The gelatin is now filtered through a
pleated filter in a hot- water funnel, and then run into
test-tubes. The tubes are sterilised on three successive
days in the steam steriliser for fifteen minutes on each
occasion. After the final steaming they are allowed
to solidify in upright or slanting positions, according as to
whether they are intended for stab or streak cultivations.
In hot weather 15 or 20 per cent, gelatin should be used.
The gelatinising power of gelatin is gradually destroyed
by heating.

Glucose Gelatin. — Two per cent, of glucose in nutrient
gelatin.

Nutrient Agar. — Fifteen grammes of powdered agar are
well boiled with a litre of nutrient broth for two to three
hours until dissolved; the water lost by evaporation is
replaced from time to time. Care is then taken to see
that the medium is faintly alkaline, after which it is
cleared with egg-albumin, as described under the prepara-
tion of gelatin. The agar is then filtered through ' Char-
din ' filter-paper or a small jelly- bag. Some workers
allow the hot agar to stand in the steam steriliser in a tall,
cylindrical vessel till the flaky particles which cause the
turbidity sink to the bottom, when the clear agar can be
poured off.

NUTRIENT MEDIA 37

Agar jelly remains solid at 40° C., and only melts com-
pletely at 99° C. ; hence this medium is well adapted for the
higher incubating temperatures. Nutrient agar is often
quite clear when hot, but is always slightly opalescent on
cooling.

Glycerin Agar. — Nutrient agar containing 5 per cent,
of glycerin.

Glucose Agar. — The addition of 1 to 2 per cent, of
glucose to nutrient agar is useful for the cultivation of
anaerobic bacteria. The tubes for this purpose are filled
two- thirds full. The medium should not be heated more
than is absolutely necessary during preparation and
sterilisation, or it becomes dark.

Urine Gelatin and Agar. — Fresh urine thickened with
10 per cent, of gelatin, or 2 per cent, of agar, with the
addition of 1 per cent, of peptone and -£- per cent, of sodium
chloride, is rendered feebly alkaline and filtered.

Peptone Water.— Ten grammes of peptone and 5
grammes of sodium chloride are dissolved in 1,000 c.c.
of distilled water; the solution is then well boiled, and
neutralised carefully in the usual manner. The solution
is again boiled and filtered. The solution is then run into
tubes, and steamed for fifteen minutes on three successive
days.

Glucose and Lactose Peptone Waters. — The addition
of 1 to 2 per cent, of glucose or lactose to peptone water is
very useful when determining the fermentative power of
organisms. The medium may be tinged with litmus, in
order to show production of acid or alkali, sufficient of a
neutral solution of litmus being added for this purpose.
These media and their corresponding broths are preferably
introduced into Durham's tubes, which consist of the
ordinary test-tubes into which small inverted tubes have
been introduced. The small tubes during the process of
sterilisation becoma filled with the medium, and then serve
as gas-holders, should the sugar be fermented with the
production of gas. If the inner tubes do not fill during
sterilisation, two grease-pencil marks should be made
on the outer tube to show the volume of air left in, or the
Durham tubes may be put in a water-bath and heated
to boiling for ten minutes, when the air bubble will dis-
appear. As a general rule the production of gas can be
observed without using an inner tube, as a few small

38 AIDS TO BACTERIOLOGY

bubbles are seen at the surface, or can be seen rising in the
liquid if the tube is gently shaken. As gas production
may have stopped at the time of inspection, it is never
wise to dispense with the inner tube.

Maltose, galactose, arabinose, raffinose, cane-sugar,
mannite, sorbite, dulcite, adonit, dextrin, starch, and
inulin, are similarly used.

Milk. — Fresh separated milk, free from preservatives,
is sterilised for twenty minutes on each of five successive
days. The medium is frequently tinged with litmus.

Milk must not be overheated, as this retards or prevents
the formation of clot when organisms that should produce
clotting are grown in it. This, perhaps, is the reason why
some brands of non- sweetened condensed milk prove
unsatisfactory for milk-tubes. Milk is always so greatly
contaminated to start with that it is unwise to rely on
sterilisation, and tubes should be incubated at blood-heat
for two days in order to ascertain which are sterile.

Potato-Tubes. — Large sound potatoes are thoroughly
scrubbed, and then with a large cork-borer cylindrical
pieces are cut to fit into test-tubes. Each cylinder is cut
into halves diagonally, the wedges are well washed in
running water for an hour, and each wedge is placed in a
test-tube. The cores of potato rest on a moist plug of
cotton- wool to keep the potato cylinder moist. The tubes
are capped, and sterilised in the steam steriliser for thirty
minutes on each of three successive days. The tubes are
left in the blood-heat incubator overnight, and any con-
taminated ones rejected.

Blood-Serum. — Blood from the jugular vein or an
incised wound is allowed to run into a tall sterile glass
vessel, with aseptic precautions. The vessel is at once
placed in a cool place without the least shaking, and
allowed to stand overnight, when a firm clot forms; the
clear serum is drawn off by a sterile glass siphon or large
pipette into sterile test-tubes, which are plugged, and laid
on a slanting surface, and the serum made to set by
heating in the hot-air steriliser to 65° C. The tubes can
then be sterilised in the usual way by steaming on three
successive occasions. All tubes should be tested for
sterility by a trial incubation before use. The serum
should have a jelly-like consistency, and an opalescent,
yellowish-white colour.

NUTRIENT MEDIA 39

Chloroform is particularly suitable for the sterilisation of
blood-serum, as it has a powerful germicidal action com-
bined with a low boiling-point, so that it can be driven off
with certainty after sterilisation is complete. (The liquid
under treatment is shaken up with chloroform, and allowed
to stand some days, when it is freed from chloroform by
prolonged heating at 62° C.)

The serum from human blood, obtained at operations
and from placentae, occasionally presents advantages
over that obtained from animals.

Modifications of Blood-Serum. — The fluid obtained
from hydroceles, cysts, or dropsical effusions, is practically
the same in composition as blood-serum.

Loffler's Medium. — Two parts of blood-serum with
one part of nutrient glucose broth. The medium is
solidified in a slanting position.

Blood-smeared Agar.— The surface of the agar in sloped
agar- tubes is smeared with blood obtained aseptically.
The tubes must not be sterilised after the blood has been
added.

Egg-Albumin.— The albumin from birds' eggs is care-
fully separated from the yolk, and treated as directed
under the preparation of blood-serum tubes. The white
from plovers' eggs yields an almost transparent medium.
Hens' eggs may (Hueppe) be themselves used. New-laid
eggs are washed in sodium carbonate solution, immersed
in 1 in 2,000 mercuric chloride solution for a short time,
thoroughly rinsed in water that has been well boiled, and
finally rinsed in strong alcohol and ether. The end is
pierced with a sterile needle, and the material to be
inoculated is introduced into the egg by means of a glass
capillary tube, from which it is blown with great care.
The hole is now closed with sterile cotton-wool. This
method of cultivation is particularly well adapted for the
cultivation of the anaerobic bacteria.

Beer- Wort. — Unhopped beer- wort is allowed to stand in
a cool place for twelve hours, filtered, steamed for one
hour, again filtered, and then sterilised.

Beer- Wort Gelatin. — -One hundred grammes of gelatin
are dissolved in a litre of unhopped beer- wort, clarified and
filtered, but not neutralised.

Silica Jelly. — This preparation is destitute of organic
matter, and is used for the organisms of ' nitrification,'

40 AIDS TO BACTERIOLOGY

which will not grow on an organic medium The gelat-
inous consistency is obtained by means of dialysed silicic
acid.

Irish Moss Jelly is used for the culture of the thermo-
philic organisms, and various other media are employed,
the composition of which is given in different monographs.

To prevent evaporation, and assist exclusion of aerial
organisms, culture-tubes may be covered with rubber
caps, or after the last steaming their mouths may be pro-
tected with gutta-percha tissue. Media should be kept
in a cool, dark place, such as in suitable- sized tins with
lids. A piece of filter-paper may be placed at the bottom
of the tin, and a few drops of clove oil sprinkled thereon.
In this way the percentage of tubes spoiling on keeping
is reduced considerably.

Desiccated media may be purchased, some of which
answer very well.

The Cultivation and Isolation of Bacteria. — The ubiqui-
tous character of bacteria renders it necessary that
those under examination should not become contaminated
with extraneous organisms. To preclude such con-
tingency, resort is had to certain devices for protection
against pro temjwre aliens. In the absence of draughts,
aerial bacteria do not move in a horizontal direction,
but merely drop. Consequently, tubes of media are not
held with the mouths up during manipulation, but in a
more or less horizontal position. Dry cotton-wool is an
effectual bacterial filter, and is used for plugging vessels.
The part of the plug entering the test-tube is never touched
with the hand by conservative workers, who leave an
ample portion of plug outside the tube for this purpose.
Many, however, regard the large plug as an archaic fetish,
and practise economy in cotton- wool by using a loosely fit-
ting plug about an inch long, which all goes in the mouth
of the test-tube, and is removed by sterile forceps and held
by the top part. It was formerly the custom to always
' flame ' a plug before reinserting it, but except when the
plug has been dropped, or otherwise exposed to con-
tamination, this ritual is not generally honoured in routine
work.

Gelatin Plate Cultures. — Three test-tubes contain-
ing nutrient gelatin are placed in warm water at about
40° C. until the contents are liquid. This temperature is

ISOLATION OF BACTERIA 41

sufficient to keep the gelatin liquid, but not high enough to
destroy the vitality of the bacteria which are to be the
subjects of experiment. The tubes are then numbered
1, 2, and 3. By means of a platinum needle, which
has been previously sterilised at red heat and allowed
to cool, after carefully withdrawing the plug, a mere trace
of the mixture of organisms under examination is intro-
duced into tube 1 and well mixed. If the material is
too coherent, attempts must be made to separate the
organisms by rubbing them with the point of the platinum
wire against the side of the tube below the surface of the
gelatin. The plug is replaced and the needle sterilised
by passing it through the flame. A platinum loop is
sterilised, and whan cool, a loopful of the gelatin transferred
from tube 1 to tube 2, and the contents well mixed, after
which two or three loopfuls from tube 2 are transferred
to tube 3. By the above dilution the number of organisms
in the third tube is probably so small as to give a satis-
factory ' plate.' It may happen that the dilution is
carried too far, in which event the .plate required is ob-
tained from the second tube. Success in this operation
is a matter of experience and judgment. When inocula-
ting tubes the cotton- wool plugs must be held between the
fingers, best between the third and fourth, using the back
of the hand, and must be carefully returned into the tube
after inoculation, without the part that goes in the tube
being allowed to come into contact with the surface of
the hand or bench. The manner in which plugs and
tubes are held is largely a matter of taste, but whatever
posture is adopted, the positions of the respective
plugs and tubes must be remembered, to avoid mixing
them.

The plug of No. 1 tube is flamed and removed, the lips
of the tube are flamed, and the contents of the tube poured
into a Petri dish which is marked ' No. 1.' The tubes
marked 2 and 3 are treated similarly. Petri dishes are
from 10 to 20 centimetres in diameter and about 1-5 or 2
centimetres deep, and have loosely fitting covers of the
same form as the dishes themselves. The colonies may
be examined and counted without removing the lid.

Esmarch's Roll Cultures. — The inoculated liquefied
gelatin is distributed in a thin layer upon the walls of a
wide test-tube by rotating the tube upon a block of ice

42 AIDS TO BACTERIOLOGY

having a horizontal surface, in which a shallow groove
has been made by means of a test-tube containing hot
water.

Agar Plates. — Tubes containing nutrient agar are
placed in a bath of boiling water until the contents are
completely melted. The bath is allowed to stand until
the temperature falls to nearly 45° C. The tubes are then
immediately inoculated and the contents poured into a
dish, as previously directed for the preparation of gelatin
plates. It is a good plan to warm the dishes or plates to
40° C. before pouring the agar, and, above all, to work
quickly, as the agar solidifies at 40° C., and after solidifica-
tion has begun to take place an even distribution of the
medium is no longer possible. Agar plates are usually
inverted in the incubator. If left upright, moisture
collects in drops on the medium and washes colonies into
one another.

Character of Bacterial Colonies. — Gelatin and agar
plates (incubated at 22° C. and 37° C. respectively) are
examined every day to ascertain the character of the
colonies. It occasionally happens that a colony contains
more than one species owing to the too close propinquity
of the original bacteria, but as a rule each isolated colony
is a pure culture. Some bacteria liquefy gelatin more or
less quickly, liquefaction being shown by a sinking and
the evident local destruction of gel.

For further study of the colonies isolated on plates they
may be inoculated into liquid or solid media. Inocula-
tions on to solid media may take the form of a ' stab ' or
' streak ' culture.

* Streak ' Cultures. — Nutrient gelatin or agar is solidi-
fied in an oblique position, so as to expose as much surface
as possible. The tube is held in a horizontal position to
prevent aerial organisms from falling in, and the plug
is carefully withdrawn with the third and fourth fingers
of the right hand, using the back of the hand. The plati-
num needle is sterilised by heating to redness in flame,
and given time to cool. A trace of the colony is taken
up on the point. The needle is carefully passed down the
tube so as not to touch the sides, and then gently drawn
along the centre of the medium, using a light but even
pressure. The noodle, on removal, is at once sterilised,
and the cotton- wool plug after flaming is returned to the

CULTURE METHODS 43

tube. The whole operation is carried out as quickly as
possible, so as to reduce the chance of outside contamina-
tion to a minimum.

* Stab ' Cultures. — A trace of the growth is picked up
on the tip of a platinum needle, which is then thrust into
the depth of a tube containing about 10 c.c. of nutrient
medium solidified in the upright position, care being
taken to introduce the wire in a central line and in a direc-
tion parallel with the sides of the tube. The tube may be
held and the manipulations carried out in the same manner
as described for a ' streak ' culture.

* Stab ' and ' streak ' cultures may be made in the same
tube, if this be filled and ' slanted ' in such a manner that
the slant ends halfway down it.

To show the production of gas, stab cultures in glucose
agar, shake cultures in gelatin, or cultures in Durham's
fermentation tubes (see p. 37), may be employed.

' Shake. ' Cultures. — A tube of gelatin is liquefied by
heating the tube in a beaker of water at 40° C. The
medium is then inoculated with the organism under
examination. The plug is replaced, and the contents of
the tube gently mixed to distribute the organisms evenly
through the medium, care being taken not to allow any of
the gelatin to touch the cotton-wool plug. The contents
of the tube are allowed to set in cold water. The presence
of organisms capable of producing gas in this medium is
shown by the formation of bubbles. At the same time,
another tube of the medium is inoculated with an organism
known to produce gas — as a control. This prevents an
error of registering this attribute as negative when really
non-production of gas is due to the use of unsuitable
medium. The test sometimes fails with gelatin made with
meat extract.

Anaerobic Cultures. — Most anaerobes will grow in a
deep stab in glucose agar or gelatin. The tube, three-
quarters full of medium, is kept in boiling water for five
minutes and then cooled. Dissolved oxygen is thus ex-
pelled and the medium softened. After the stab is made,
the top portion of the medium is gently warmed to seal
the top of the needle track.

Air may be excluded from a culture in a fluid medium
by running a layer of sterile vaseline or olive oil (about a
centimetre thick) on to the medium.

44 AIDS TO BACTERIOLOGY

Buchner's Tube. — A large test-tube, having a constric-
tion a little above the bottom, and fitted with a rubber
stopper, well vaselined. The inoculated tube is placed in
the Buchner's tube, and rests on the constricted portion.
Some strong aqueous solution of pyrogallic acid is then
run into the outside tube, followed by an equal volume of
20 per cent, caustic potash solution. Without allowing
the pyrogallic acid and potash to mix, the rubber stopper
is quickly inserted, and the solutions in the tube below
the constriction mixed. Stoppered glass bottles may be
used for the purpose.

In FrdnkeTs method a large, strong test-tube contain-
ing the medium is fitted with a rubber cork, through
which pass two glass tubes, bent at right angles just above
the stopper, and the ends drawn out. One tube should
reach almost to the bottom of the tube, and the other just
through the cork. After sterilisation and inoculation,
hydrogen is passed through the longer tube and escapes
by the shorter one. When all the air has been expelled
the tubes are sealed in a Bunsen flame, the shorter tube
being done first.

Bullock's apparatus consists of a large bell- jar with
ground flange, which can be luted to a plate of ground-
glass with a suitable grease or resin ointment. Through
the top, two tubules with ground stoppers, and provided
with stopcocks, pass. By means of these hydrogen can be
passed through the bell- jar to displace the air, when the stop-
cocks are closed. A dish of alkaline pyrogallol may be placed
at the bottom to absorb oxygen in case of a slight leak.

Discrete colonies of organisms may be obtained by
smearing the infected loop over the surfaces of three or four
slanted media tubes in turn, without reinfecting. The
tubes are incubated in a wide-mouthed stoppered bottle
with ample volumes of pyrogallic acid and soda~solutions.
One gramme of the former should be allowed for every
100 c.c. of air space.

Animal Inoculations. — Animal inoculations may be
required, among other reasons, (a) to enhance the viru-
lence of an organism which has become attenuated through
culture on media — the infective agent may be injected
alone, with another pathogenic organism, or with a toxin
to lower the animal's resistance; (b) to identify an organ-
ism; (c) to obtain a pure culture.

ANIMAL INOCULATIONS 45

Rabbits, guinea-pigs, white mice, and white rats, are the
animals generally used. The first two may be injected
subcutaneously or intraperitoneally. The hair on the
abdomen, between the scapulae, or near the root of the
tail, whichever part be chosen for the injection, is clipped,
and the skin rubbed with cotton- wool soaked in 1 in 1,000
mercuric chloride solution. For a subcutaneous injection,
the skin is pinched up and the needle inserted. The
needle is run in for its whole length when a large amount
is to be injected. Intraperitoneal injections are made in
the lower part of the abdomen; the abdominal walls are
pinched up, and the needle passed through the fold and
then withdrawn until the point is felt to be free in the
abdominal cavity, when the contents of the syringe are
emptied. Care must be taken to avoid injuring the
intestines. A special curved needle, with the hole about a
quarter of its length from the point, is frequently used for
intraperitoneal injections.

The large auricular veins of the ear render the rabbit a
suitable animal for intravenous injections. The vein
selected is rendered prominent by lightly pinching the
base of the ear.

For a ' pocket inoculation ' a small incision is made in
the skin, and, the latter having been separated from the
muscles by inserting the point of a pair of scissors and
slightly opening them, the tissue is inserted in the cavity.
The wounds left after inoculation are closed with collodion,
collodion and wool, or with one or two sutures.'

Mice are generally inoculated on the back, at the root of
the tail. Inoculations may also be made into the anterior
chamber of the eye, by rubbing infected material into a
scarified surface, or by the introduction of the material in
collodion sacs.

Examination of the Dead Animal. — As soon as pos-
sible the body is pinned on a board, the dorsal surface down.
The forceps, scissors, and scalpels, should be sterilised by
boiling in water containing a little sodium carbonate. The
animal and board are well soaked in disinfectant solution,
and the hair on the abdomen shaved or clipped. The
abdomen is seared with a hot iron, and an incision made
from the top of the sternum to the pubes; lateral incisions
are made, and the skin reflected and pinned out.

A fresh set of instruments is used for the next

46 AIDS TO BACTERIOLOGY

incisions, and a third set for removing the organs. The
material from which cultures are to be made depends on
the organism suspected. The site of inoculation, the
spleen, or the blood, may furnish the organism in most
abundance. Before any organ is opened it is first seared
with the cautery. Blood and peritoneal fluid may be
collected in sterile capillary pipettes. Blood is taken from
the right ventricle. After dissection the animal is
drenched with disinfectant, and, together with the board,
is burnt at once. The greatest care must be taken to
prevent the dissemination of infectious matter, and in
the event of any material being dropped, it must be
immediately swabbed up with disinfectant.

Blood Preparations. — Small quantities of blood are
obtained by pricking a finger or lobe of the ear with a
bayonet-pointed needle after sterilisation of the skin
by rubbing with alcohol and ether. For a Widal reaction
the blood is taken up in a capillary pipette. For other
&erum tests Wright's capsules are used. For blood-films,
the exuded drop of blood is touched with the edge of a
microscopical slide, and then brought in contact with an-
other slide, near its end. When the drop has spread across
the slide, the first is gradually drawn or pushed across the
horizontal one. The thickness of the film can be varied
at will by altering the angle at which the top slide is drawn
across the other. For determining the nature of organisms
in septicaemia, larger quantities of blood are required —
up to 5 or 10 c.c. Such blood is taken, with aseptic pre-
cautions, from a superficial vein (the median basilic or
median cephalic veins are convenient) with a sterile glass
syringe, a tourniquet being applied to produce venous
congestion. The blood is introduced in quantities of \ c.c.
into agar plates or broth-tubes.

CHAPTER IV

THE MICROSCOPIC EXAMINATION OF
BACTERIA

LIVING bacteria are observed in ' hanging drops,' and
dead bacteria in film preparations or in sections of tissue.
For bacteriological purposes, cover-glasses | inch in

HANGING-DROP PREPARATION 47

diameter and of No. 1 thickness are used. Cover-glasses
may be round or square, as preferred. They are cleaned
with sulphuric acid and potassium bichromate, and
kept, when clean, in alcohol, from which they are removed
with forceps, and either wiped with a clean rag or passed
through the flame to burn away the alcohol. To obtain
a satisfactory preparation, the cover-glass must be free
from grease. On a clean cover-glass or slide a minute
droplet of water or other liquid can be evenly spread,
whereas in the presence of grease it will run into pools.
A satisfactory film cannot then be expected, and there is a
probability of its washing off during manipulation. A
cover-glass should never be wiped with a circular or rotary
motion, but the wiping rag should be gently drawn across
it, taking care, in the case of a cover-glass, that the
pressure on both sides of it is equal.

Hanging-Drop Preparation. — Motility is most evident
in young cultures, and broth or agar cultures of twenty-
four hours old are most suitable. A hollow-ground slide
is passed through a Bunsen flame and then laid on a
clean bit of filter-paper, excavated side up. A ring of
vaseline is painted round the outside margin of the
excavation with a match-end. A clean cover-glass is
passed through the flame, laid on a clean flat surface, and
a droplet of a broth culture placed on the centre (when
a culture from a solid medium is used, a droplet of sterile
broth or water is placed in the centre of the sterile cover-
glass and inoculated with a minute trace of growth).
The excavated slide is lifted, turned over, and gently
lowered over the cover-glass, so that the drop of culture is
in the centre of the well and the ring of vaseline forms
a seal. The slide is now turned over so that the cover-
glass is uppermost, and placed under the microscope. For
organisms of the size of the typhoid bacillus a -^ objective
is suitable, but when a higher power is to be used, the
edge or centre of the droplet should first be centred with
a low-power objective. The light should be diminished,
either by nearly closing the diaphragm or by lowering
the condenser. Neither the Brownian movement (p. 2)
nor movement due to currents in the fluid should be
confounded with motility, in which the movement of
individual bacteria is independent, progression taking
place in different directions. Conversely, motile bacteria,

48 AIDS TO BACTERIOLOGY

particularly in cultures more than a day old, have a rest-
ing stage, when no motility is visible.

Intra-Vitam Staining. — A drop of a fluid culture is
placed on a sterile slide and covered with a sterile cover-
glass. A drop of stain is placed in contact with the edge
of the cover-glass. On application of a piece of filter-
paper to the edge diametrically opposite, the preparation
is irrigated with the stain. Non-toxic stains such as
neutral red or methylene green are used in 0'5 per cent,
aqueous solution. Other reagents may be similarly applied.

Negative Staining.— Burri's Indian-Ink Method. — Liquid
Indian ink (Chin-chin, Giinther and Wagner, etc.) is
sterilised either by heating in an autoclave or allowing
it to stand for twenty-four hours mixed with one twenty-
fifth its volume of tincture of iodine and then centrifuging.
A drop of this is placed on a microscope slide, and by the
side of it a drop of the culture to be examined. (Dilution
of the ink with from one to six times its volume of water
is sometimes necessary.) Then the edge of another micro-
scope slide is allowed to rest on the two drops, which mix
and run along the line of junction of the slides. The second
slide is drawn across the lower one to make a smear as for
a blood-film (p. 46). Organisms show up white on a black
background.

The Staining of Micro-Organisms. — Bacteria take up
the basic anilin dyes with great avidity, and in some
cases peculiarity in respect of a certain staining method
serves for identification.

Concentrated alcoholic solutions of stains are prepared
by allowing a large excess of the dye to digest for some
time in strong alcohol, shaking the solution from time to
time. The concentrated solutions are then filtered and
preserved in stoppered bottles. To increase the staining
properties, certain reagents (phenol, anilin, and alkalies)
are employed as mordants.

Stains should be filtered before use, otherwise granules
of colouring matter may be deposited upon the preparation.

Ehrlictis Anilin-Gentian Violet :

Saturated alcoholic solution of gentian violet 11 c.c.
Saturated aqueous solution of anilin . . 100 c.c.

The anilin solution is prepared by shaking about 5 c.c. of
colourless anilin with 100 c.c. of distilled water for some

STAINING METHODS 4$

time, and filtering the solution through a wet filter-paper.
Gentian violet can be replaced by 11 c.c. of saturated
alcoholic solution of fuchsin or methyl violet.

The prepared stain should not be kept longer than two
weeks.

Carbol -Gentian Violet :

Saturated alcoholic solution of gentian

violet . . . . . . . . . . 10 c.c.

Five per cent, phenol ... .. .. 100 c.c.

ZiehVs Carbol-Fuchsin :

Fuchsin . . . . . . . . 1 gramme.

Phenol . . . . . . . . 5 grammes.

Absolute alcohol . . . . . . 10 c.c.

Distilled water 100 c.c.

The fuchsin is dissolved in the alcohol, and the phenol,
previously dissolved in the water, is then added.

For ordinary cover-glass preparations this solution is
diluted with water in the proportion of 1 : 6.

In staining for tubercle bacilli, it must be remembered
that this dye sometimes loses its staining properties with
age, and it shouldjbe tested on a sputum known to contain
the bacillus in large numbers.

Lo filer's Methylene Blue :

Saturated alcoholic solution of methy-

lene blue . . . . . . . . 30 c.c.

Caustic potash solution (1 : 10,000) .. 100 „

This solution keeps well.

Kuhne's Carbol- Methylene Blue :

Methylene blue . . . . . . 1-5 grammes

Absolute alcohol . . . . . . 10 c.c.

Five per cent, aqueous solution of

phenol . . . . . . . . 100 c.c.

Nicolle's Garbol-Thionine Blue :

Saturated solution of thionine blue in

90 per cent, alcohol . . . . . . 10 c.c.

One per cent, aqueous solution of

phenol . . . . . . . . 100 „

Eosin : | to 1 per cent, solution in water or alcohol.

So AIDS TO BACTERIOLOGY

Bismarck Brown [(&) stain in Neisser's method for
diphtheria] :

Bismarck brown . . . . . . 2 grammes.

Distilled water (boiling) . . . . 1 litre.

Filter.
Pugh's Stain for Klebs-Lomer bacilli, see p. 111.

Leishman (p. 186), Jenner, picrocarmine, and Ehrlich-
Biondi triple stains are best bought ready prepared,
either in solution or in tablet form. All stains should be
kept in the dark when not in use.

Cover-Glass Preparations. — A small droplet of water is
placed in the centre of a clean cover-glass by means of
a sterile looped platinum wire. The mouth of the culture-
tube from which the preparation is to be made is singed
in the Bunsen flame. The plug is loosened by a rotary
motion, and partially withdrawn. An inoculating needle
(straight in the case of a streak culture, looped for a
broth culture) is heated to redness, the lower part of the
rod being also heated. The needle is held between the
right thumb and forefinger, and the plug is withdrawn
and held by the ring and little fingers of the right hand.
A trace of the growth (preferably from the margin in
the case of a streak culture, as the growth is youngest
there) is picked up. The needle is withdrawn and the
plug replaced. The growth is rubbed up with the drop
of water on the cover-glass, and spread over the surface
thereof. The needle is sterilised. The cover-glass may
be allowed to dry spontaneously, or may be held between
the fingers high over the flame. The film is now fixed
by passing the cover-glass, held in forceps, three times
through the Bunsen flame at the same rate as a clock's
pendulum swings. This fixing insures the film being
thoroughly dry, coagulates albuminous material, causing
adhesion of film to the glass, and may tend to diminish
the staining capacity of extraneous matter. A drop or
two of a filtered stain should now be dropped on the film,
or the cover-glass may be floated face downwards on the
stain contained in a watch-glass. The stain is allowed
to act for from two to ten minutes, according to the
preparation used. (The student should ascertain the
length of time which gives best results by experiment.)
To quicken the staining process, as is necessary in the

COVER-GLASS PREPARATIONS 51

case of some organisms, by using hot staining solution,
the cover-glass, well covered with the stain, is held by
forceps over a low gas-flame until steam just begins to
rise from the liquid, when the source of heat is removed*
This treatment is repeated at frequent intervals. A
better method is to float the cover-glass face downwards
upon the staining liquid, which has just previously been
heated in a small dish until the steam begins to rise. Great
care must be taken not to allow the stain to boil, as this
causes a precipitation of colouring matter, which renders
the preparation useless. The cover-glass is then well
rinsed in running water until no more colouring-matter
conies away. The cover-glass is blotted between filter-
paper and allowed to dry spontaneously. Sorop workers
make their preparations on the slide instead of a cover-glass.

A small drop of a thick solution of Canada balsam in
xylol is placed in the centre of a clean glass microscope
slip, and the cover-glass, prepared surface downwards,
deposited on the drop of balsam, which then spreads out.
The preparation can now be observed by placing a drop
of cedar oil on the top of the cover-glass, and examining
with the oil-immersion lens. After examination the cedar
oil on the cover-glass is carefully absorbed with filter-
paper. After a few days the balsam will become hard.

if a permanent preparation is not required, the cover-
glass can be examined immediately after washing off the
excess of stain by placing on a glass slip, taking care to
dry the top surface of the cover-glass before applying the
drop of cedar oil, or may be dried and examined in cedar
oil or liquid petrolatum.

Smear Preparations. — A cover-glass is brought in
contact with the freshly-cut surface of the organ, such
as the liver or spleen. Another method is to press the
material between two cover-glasses, which are then
separated by sliding them apart, leaving a thin layer of
material on each. This method is particularly applicable
to blood and sputum. The smears are air- dried and
stained, as described under cover-glass preparations.
(For preparation of blood films see p. 46.) Smears of
blood, pus, and organs may be stained by Leishman's or
Jenner's methods (see p. 186).

'Impression' Cover-Glass Preparations. ('Contact*
Preparations). — A cover-glass is held with a pair of forceps

52 AIDS TO BACTERIOLOGY

over a colony (which should not exceed 2 millimetres in
diameter), in a slanting position, with one edge resting on
the nutrient medium, then allowed to sink gradually
down over the colony, and very gently pressed. The
cover-glass is carefully lifted with a needle, and allowed
to dry spontaneously. The preparation is ' fixed ' and
stained. This method shows the manner of growth and
the arrangement of the organisms.

Staining of Spores. — In ordinary cover-glass prepara-
tions spores resist the stain. All unstained spots are not
spores: they may arise from faulty staining due to air-
bubbles, the use of old staining solutions, or, in the case
of old cultures, the organisms may have become de-
generate «tnd vacuolated.

Heat Method. — A cover-glass preparation is passed
through the flame twelve times in ' fixing,' stained for a
few minutes with warm Ehrlich's gentian- violet or Ziehl's
fuchsin solution, and then well washed in water. The
heating destroys the power of the organisms to take up
the stain, leaving only spores stained.

Neisser's Method. — The cover-glass preparation, made
in the usual way, is stained with warm carbol-fuchsin
solution for about ten to twenty minutes. It is best to
float the cover-glass on the surface of the stain contained
in a small dish on a sand-bath or piece of asbestos card-
board warmed with the Bunsen. The cover-glass is
removed, washed in water, decolorised for a few seconds
in a 3 per cent, alcoholic solution of hydrochloric acid,
well washed in water, counter-stained with Ldffler's
methylene blue for three minutes, washed in water,
blotted, dried, and mounted. The bacilli will be stained
blue and the spores red.

Moeller's Method. — The films are prepared and fixed,
and then treated with — (a) absolute alcohol, two minutes;
(6) chloroform, two minutes; washed thoroughly; (c) 5 per
cent, chromic acid, one minute. They are stained in
warm Ziehl's fuchsin for ten minutes, decolorised in 1 per
cent, sulphuric acid for a few seconds (this has to be done
with care), washed, counter- stained with Loffler's blue for
two or three minutes, again washed, dried, and mounted.
The spores are stained red and the bacilli blue.

Flagellum Staining. — Flagella possess no affinity for
stains unless previously mordanted. The cover-glasses

FLAGELLUM STAINING 53

must bo absolutely dean, and the growth be well diluted
before spreading, to render individuals sufficiently isolated.
A trace of an eighteen to twenty-four hour culture of the
organism is very gently mixed with a few drops of tap-
water in a watch-glass. A minute drop of water is placed
on a clean cover-glass which has been well heated over a
Bunsen flame and allowed to cool, and a very small loopful
of the bacterial emulsion is added. The cover-glass is
spread at once, and the material thereon should be suffi-
ciently small to cause the film to dry immediately after
spreading. The cover-glass is held in the fingers and
passed three times through the flame at such a rate that
the heat can be endured by the fingers.

McCrorie's Night Blue Method. — Solution A. — Dissolve
0-5 gramme of McCrorie's night- blue in 20 c.c. of absolute
alcohol.

Solution B. — Dissolve 1 gramme of tannic acid in 20
c.c. of hot water. Add 1 gramme of alum dissolved in
20 c.c. of cold water.

Add A to B slowly, shaking gently all the time. Filter.

The stain is allowed to act for two minutes in a warm
place.

M uir's Modified Pit field Method.

A. The Mordant.—

Tannic acid (10 per cent. aq. sol., filtered) 10 c.c.

Sat. aq. sol. mercuric chloride . . . . 5 „

Sat. aq. sol. alum . . . . . . . . 5 ,,

Ziehl's carbol-fuchsin . . . . . . 5 „

A precipitate forms which may be allowed to deposit
or centrifuged.

The clear solution is removed and will keep a week.

B. The Stain.—

Sat. aq. sol. alum . . . . . . . . 10 c.c.

Sat. alcoholic sol. gentian violet . . . . 2 „

This keeps two days.

The film is flooded with the mordant and steamed for
one minute. After well washing in water for two minutes,
it is very cautiously dried, flooded with the stain, steamed
for one minute and well washed in water.

Capsule Staining. — By considerably diminishing the
light, capsules may be observed in fresh preparations.

54 AIDS TO BACTERIOLOGY

But the clear haloes sometimes seen round bacteria in
albuminous material must not be confused with capsules.
Bum's India ink method and Gram's method often show
the capsules very well.

Richard Muir's Later Method. — A thin film after drying
is stained with warm carbol-fuchsin for 30 seconds,
washed slightly in alcohol and again thoroughly in water.
It is placed in the following mordant for a few seconds:

Sat. sol. mercuric chloride . . . . . . 2 parts

Tannic acid (20 per cent.) solution . . . . 2 „

Sat. sol. potash alum . . . . . . . . 5 „

After well washing in water, the preparation is treated
with methylated spirit for about a minute and should
then be pale red. After thorough washing in water
and counter-staining with methylene blue for 30 seconds,
the film may be dehydrated in alcohol, cleared in xylol
and mounted, or may be simply dried with filter paper.
The bacteria are of a deep crimson, and the capsules blue.

The Treatment and Staining of Sections. — The tissue is
first ' hardened,' and then cut into sections with some
form of microtome.

Hardening of Tissues. — The most satisfactory harden-
ing reagent is alcohol. It is preferable to pass through
increasing strengths of alcohol — e.g., 50 per cent., 75 per
cent., and finally absolute — the tissue remaining in each
for twenty-four to forty- eight hours. The tissue is cut
into pieces 10 to 20 millimetres square and 5 to 10 milli-
metres thick; these are immersed in the alcohol, which
may be changed once. If absolute alcohol alone be used,
it may be contained in a wide-mouthed bottle, in the cork
of which are fixed several needles. The pieces of tissue
are placed on the needles in such a manner that, when
the cork is fixed in the mouth of the bottle, the pieces of
tissue are just beneath the surface of the alcohol. The
alcohol gradually abstracts the water from the tissue, and
as that containing the water sinks to the bottom, fresh
alcohol constantly comes in contact with the material.
Tissues containing much water are naturally more diffi-
cult to harden than those containing little.

Before hardening in alcohol, it is usual to ' fix ' the
tissues, for the better preservation of the tissue element
A saturated solution of corrosive sublimate in water, to

TREATMENT OF SECTIONS 55

which 1 to 2 per cent, of acetic acid may be added at the
time of using, or 10 per cent, formalin, are suitable for
the purpose. The pieces of fresh tissue are placed in
either solution for twelve to twenty-four hours, washed in
running water, and then placed in absolute alcohol, or
passed through increasing strengths of alcohol as detailed
above.

After hardening, the tissue may be preserved in 70 per
cent, alcohol. Miiller's fluid (potassium bichromate) and
chromic acid are not suitable for hardening tissues for
bacteriological work.

After hardening, the tissue is embedded, in order to
prepare it for the section- cutting machine.

The Freezing Method. — The tissue can be frozen without
preparation beforehand, but where it has been hardened
it must be first freed from alcohol. The pieces of tissue
are placed in a wide-mouthed bottle, a funnel is stuck in
the mouth of the latter, and a stream of water from a
tap allowed to run in for one to two hours. The tissues
are then soaked in a mucilage of cane-sugar and gum
acacia (to which a little carbolic acid or a piece of thymol
should be added to prevent the growth of bacteria and
moulds) for twenty-four hours, and are then ready for
cutting. A piece of tissue is placed on the plate of the
freezing-microtome, a little mucilage added, so that it is
surrounded with this, and the whole frozen, the freezing
being carried out by spraying the under-surface of the
plate with ether. This is done by filling the bottle of the
apparatus with ether and working the bellows. The
sections are cut by working the knife, which should be
moistened with water, and the sections as cut are re-
moved with a moistened camel' s-hair brush, and immersed
in a dish of tepid water for an hour to remove the gum.
From time to time the bellows are worked to keep the
mass frozen. Compressed carbon dioxide is now often
used instead of ether; the jet of escaping gas impinging
on the under surface of the plate quickly freezes the
section. After the washing with water the sections may
be stained, or may be preserved in 70 per cent, alcohol
for future examination.

The freezing process is indicated when rapid production
is essential. The sections stain readily. It is frequently
used during the actual progress of operations. It has

56 AIDS TO BACTERIOLOGY

certain disadvantages — the cellular structure may be dis-
torted, the sections are not very thin or regular, and
delicate tissues are apt to be torn.

Embedding in Paraffin. — This method gives the thinnest
sections and preserves the structures. The material,
after hardening, is placed in absolute alcohol for twelve
to twenty-four hours, and then in pure xylol until it
looks clear. Chloroform may be used instead of xylol.
After this it is placed in a bath of melted paraffin wax,
in which it remains for six to eighteen hours, according
to the size, until thoroughly impregnated with the melted
paraffin. The paraffin is kept in the melted state in a
special bath or hot- water oven, the temperature of which
is regulated by a thermo-regulator: Hearsons make a
special form with ' Excelsior ' gas regulator. As regards
the paraffin wax to be used, opinions differ, but probably
one having a fairly high melting-point — e.g., 50° to 52° C.
— is the best all round. Some tissues, such as skin, must
not remain longer than is absolutely necessary in the
melted paraffin, or they become hard and friable, and
will not cut. After impregnation the material has to be
embedded; a paper mould or small cardboard box (such
as a pill-box) is about one- third filled with melted paraffin
wax; the prepared pieces of tissue are laid on the centre
of the wax layer, then more melted paraffin wax is poured
on in such a way as to enclose the material in the centre
of a small block of wax. When set, which is preferably
hastened by immersion in cold water, the block is trimmed
to a suitable form with a knife, and cemented to the
carrier of the microtome by softening the base of the
mass with a match-flame, and melting the margins with
a hot wire. The sections are cut without any moistening
fluid.

The sections are next mounted on slides. This is done
by placing them in a dish of warm water (a pastry- tin
does well, and may be warmed over the Bunsen), the
temperature being so adjusted that the paraffin becomes
softened, but not melted, so that the sections become flat.
A slide is introduced into the water under a section, which
is then lifted up on it, being fixed and adjusted by means
of a needle. The water is then tilted off, and the specimen
allowed to dry for not less than two to three hours in
the warm incubator. If the sections be thin, they adhere

TREATMENT OF SECTIONS 57

sufficiently firmly to the slide to allow staining, etc.,
without floating off. But if they be thick, or if the stain-
ing has to be prolonged, it is preferable to first smear the
slide with egg-albumin mixture (egg-white beaten up
with a little water, mixed with an equal volume of glycerin,
and sodium salicylate, 1 gramme per 100 c.c., added as
a preservative, and strained) before picking up the section
on it.

Before staining, the slide with section is immersed in
xylol (best in a suitable pot or glass cylinder) for two
minutes to dissolve out the paraffin, then in absolute
alcohol to remove the xylol.

The Staining of Bacteria in Sections. — The following
procedure is common to most methods: The sections are
rinsed with distilled water to remove alcohol, and then
subjected to the action of the stain for a time varying
from a few minutes to several hours. The time is in
some cases shortened by warming the staining solution.
The sections are next washed, preferably in distilled
water, and then in some instances decolorised with a
suitable reagent; they are again washed, then counter-
stained if necessary. The sections are now dehydrated
with alcohol, and then cleared with xylol, cedar oil, or oil
of cloves. Xylol or cedar oil is preferable to oil of cloves
as a clearing agent, as it has no solvent action on the
stains, and the former does not resinify on exposure to
the air, and evaporates without leaving a deposit. Great
care must be taken to remove all the water from the
section by means of absolute alcohol before transferring
to the xylol or cedar oil, otherwise the section will not
properly clear.* After remaining in the xylol for about
two or three minutes, the section, if unattached to the
slide, is removed by means of a section-lifter, and then
laid out flat by careful manipulation with two small
pointed glass rods or needles on a clean glass slide; the
excess of clearing agent is removed by careful blotting,
with firm pressure, with two or more thicknesses of filter-
paper. A drop of thick solution of Canada balsam in

* Oil of cloves will clear out of methylated spirit, but for xylol
or cedar oil absolute alcohol must be used. For many purposes
methylated spirit may be substituted for absolute alcohol, but
the ordinary commercial methylated is unsuitable, as it becomes
milky on the addition of water. The old methylated spirit
(alcohol and wood .spirit) must be used.

58 AIDS TO BACTERIOLOGY

xylol is dropped on the section, and a cover-glass laid on
in such a way that the drop of balsam covers up the
section, and extends over the whole under-surface of the
cover-glass, as in the case of simple cover-glass prepara-
tions. The preparation is now ready for examination
with the oil-immersion lens. If the section is fixed to the
slide, as is the case with paraffin sections, all the manipu-
lations are carried out on the slide, the slide being flooded
with the stains and various reagents, or immersed in pots
containing them.

Loffler's Method. — The sections are stained in L6 flier's
methylene blue for from ten to sixty minutes, then rinsed
in distilled water and slightly decolorised by immersing
in a 0-5 per cent, solution of acetic acid for a few seconds.
The sections are now washed, dehydrated in absolute
alcohol, cleared in xylol, transferred to the slip, blotted,
and mounted as usual. Carbol-thionine blue may also be
used, and often differentiates better than Loffler's
blue.

Kuhne's Method. — The sections are placed in Kiihne's
carbol methylene blue for thirty minutes or longer, then
washed in water, and very carefully decolorised in very
dilute hydrochloric acid (20 drops of strong acid in 100 c.c.
water). Thin sections only require to be immersed for
two or three seconds. The sections are at once trans-
ferred to an alkaline solution (10 drops of a saturated
solution of lithium carbonate in 10 c.c. of water), washed
in water for a few minutes, dehydrated in absolute alcohol
tinted with methylene blue. The sections are now placed
in anilin oil, which also contains a little dissolved methy-
lene blue. After being washed in colourless anilin, then
in xylol, they are mounted, as usual, in balsam.

Ziehl-N eelsen Method. — Where sputum is to be examined,
a little of the material is spread on a microscope slide.
If any small yellow caseous particles are present, these
should be selected, otherwise the thick portion of the
sputum should be used. After spreading, drying, and
fixing, filtered carbol -fuchsin solution is dropped on the
film, and the slide is heated over a Bunsen flame till
steam just rises, care being taken to prevent the stain
boiling. The stain is allowed to act for five minutes,
fresh stain being added as evaporation takes place. The
preparation is now decolorised by dipping alternately

STAINING OF SECTIONS 59

in 25 per cent, sulphuric acid and in water until the film
is practically colourless after the last rinse in water. The
film is now well washed in water, and counter- stained
with Loftier' s methylene blue for one minute, again
washed in water, and allowed to dry. A drop of cedar
oil is added, and the film examined with the oil-immersion
objective direct. Any acid-fast organisms present will be
stained red.

The method for staining the tubercle or leprosy bacillus
in section by this process is as follows: (1) Stain the
sections in warm carbol-fuchsin solution for ten minutes.
(2) Rinse in water. (3) Decolorise in 25 per cent, sulphuric
acid and in water, transferring from one to the other
alternately until almost decolorised; a faint pink does no
harm. (4) Rinse in water. (5) Counter-stain in Loftier' s
methylene blue solution for three minutes. (6) Dehydrate
in absolute alcohol for half to one minute. (7) Clear in
xylol or oil of cloves for five minutes, transfer to slide,
blot off excess of clearing agent, and mount with a drop
of balsam.

Gramas Method. — Grain's method can be applied
equally well to cover-glass preparations and to sections.
The process is as follows: (1) Stain the film in Ehrlich's
anilin gentian violet or carbol gentian violet for five
minutes, or a section for ten to thirty minutes, and
drain off the stain. (2) Immerse the preparation
without washing in iodine solution (1 gramme of iodine
and 2 grammes of potassium iodide dissolved in 300 c.c.
of water) for one to two minutes, when the colour changes
to a dirty brown. (3) Wash in alcohol until no more
colour comes away. (4) Counter-stain in eosin or Bis-
marck brown. Cover-glass specimens can now be washed,
dried, and mounted, while sections require to be further
treated as follows: (5) Dehydrate in alcohol. (6) Clear
in xylol or oil of cedar, transfer to the slide, if necessary,
with the section-lifter, lay out flat, blot off the excess of
clearing agent, add a drop of balsam, and mount. (Cover-
glass preparations of pure cultures do not require counter-
staining. After (3) rinse in water, dry, and mount.) All
bacteria do not retain their stain when thus treated.
Those which after the alcohol treatment, i.e. (3), are still
stained are referred to as ' Gram -positive,' while those
which are decolorised are known as ' Gram-negative.'

60 AIDS TO BACTERIOLOGY

Gram-GilntTier Method. — The section is treated with
a 3 per cent, solution of hydrochloric acid for a few
seconds after the first alcoholic washing. By this treat-
ment cleaner and brighter preparations are obtained.

CHAPTER V
THE ACID-FAST ORGANISMS

MOST organisms when stained with carbol-fuchsin are
readily decolorised by weak solutions of mineral acids.
The tubercle, leprosy, and smegma bacilli are, however,
notable exceptions, and retain the stain even after treat-
ment with 25 per cent, sulphuric acid or with 30 per cent,
nitric acid. This persistent retention of the stain is also
shown by some saprophytes (vide p. 74), and by some
members of the Streptotrichece. It is probable that the
degree of resistance of streptothrices to decolorisation is
directly proportionate to the vitality of the organism.
A pathogenic streptothrix from an old lesion or from
one that has been vigorously treated with antiseptics will
probably be decolorised, and will be Gram-negative,
whereas acid-fast bacteria are Gram-positive. It is
believed that some strains of the tubercle bacillus arc
naturally not acid-fast. In young cultures of tubercle
bacilli some, presumably immature, rods are not acid-
fast while older ones are.

The Tubercle Bacillus.

Bacillus tuberculosis is a non-motile, slender rod often
slightly curved, with rounded ends. It is from 2-5 to
5 /LI long and 0-2^ thick (see also p. 159). When stained
it often shows unstained areas (' beading ') which a
minority of observers consider to be spores. In the moist
state the thermal death-point is 60° to 65° C. Milk which
has been kept at 70° C. for thirty minutes (Pasteurisation)
is regarded as safe, but there is some question as to whether
tubercle bacilli are not protected by the pellicle that forms
on milk heated in an open vessel. The Board of Agri-
culture recommend an exposure to 85° C. for fifteen
minutes for rendering creamery products harmless.

TUBERCLE BACILLUS 6l

Culture. — The organism is aerobic and facultatively
anaerobic. On ordinary media growth is either absent
or scanty. It grows best at blood-heat, growth being
extremely slow; no appreciable evidence being shown
under four to six weeks. Primary cultures are made on
Dorset's egg medium, glycerin agar cum egg-yolk, or
glycerinated potato. In glycerin broth it develops as
a floating pellicle. On glycerin agar it gives a cream or
brownish-yellow film which is dry and wrinkled. A pure
culture can be obtained by the inoculation of tubercular
sputum into a guinea-pig. After three to six weeks the
animal is killed, and matter from the tubercles is streaked
over glycerin potato (a semicylinder of potato in a Roux's
potato tube, the bulb being filled with 5 per cent, glycerin).
In six or eight weeks a fair number of the tubes will show
a growth. The organism does not liquefy gelatin. On
repeated subculture it becomes longer and thicker; clubbed
and branching forms may develop, which leads some to
class the bacillus with the trichomycetes. As a sapro-
phytic habit is developed the virulence is diminished,
but can be restored by passage through an animal.

Staining Reactions. — Ordinary stains have little effect
on the organism, but it stains well by Gram's method
and the Ziehl-Neelsen method. Much concluded that
three forms of the organism exist: (1) The ordinary
'acid-fast' bacillus; (2) a bacillus that is not acid-fast;
and (3) free granules also not acid-fast. Whether these
last two forms are degenerate organisms or resistant
types remains unproved. At any rate it appears un-
questioned that the non-acid-fast granules on injection
into animals produce tuberculosis due to the ordinary
acid-fast bacillus.

In addition to being acid-fast, the tubercle bacillus is
also " alcohol-fast," a property that serves to distinguish
it from the smegma and similar bacilli which lose their
stain in alcohol treatment. Inasmuch as smegma bacilli
may occur in urine, sputum, and other material frequently
examined for tubercle, it is advisable to apply alcohol
to fuchsin- stained preparations as a routine process,
especially as it can be combined with acid treatment.
Instead of using 25 per cent, sulphuric acid the fuchsin
preparation is decolorised in acid alcohol (3 per cent,
hydrochloric acid in alcohol) till almost white.

62 AIDS TO BACTERIOLOGY

Channels of Infection. — The sputa of consumptives are
almost certainly responsible for most infections. The
tubercle bacillus is, for a non-sporing organism, tenacious
of life, and when sputum is allowed to dry, especially
when protected from light and air, may easily be dis-
seminated in dust. In the acts of coughing, sneezing, and
talking, minute droplets of fluid are disseminated. Fliigge
has shown that these droplets may remain in the air for
some time, and also that, when from consumptives, they
often contain tubercle bacilli. The inhalation of either
tubercle-laden dust or droplets of saliva from consump-
tives must be regarded as a standing menace. Infection
may take place through the alimentary tract, especially
in infants, by the ingestion of tuberculous milk or meat.
Flies can carry the disease. A phthisical patient may
swallow his own sputum, and thus infect his alimentary
tract. Von Behring considers that tubercle bacilli taken
in milk during infancy may remain dormant for years,
and then set up infection. Accidental inoculations
through the skin with tuberculous material, principally
the result of post-mortem examinations (' pathologists'
warts '), are seldom serious. Lupus is presumably acquired
by inoculation through a wound or abrasion of the skin.

Hamburger and Schlossmann practically agree that
90 per cent, of all children up to the completed twelfth
year are infected.

Autopsy statistics show tuberculous lesions in 58 per
cent, of adults according to Beitzke. Other statisticians'
figures run up to 90 per cent, and more.

Pathogenesis. — Scarcely any portion of the human
frame is immune to tubercle. Whooping-cough, pneu-
monia, typhoid, measles, influenza, diabetes, and syphilis,
predispose to the disease, as do also intemperance, poverty,
uncleanliness, overcrowding, and lack of ventilation.
Trades in which operatives are exposed to dust of an
irritating kind, which are carried on in overcrowded,
hot, or badly-ventilated rooms, in which workers adopt
a cramped posture, or in which exposure to quick alterna-
tions of temperature is frequent, show a heavy mortality
from the disease.

The disease is not hereditary in the true sense of the
word. An infected child of a tuberculous mother is gene-
rally the subject of postnatal infection. Nevertheless,

TUBERCLE BACILLUS 63

intra- uterine or placenta! infection does occasionally occur.
Although human semen may contain tubercle bacilli.
Jordan considers that transmission by the male parent
is very unlikely to occur, and there is only one recorded
case of an infected ovum. A ' tendency ' to the disease may
be inherited, but it is more logical to attribute the occur-
rence of the disease in a family to the intimacy of family
relations allowing abundant opportunity for infection.

The lesions produced in tuberculosis have more or less
similarity to those of leprosy. The little yellowish
nodules or tubercles (to which anatomists first applied the
term ' tuberculosis ') are non- vascular, and vary in size
from that of a pin's head up. They consist of vast
aggregates of cells (hence the term ' Granulomata '). In
the centre of a young tubercle a mass or masses of pro-
toplasm are found (' giant cells '). (Giant cells are much
less common in leprosy.) In the giant cell is a ring of
tubercle bacilli, and around this zone and arranged round
the periphery is a ring of nuclei. Around the giant cells
epithelioid cells with large nuclei are found, and around
these a collection of lymphoid cells.

Although tuberculosis is best known as a pulmonary
disease, the glands, skin, bones, peritoneum, urinary
organs, meninges, etc., are also frequently attacked.
Widely diverse findings are recorded of the frequency with
which the tubercle bacillus occurs or is found in the
blood. Minchin assumes tubercle bacilli in the host to
be suspended in ' shut-away fluid ' which protects them
from the influence of germicides and sera.

Bovine Tuberculosis. — Cattle, particularly those kept
in insanitary sheds, are very liable to tuberculosis. The
bovine organism may reach others than those in contact
with the animal, through the medium of the milk or
flesh. When the udder is affected, the bacilli will prob-
ably be found in the milk, but even when the udder is
free from tuberculosis, the bacilli may find their way
into the milk, from the faeces, uterine secretion, or sputum,
according to the locality of the infection, unless precau-
tions are taken during milking. The bovine bacillus is.
shorter, thicker, and less readily cultivated than the
human one. These considerations, with others, led Koch
in 1901 to question the communicabiiity of the bovine
tubercle to man. A Royal Commission was appointed in

64 AIDS TO BACTERIOLOGY

the same year, and they arranged human tubercle bacilli
in two classes — those growing with difficulty on certain
artificial media, which they called dysgonic, and those
growing readily on the same media, which they called
eugonic. Experiments with dysgonic bacilli of human
origin gave results identical with those made with bovine
bacilli. In a few cases both the human and bovine types
may be found together. As a rule, pulmonary lesions in
man are due to the human type of bacillus, but sometimes
the bovine tubercle bacillus is solely responsible. (Kossel
found two cases in 709 cases of phthisis.) See p. 265.

The majority of cases in which the bovine tubercle
bacillus produces lesions in man are cases of alimentary
tuberculosis: cervical gland and primary abdominal
tuberculosis. In the latter class of cases at least the
tubercle bacillus has unquestionably been swallowed.
The Commissioners stated their conviction that a certain
number of cases occurring in the human subject, especially
in children, are caused through the introduction into the
body of bovine bacilli, the majority of cases being caused
through tuberculous milk.

As long ago as 1886,* Harries stated from clinical
observations that lupus was not due to tubercle bacilli
as then known to infect human beings, and a few years
ago the opinion that lupus was an infection with the
bovine type of tubercle bacillus gained credence. Nine
out of the twenty cases of lupus investigated by the
Commission proved on culture to be bovine tubercle
bacilli, though generally they were of feeble virulence.
Park and Krumwiede found that from 6 to 10 per cent,
of all deaths due to so-called surgical tuberculosis were
caused by bovine tubercle bacilli, the cases of tuberculous
adenitis and abdominal tuberculosis of children being
more often caused by the bovine than by the human type.
The same was found true, though to a less extent, in
adults. Stiles says that 90 per cent, of surgical tuber-
culosis seen in Edinburgh was due to bovine tuberculosis
and 90 per cent, of the gland cases were due to infection
through the tonsils by milk. Some cases of tuberculous
joints which show no improvement under human tuberculin
treatment rapidly undergo cure with bovine tuberculin.

* ' Lectures on Lupus ' : A, Harries, M.D., and C. Campbell,
M.D. Bailliere, Tiudall and Cox. 1886.

BOVINE TUBERCULOSIS 65

The Royal Commission did not conclude that the
human and the bovine types represent two distinct organ-
isms, but preferred to regard these two types as varieties
of the same bacillus, and the lesions which they produce,
whether in man or in other mammals, as manifestations
of the same disease. Dr. Arthur Eastwood, the bacterio-
logist to the Commission, after mention of the remark-
ably stable characters of the viruses, says: ' I find that
underlying all the mammalian viruses which I have
investigated there is an essential unity of characteristics,
the differences observed being differences of degree but
not of kind. On artificial culture media they all grow
in the same way, though they differ quantitatively in
the amount of growth yielded. In the tissues of suitable
experimental animals they all produce lesions histologically
characteristic of mammalian tuberculosis, though they
differ in the intensity of the tissue changes which they set
up under similar experimental conditions.' As the
Commission have conclusively shown that many cases
of fatal tuberculosis in the human subject have been
produced by the bacillus known to cause the disease
in cattle, they are emphatic that the possibility of such
infection cannot be denied. Of fifty-nine cases of tuber-
culosis in pigs investigated by the Commission fifty yielded
the bovine virus, three the human, five the avian, and
one a mixture of the bovine and avian.

Butter, skimmed milk, and butter-milk will contain
tubercle bacilli if made from tuberculous milk. The use
of the mixed milk of a herd, although perhaps reducing
the risk, does not entirely remove it. Desirable though
it would be to prohibit the use of meat from animals
suffering from any manifestation of tuberculosis, economic
considerations have to be regarded. It is found that
the fat and muscular tissues are seldom involved, the
lungs, liver, and pleura (' grapes ') being the organs most
often diseased. The Royal Commission on Tuberculosis
(1898) recognised the fact that tubercle bacilli are seldom
encountered away from diseased areas, and recommended
that, provided the carcasses b^ otherwise healthy, in the
restriction of lesions to the lungs, thoracic lymphatic
glands, liver or pharyngeal lymphatic glands, the non-
tubercular portions of the carcass should not be con-
demned.

5

66 AIDS TO BACTERIOLOGY

In the German Freibanks, after removal of tubercular
areas, the meat is sterilised, and then stamped and sold
as inferior. It has been shown that the tubercle bacillus
is not destroyed, if in the centre of a joint of meat
over 6 pounds in weight, by the ordinary method of
cooking.

The Local Government Board has ordered that ' strip-
ping ' (removal of ' skin ' from inside of the ribs or flanks,
with a view to effacing signs of the disease), in the case
of foreign dead meat, shall be sufficient reason for con-
demnation. It must not be overlooked that, after re-
moving tubercular organs, a butcher may use the same
knife for cutting up a carcass, and thus infect healthy
meat.

Tuberculosis is rare in new-born calves, and, if removed
from a tubercular mother at birth and properly treated,
they will not, as a rule, develop the disease. In Bang^s
method for eliminating tuberculosis, the herd is tested
with tuberculin, those that react or which are suspicious
being isolated. The herd is divided into two sections,
which are separated from one another, and have separate
attendants and separate buildings; they are, however,
allowed to mix when out in the fields. Every six months
the healthy side is tested with tuberculin, and any beasts
that are found to react are placed on the infected side,
while all calves are placed on the healthy side. The
animals on the tuberculous side which are obviously tuber-
culous are got rid of, but those that are apparently healthy
are used for breeding. Ostertag's method consists in the
elimination of cows suffering from ' open ' tuberculosis
(i.e., where there are open tuberculous lesions in organs
with means of external communication, such as in the
udder, lungs, intestines, and urino-genital organs) from
the others.

Avian Tuberculosis. — Fowls, pheasants, turkeys, and
pigeons suffer from the disease, which attains an extra-
ordinary virulence in insanitary fowl-houses. Ducks and
geese are immune. Whereas growth of the human bacillus
ceases at 41° C., the avian organism grows well at 43° C.
Its cultures on solid media are softer, more greasy, and
less tightly packed than those of human tuberculosis.
The rabbit is readily infected with it, mice, horses and
swine are susceptible, while the guinea-pig shows much

TUBERCULIN 67

greater resistance. Inasmuch as bacilli with typical avian
characteristics have been obtained from human cases,
it seems highly probable that avian tuberculosis may be
a source of infection.

Loswenstein found that the ovary was always diseased
in affected fowls, and the bacilli were also present in the
yolk and in the membranes of the ovum. He found that
even boiling eggs to hardness did not destroy the vitality
of the bacilli and thought human beings were infected
through eating raw or imperfectly cooked eggs. On the
other hand, the Commission, comparing the mammalian
with the avian viruses, found differences not merely of
degree but of kind, though sometimes lesions of a chronic
type are produced that are indistinguishable from infec-
tions set up by mammalian bacilli. In their final Report
they dismissed the infection of man from tuberculosis
in birds as a negligible factor.

Tuberculin. — Koch's Old Tuberculin is prepared by
growing tubercle bacilli in glycerin veal broth. A copious
film formation being needed, flat flasks are used, so that
a comparatively large surface is exposed to the air. After
six to twelve weeks the culture is evaporated to one-tenth
its bulk on a water- bath, and then filtered through a
Pasteur filter.

The injection of 0-002 c.c. into a tuberculous person gives
rife to laboured breathing, malaise, and pyrexia, great
inflammatory reaction and necrosis occurring round the
tubercular focus. If injected into a patient in whom
phthisis is dormant, it is very apt to cause the old trouble
to break out afresh.

In the diagnosis of tuberculosis in cattle it is very valu-
able, the failures being only about 2 per cent. The injec-
tion of 0-1 to 0-2 c.c. causes a rise of temperature of
2° to 3° F. above the normal in from eight to twelve hours.

Koch's New Tuberculin is prepared by drying and
pounding young and virulent tubercle bacilli, and ex-
tracting with water; the emulsion is then centrifuged.
The supernatant fluid (TO) is now discarded. The
residue left in the centrifuge is dried, triturated and centri-
fuged as before, these processes being repeated until
hardly any solid residue is left. The whitish, opalescent
liquids resulting from these operations are mixed, and
constitute TR.

68 AIDS TO BACTERIOLOGY

TR possesses immunising power with but little reaction.
The fluid TR is made to contain 2 milligrammes of solid
matter in the cubic centimetre. This is diluted with
sterile salt solution to the required strength. If reaction
occurs, this must be further diminished. Injections
are made every other day with slightly increasing doses,
so that there is never a rise of temperature of over 1° F.

Koch's Bazillenemulsion is a suspension of pulverised
bacilli in equal parts of water and glycerin.

The reaction of a tuberculous individual to tuberculin
is due to anaphylaxis. It differs from most anaphylactic
reactions as it is accompanied by rise and not fall of
temperature. A dose of tuberculin, however, does not
sensitise the animal to a second injection, this preparation
stage taking place in the tuberculous tissues.

The sera of animals immunised in various ways have
been employed in treatment, but only with occasional
success.

Notification. — Tuberculosis in man is compulsorily
notifiable throughout England and Wales. Tuberculosis
with emaciation in any bovine animal and tuberculosis
of the udder in cows are notifiable under the Tuberculosis
Order of 1913.

Segregation (Newsholme's Law).— International statis-
tics show reason for believing that the reduced mortality
from tuberculosis is largely due to the increasing extent
to which advanced cases are treated in general institu-
tions, and thus segregated from their families, instead of
receiving outdoor relief and thus infecting them.

The term ' pseudo-tuberculosis ' has been applied to a
number of conditions the common feature of which is the
presence of tubercle-like nodules in the tissues. Such
may be caused by pathogenic streptotrichece, yeasts and
moulds, parasitic worms, etc. See also B. pseudo-tuber-
culosis (p. 125), and Johne's bacillus (p. 70).

Laboratory Diagnosis. — Sputum. — The Ziehl-Neelsen
method is used (p. 58). Although tubercle bacilli are
resistant to putrefactive changes, a little 5 per cent,
phenol should be added when the examination is delayed.
Where it is advisable to concentrate the specimen's
content of tubercle bacilli, one part of ' antiformin ' (a
mixture of equal parts of Liquor sodee chlorinates [B.P.]
and 15 per cent, caustic soda solution) may be added to

LABORATORY DIAGNOSIS 6g

from four to six parts of sputum caccording to the con-
sistence of the latter. The resulting mixture is shaken
well and allowed to stand for two or three hours till
solution occurs, which may be hastened by placing the
mixture in the incubator. On centrifuging, the tubercle
bacilli are obtained concentrated in the sediment, which
is generally very slight. Other acid-fast organisms are
occasionally met with (p. 74) in sputum. Negative
evidence is only of value when repeated examinations
have been made.

Milk.— See p. 222.

Urine. — To exclude the smegma bacillus (pp. 61, 73),
the urine may be drawn off with a catheter. The urine
should bo allowed to stand in a conical glass for twenty-
four hours, or should be centrifuged. (Russ's electrical
method (p. 10) often reveals the presence of tubercle
bacilli when centrifugal methods fail to do so.) Should a
catheter not be used, the meatus urinarius and the labia
(or glans penis) should be cleansed by sponging. The
first portion of urine should be discarded. The film is
stained as for sputum, but after the acid treatment is
put in absolute alcohol for one minute (or, after staining, is
put in alcohol containing 3 per cent, hydrochloric acid for
ten minutes — Housell's method) to decolorise any smegma
bacilli, washed in water and counter-stained. Inoculation
of guinea-pigs may also be used.

Agglutination Reaction. — The serum of tuberculous
cases agglutinates the tubercle bacillus, but the technique
is difficult. Special cultures of the tubercle bacillus have
to be employed, or dried and triturated cultures may be
used (Koch).

Tuberculin Reaction. — See p. 67.

Von Pirquefs Cutaneous Reaction consists in the appli-
cation of tuberculin to a scarified surface. In twenty-
four hours red papules arise. It gives positive results in
healed cases of adults as well as in those where the disease
is active. It has more significance with children under
three years. Lapage says the method gives a lower
percentage of results than the subcutaneous, and that a
single negative result is not of much value. Lapage's
statistics on this method show that at the end of the school
age between 50 and 60 per cent, of children have become
infected with tuberculosis.

yo AIDS TO BACTERIOLOGY

Mortis Ointment. — An ointment of tuberculin in lanolin.
The reaction is similar to that of von Pirquet.

Calmette's Ophthalmo-TubercMlin Reaction.— A solution
of old tuberculin freed from glycerin by precipitating
with alcohol is instilled into the conjunctival sac. In
a tuberculous subject a congestion of the conjunctiva
takes place. Calmette does not consider it of any value
in prognosis, and it is suggested by some as not safe
to use,

The Opsonic Index. — A healthy person's blood is cen-
trifuged in normal saline solution containing sodium
citrate. The leucocytes deposited are centrifuged with
normal saline. An emulsion of moist dead tubercle bacilli
(such as can be purchased) in 1-6 per cent, salt solution is
prepared. Equal volumes of washed leucocytes, bacterial
emulsion, and the patient's serum are drawn up into a
capillary pipette, and then blown out, mixed, and drawn
up into the pipette. The tip of the pipette is sealed in a
flame, but the blood must not be heated, as this destroys
the opsonic power, and an identical mixture is made,
using normal serum. The pipettes are incubated at blood-
h3at for fifteen minutes, and blood films are then prepared
from each and stained. The number of tubercle bacilli
in at least fifty polymorphonuclear cells is counted. The
index is found by dividing the number of organisms in
the specimen by the number in the control. Experience
and care are necessary to obtain comparable results. It is
general to examine the blood before and after some dis-
turbance of the focus, by walking and breathing exercises
in the case of pulmonary affections, or the application of
a Bier's bandage or massage if the focus be localised and
approachable. If fever be present, an inverse relation-
ship of opsonic curve to temperature suggests, tubercular
trouble.

Johne's Bacillus.

During life, Johne's -disease, a complaint peculiar to
the ox and sheep, is sometimes mistaken for tuberculosis.
The chief symptoms are diarrhoea and wasting. ' The walls
of the diseased bowel contain large numbers of an acid-
fast organism, ^morphologically indistinguishable from
the tubercle bacillus. Twort says that in the first genera-
tion the organism grows long, with branching and club

LEPROSY BACILLUS 71

formation. In subcultures it is smaller, being in the
second or third generation about the same size as the
tubercle bacillus. Tt will not grow on ordinary media,
but Twort not only devised a medium (of glycerinated
eggs mixed with dried, dead, acid-fast bacilli) but also
prepared an effective diagnostic vaccine from the cultures.
Twort and Ingram incline to the view that the causative
organism of Johne's disease in sheep is the same as that
of cattle. Besides the animals mentioned only the goat
seems affected by injections of the organism. Infected
cattle are said to react to avian tuberculin.

The Leprosy Bacillus.

Morphology. — TheJ5. leprce is a long slender rod, usually
straight, with more or less pointed extremities. So far
as is known, it is non- motile, and produces no spores.
It is Gram-positive and acid-fast, although it stains more
rapidly and decolorises more quickly than the tubercle
bacillus. Large numbers of the bacilli are found, but a
large proportion, especially in the older lesions, are dead.
By Ziehl's method, using 20 per cent, nitric acid, and
counter-staining with polychrome blue, young bacilli are
stained red, older bacilli violet, and granular bacilli blue.

Kedrowsky found a diphtheroid leprosy organism which
was not acid-fast. Bayon, the Research Bacteriologist
at Robben Island, thinks it to be a stage in the develop-
ment of the typical bacillus. A disease similar to leprosy
may be found in rats in most parts of the globe. Some-
times this runs to a glandular type, sometimes to a skin
affection; acid-fast bacilli indistinguishable from typical
leprosy bacilli are present in enormous numbers, and
Dean has found an organism similar to Kedrowsky' s
bacillus in cultures.

Culture. — Peptone glycerin serum, human blood-serum,
fish broth, placental juice glycerin agar (Bayon), symbiotic
culture with amoebsc (Clegg), and eggs have been stated
to allow the growth of the bacillus.

Inoculation.— Several workers have reported successful
attempts to inoculate man, monkeys, and white mice,
but the results are much criticised. Bayon, however,
says that Kedrowsky 's diptheroid produces leprous
lesions in the rabbit, rat, and mouse, and that the result-
ing lesions are similar to those caused by certain strains

72 AIDS TO BACTERIOLOGY

of human tubercle. Couret says that multiplication of
the leprosy bacillus occurs in inoculated fish, frogs, turtles,
and other cold-blooded animals.

Pathogenesis. — Two types of leprosy are recognised:
the nodular (or tubercular), in which the new formation
has its seat in the skin or mucous membrane; and the
anaesthetic (lepra anaesthetica), in which the nerves are
chiefly affected. In the skin variety the hands and face
are mostly affected, and larger or smaller swellings appear
(red or blue in colour), which become hard. These
tubercles consist of granulation tissue, and may ulcerate
and cicatrise, producing great deformities. In the
anaesthetic form the nerve trunks become the seat of the
granulations in the interstitial connective tissue. The
spindle-shaped swellings compress and separate the nerve
fibres. Besides the anaesthesia, other evidences of inter-
ference with nerves, such as eruptions, alterations in
pigmentation, and ulceration, frequently occur.

The leprosy bacillus has been found in most of the
tissues and viscera, though it occurs more in the 'liver
and spleen than in the kidneys and brain. Tt has been
detected in the blood during febrile paroxysms in the later
stages. Enormous numbers are found in cutaneous and
other nodules, and in the discharges therefrom. Stitt
says the earliest lesion is probably a nasal ulcer at the
junction of the bony and cartilaginous septum. Scrapings
from this ulcer may give an early diagnosis.

Lepers frequently react to Old Tuberculin and give
positive Wassermann tests. The significance of these
findings is uncertain, but it has been suggested that nothing
more than coexistence of tuberculosis or syphilis re-
spectively is indicated. Fletcher (Jour. Hygiene, July,
1915) says that lepers give negative luetin reactions and
thinks the positive Wassermann due to a cause other than
syphilis. According to Stanziale and Serra the Wasser-
mann reaction is positive in rabbits which have been
successfully inoculated with Kedrowsky's diphtheroid.

Channels of Infection. — The use of fish, especially in
the putrid condition, was considered by Hutchinson to
be a causative factor, but the disease occurs among the
Basutos, who never eat fish, and among the vegetarian
Brahmins. David Walsh modifies the idea by pointing
out that dried fish, a common article of diet amongst

LEPROSY BACILLUS 73

Eastern nations, may be contaminated by the nasal dis-
charges of lepers, the fish being merely the passive agent
whereby transmission is effected. Deficiency of salt has
been suggested as conducive to the disease. Insects —
e.g., flies, fleas, lice, the itch parasite, etc. — may perhaps
disseminate the disease, and Goodhue claims to have found
the bacilli in the gnat and bed-bug. MacLeod thinks the
most common mode of invasion is via the nasal mucosa
and upper respiratory tract. The bacilli may gain an
entrance through the mouth and infect the tonsils, and
they have been found in the sputum; the genital organs
and the skin may also allow invasion. The remarks
made about the inheritance of tuberculosis seem to apply
to leprosy. Although contagious, the extent to which
it is propagated by this means is said to be exceedingly
small.

The resolutions of the British and Colonial delegates
to the International Conference on Leprosy at Bergen
(1909) included inter alia the following: Leprosy is spread
by direct and indirect contagion from persons suffering
from the disease. Leprosy is most prevalent under con-
ditions of personal and domestic uncleanliness and over-
crowding, especially where there is close and protracted
association between the leprous and non-leprous. In
leprosy an interval of years may elapse between infection
and the first recognised appearance of disease. It is a
disease of long duration, though some of its symptoms
may be quiescent for a considerable period and then
recur. The danger of infection from leprous persons is
greater when there is discharge from mucous membranes
or from ulcerated surfaces.

The Smegma Bacillus.

The smegrna bacillus closely resembles the tubercle
bacillus in size and shape, and also in being acid-fast
and Gram-positive. It is found in the preputial secretion
and between the labial folds of the vulva. It occurs on
the skin, and also, it is stated, in the ear, on the tongue
and teeth, in the sebaceous secretion, and perhaps in the
sputum. It exhibits a marked preference for those
secretions containing fatty matters. As found on the
bodies of lower animals, variations in appearance were

74 AIDS TO BACTERIOLOGY

described by Cowie, and the name is now generally held
to include a number of allied organisms. Inoculation of
animals gives negative results. Czaplewski found it to
grow, though with difficulty, on serum, glycerin agar,
and in broth. Subcultures grow more freely. Muir and
Ritchie first showed that after staining and decolorising
with acid in the Ziehl-Neelsen method, a minute's ex-
posure to alcohol sufficed to remove the red stain from
smegma bacilli, and thus to prevent their being mistaken
for tubercle bacilli of which there is sometimes a risk,
especially in the examination of urine. In practice,
HouselPs combination of alcohol with the acid is most
frequently used (p. 61).

Pappenheim's solution* has been recommended for
decolorising acid-fast bacilli other than tubercle after
fuchsin staining and washing, but it appears to have little,
if any, advantage over acid alcohol.

Other Acid-Fast Organisms.

A number of acid-fast bacilli have been found in milk,
butter, manure, and grass. Most of them grow freely on
ordinary media at room temperature, often producing a
pigment, yellow or brown. The best known among them
are the Timothy-grass bacillus (p. 159) and the ' Mist-
bacillus ' of manure. Some are pathogenic to guinea-
pigs, and the lesions produced may simulate those of
tuberculosis to some extent.

Petri and Rabinowitch's ' butter bacillus ' is found in
butter fairly frequently, and when intraperitoneally
injected with butter into guinea-pigs produces nodules
that may be confused with tubercles.

Acid-fast organisms have been found in sputum when
tuberculosis could be negatived on clinical and other
grounds. A hint as to their true character may be
sometimes obtained from the very small number seen on
prolonged search. Doubt as to identity should also be
expressed when the shape and size are not those usually
found in the case of the tubercle bacillus. Acid-fast
bacilli have been described in bronchitis and pulmonary

* Pappenheim's solution, 1 part of corallin (rosolic acid) in
100 parts of absolute alcohol, to which methylene blue is added
to saturation; 20 parts of glycerin arc then added.

SPORE-FORMING PATHOGENIC ORGANISMS 75

gangrene. Wyatt Wingrave (Medical Press, 1014, 32)
says simple acid-fast bacilli are found in chronic ear
discharges, atrophic rhinitis, faeces, lingual accumulations,
and in fact in any situation where bacilli grow in the
presence of fat." Sanguinetti has found them in distilled
water.

CHAPTER VI
SPORE FORMING PATHOGENIC ORGANISMS

OF the many organisms recognised as distinctly patho-
genic, only six are known to produce spores — viz., B. an-
thracis, B. tetani, B. botulinus, the bacillus of malignant
oodema, and two organisms pathogenic for animals alone —
B. Welchii and B. (Clostridium) Chauvcei. With the
exception of B. anlhracis, they are anaerobes.

The Anthrax Bacillus.

Morphology. — B. antliracis varies in length from 5 to
6//, in breadth from 1 to 1'5 /n. It is aerobic, and faculta-
tively .anaerobic. It is not motile, is usually straight,
and has square ends, which are characteristic. In the
blood, where it occurs singly or in short chains, the ends
of the bacillus are slightly convex; on cultivation they
become slightly concave, but neither modification dispels
the characteristic squareness of appearance. Round and
oval involution forms are often seen in old and attenuated
cultures. In the fresh state the threads do not show
segmentation; this is seen only on staining.

In cultures chains of great length are formed. When
the bacilli have a supply of oxygen, and the temperature
is between 20° and 38° C., ellipsoidal central spores are
developed. Spores are not found during life, being only
developed after death, when the bloody discharges come
in contact with air. A variety, first obtained by Behring,
which is sporeless for many generations, is produced by
culture at 42° C., or on nutrient gelatin containing 0-1
per cent, of phenol.

Culture.— On a gelatin plate small spherical colonies
develop in the depth, which consist of closely twisted
bands of bacillar chains. When the growth reaches the

76 AIDS TO BACTERIOLOGY

surface, spikelets at once begin to radiate over this in
wavy convolutions, liquefying the gelatin. This stage is
usually reached in two days, and is most characteristic.
In the gelatin stab, growth takes place along the needle
track, fine branching filaments often growing out into
the gelatin (' inverted fir-tree growth '). Liquefaction
commences at the top of the stab, proceeding downwards
in a horizontal plane, upon which a mass of bacilli rest,
leaving the gelatin above clear and liquid. In broth a
flocculent growth forms at the bottom of the tube, the
bulk of the broth remaining clear and no pellicle forming.
On agar a thick, grey-white, sticky growth takes place,
and on potato a considerable white growth, both usually
containing a large number of spores. Blood-serum is
slowly liquefied. An alkaline reaction is generally favour-
able to the growth of this organism, although it grows well
on potatoes, which are normally acid The bacilli stain
well by Gram's method.

Pathogenesis. — In the human subject the disease
appears as — (1) malignant pustule, a localised swelling
caused by direct inoculation, and (2) ivoolsorter* s disease,
a general infection caused through inhalation of spores
during the handling of wools, hides, and fleeces. Infec-
tion may also take place through the digestive organs
(intestinal anthrax). A single case of the lesion appearing
in the larynx is reported. In cattle the disease is known
as ' Siberian plague ' or ' splenic fever,' the latter cog-
nomen being due to the much swollen and soft condition
of the spleen found in infected animals. In man, enlarge-
ment of the spleen is less marked, and the disease appears
to take the form of a toxaemia.

It is pathogenic to the following animals, which are
arranged roughly in order of their susceptibility: mice,
guinea-pigs, rabbits, sheep, cattle, horses, man, goats, and
pigs. Algerian sheep, dogs, frogs,* and white rats are
immune.

In susceptible rodents there may be considerable in-
flammation around the seat of inoculation, and the sub-
cutaneous connective tissue may be distended with a
bloody gelatinous exudation. If the tissue is examined
microscopically, the blood is found to be full of bacilli,

* Unless the frog is warmed to 37° C.

SPORE-FORMING PA THOGENIC ORGA NISMS 77

which in some places may have so distended the capil-
laries as to have ruptured them and escaped into the
surrounding tissue. Anthrax, once introduced, may
become endemic in a field in the following manner: The
infected animal dies; the bacilli in the bloody discharges
that come in contact with the air develop spores, which
may be blown about on to the surrounding soil, and remain
dormant there for long periods. Animals feeding on
grass growing about this spot would be liable to infection.
The bacilli, if developed, might be killed by the gastric
juice, but the spores could withstand its action and enter
the circulation.

Pasteur suggested that after the burial of an animal,
worms might bring the spores to the surface. Klein
found that all bacilli and spores are killed within a week
by putrefaction if the body be left intact. Outbreaks
among horses and cattle have occurred through infected
oats and Unseed cake. The bacillus has been found in
effluents, and cattle drinking therefrom have been in- .
fected. In one case, foreign bone manure conveyed the
bacillus to a workman. The chief danger to operatives
in industries involving the handling of wool, hides, and
horsehair appears to lie in the small clots of blood on
the same. The Bradford Anthrax Investigation Board
urge that all blood-stained material should be thrown out
before any process of combing takes place.

A highly satisfactory immunity, which, however, is
transitory, seldom lasting more than a few months, may
be conferred upon susceptible animals by injection of_a
culture attenuated by growth at 42-5° C. for fifteen days,
followed twelve days later by a second culture attenuated
by ten days' growth at the same temperature (Pasteur's
vaccine).

Cases of anthrax in animals are dealt with under the
Anthrax Order of 1899 issued by the Board of Agricul-
ture.

Serum Treatment. — Marchoux vaccinated sheep with
an attenuated culture, and then injected them with'in-
creasing doses of virulent cultures. Sclavo employs the
ass, immunising by the same method, and the antiserum
so prepared has been used with success in the treatment
of anthrax in man. The serum is bactericidal rather than
antitoxic.

78 AIDS TO BACTERIOLOGY

Disinfection. — No danger is apprehended from the
handling of wools from Australasia and the Argentine,
but precautions are necessary when dealing with horsehair
from China, Siberia, and Russia, and with Persian wool.
While the vegetative forms are quickly killed by heat and
disinfectants, 5 per cent, phenol acting for at least twenty-
four hours is required for the destruction of the spores,
and the disinfection of hides, hair, and wool is therefore
difficult. For hides and skins the use of formaldehyde is
impracticable, since it so injures the consignments as to
prevent them being turned into good leather. Legge
thinks it doubtful if there is any way in which hides to
be afterwards tanned can be effectively disinfected.

Constant Ponder concludes the best method for de-
stroying the infection on hides to be that devised by
Seymour Jones. The process involves treating the hides
for twenty-four hours in a soak containing 1 per cent, of
formic acid and 0-02 per cent, mercuric chloride, and
then transferring them to the ordinary brine pit. This
does not interfere with the subsequent processes involved
in transformation into leather. Hewlett found the pro-
cess to be satisfactory provided the strength of the solution
was doubled or trebled when dealing with horsehair.
Forty-eight hours' exposure to a solution containing 2 per
cent, hydrochloric acid and 10 per cent, sodium chloride
(Schattenfroh method) is also satisfactory (Lancet, August
7, 1915).

Webb and Duncan's experiments seem to show that,
leaving out of consideration white or grey hair, which is
liable to change colour, no injurious effect is produced on
horsehair by steam disinfection, provided the temperature
does not exceed 218° F. — a comparatively low tempera-
ture for efficient disinfection. Legge concludes that, to
secure certain destruction of all anthrax spores in horse-
hair, absolute reliance cannot be placed on either steam
disinfection (within the limits in which it can be applied)
or simple boiling.

In the Home Office Regulations in respect of processes
involving the use of horsehair from China, Siberia, or
Russia, no manipulation except opening or sorting is
allowed until the hair has undergone disinfection. Dis-
infection may be accomplished either by exposure to a
temperature of not less than 212° F. for at least half an

SPORE-FORMING PATHOGENIC ORGANISMS 70

hour, or by exposure to a disinfectant which has been
certified as effective for the destruction of anthrax spores,
and approved by the Secretary of State. Eurich, the
bacteriologist to the Anthrax Investigation Board, said
(1909) that no trustworthy method of sterilising the wool
before washing had been found. In 1913, Eurich and
Willey conducted experiments in regard to the use of
steam as a disinfectant, as a result of which their Board
agree that disinfection by steam cannot be applied to
ordinary wool or hair except under conditions that would
stop any trade in the sorts so treated. The method can,
however, be applied to blood-stained material that has
been sorted out or otherwise separated from the bulk,
so that blood-stained material need no longer be regarded
as absolute loss, but as a waste product.

A process of destroying carcasses involves solution of
the entire animal in sulphuric acid. The Anthrax Order of
the Board of Agriculture (1911) provides for prosecution
for causing an effusion of blood, and no one may burn,
bury, or otherwise dispose of the carcass without the
sanction of the local authority.

The Tetanus Bacillus.

Morphology. — B. tetani is a straight, slender, rod 4 ju,
long, with rounded ends. It is slightly motile, possessing
many peritrichic ilagella. Especially in old cultures, long
filaments are formed which show no spores. Spherical
spores are frequent, which, being located at one end, and
being thicker than the bacillus, gives the ' drumstick
appearance.' The bacillus is Gram-positive.

Culture. — The tetanus bacillus is a ' strict ' anaerobe,
although Jordan states that a degree of tolerance to
oxygen can be established, and that it will grow in a
mixed culture when air is admitted. In glucose-gelatin
or glucose-agar stab culture, a feathery radiated growth is
formed with slight gas-formation. Gelatin is liquefied.
All cultures possess a characteristic smell, suggestive of
an ill-kept stable. The organism grows well on blood-
serum without liquefaction.

Theobald Smith has shown that the bacillus will grow
in ordinary broth in a fermentation tube if a piece of
sterile tissue, such as a piece of liver, kidney, or spleen
of guinea-pig or rabbit, be inserted where open bulb and

8o AIDS TO BACTERIOLOGY

closed arm meet. No precaution for exclusion of oxygen
is necessary, and spores are said to develop in a day or
little longer. Embleton says B. tetani will grow in broth
along with Staphylococcus aureus and need no precaution
to exclude oxygen.

Tetanus spores, especially if in a dry condition, keep
their virulence for an indefinite time. Much uncertainty
exists as to the temperature necessary to kill them. It
seems probable that sometimes they withstand boiling
for five minutes and require heating in an autoclave for
their certain destruction. Embleton says they can stand
almost a dull red heat. The resistance of the spores to
disinfectants is also high. They survive 5 per cent, phenol
solution for fifteen hours.

Channels of Infection and Pathogenesis. — The tetanus
bacillus and its spores are found very frequently (90 per
cent, of samples) in cow and horse dung and less rarely
in human fseces (5 per cent, of samples, Andre wes). It
is constantly present in manured land. Unless some
degree of penetration into the tissues is effected, the
disease does not develop. If only a scratch, abrasion, or
superficial cut be suffered, infected material obtaining
access thereto seems never to become pathogenic. If
manured soil, however, be injected into a laboratory
animal, or a stableman or gardener gets wounded with a
rusty nail or broken glass, the disease may be produced.
The disease is more prevalent in some districts than in
others, and by following the statistics of French veterinary
surgeons, Bazy (Medical Press, 1915, p. 315) has been
able to mark out certain ' accursed fields ' for tetanus
just as can be done for anthrax. The disease is prevalent
among horses and men on battlefields. The incidence
varies with the state of the ground fought over. In the
present war the disease is non-existent among troops
engaged on uncultivated areas round the Dardanelles just
the same as there has been no tetanus in naval cases of
wounds. On the highly cultivated grounds of Flanders and
France, especially in the Aisne region, cases were not infre-
quent. Bazy's statistics bear on 10,396 wounded; of this
number 129 developed tetanus, of which 90 proved fatal.

Umbilical tetanus is common among newborn infants
in some countries. Puerperal tetanus and tetanus super-
vening after operations are now rare. Impure vaccine

SPORE-FORMING PATHOGENIC ORGANISMS 81

lymph and Fuller's earth have occasioned tho disease.
The spores can on occasion live through the operations
involved in the manufacture of gelatin, and injection of
contaminated gelatin has produced tetanus.

The organism is pathogenic to man, the horse, guinea-
pig, mice, and rabbit; birds are but slightly susceptible.
The bacilli remain in the locality of the site of inoculation
and in the nearest lymph glands, symptoms being caused
by absorption of the toxins. In the development of the
disease, damage to local tissues and prevention of phago-
cytosis are the most important factors, the latter being
allowed by the extraneous organisms which gain access
with the tetanus bacilli. The spore, if freed from toxin
by simple washing, is readily disposed of by phagocytosis.

Usually fifteen days elapse between infection and the
appearance of symptoms, but they may occur in man in
two days, or not till after twenty-seven days.

It is rarely that drumstick spores can be found in
material from a wound, and should any be found, the
existence of other Gram-positive bacilli growing terminal
spores must not be overlooked.

Toxins.— Grown anaSrobically in glucose broth, the
tetanus bacillus produces a powerful extracellular toxin,
of the constitution of which little is known. There is a
substance producing the characteristic spasm (tetano-
spasmin), a hsemolysin (tetanolysin), and some albumoses
to which Sidney Martin ascribes the fever of tetanus.
The toxin is readily destroyed by heat and light, and di-
minishes in toxicity with keeping. Tetanus toxin is rapidly
fixed by the tissues of the central nervous system, and
travels from the wound by way of the nerve trunks.

Antitetanic Serum. — In Roux and Vaillard's process,
virulent tetanus bacilli are cultivated in broth in an
atmosphere of hydrogen. After about fourteen days'
growth, the culture is filtered through a porcelain filter
to free it from bacilli. Injections are then made into
horses with this toxin daily, subcutaneously or intra-
venously, starting with 1 c.c. of iodised toxin, gradually
increasing the dose until the pure toxin can be injected
without danger, the treatment lasting about three months.
The serum is standardised. The results attending the
use of this serum have been less satisfactory than those
obtained with antidiphtheria serum, as the disease is only

6

82 AIDS TO BACTERIOLOGY

recognised when considerable absorption of toxin has
occurred. Its use as a prophylactic is undoubted.
Even in those cases where the disease is firmly established
the serum is often still of service. In field ambulances,
etc., where surgeons systematically give prophylactic
injections, Bazy says the mortality is only one- third of
that where it is only given to suspicious cases.

The Bacillus of Malignant (Edema.

Morphology. — B. cedematis maligni is a motile rod,
about 4 IJL long and Ol ^ broad, with several flagella. It
has a tendency to grow in long filaments. It forms
spores in a more or less central position, but not when in
filamentous form. It stains readily with the ordinary
dyes, but is Gram-negative.

Culture. — Distinction from the anthrax bacillus is also
seen on cultivation. It is a strict anaerobe, and grows at
room temperature. Development is accelerated by the
presence of 2 per cent, of glucose. Gelatin is liquefied,
and a foul-smelling gas is produced in both gelatin and
agar stab cultures. Blood-serum is liquefied, but there
is no visible growth on potato.

Distribution and Pathogenesis. — Malignant oedema has
followed subcutaneous injection of musk, and the organism
has been found in musk-sacs. It may follow castration
in the horse or parturition in cattle. The organism
occurs in the soil, dust, and in the human intestine.

The bacillus may be the exciting cause of gangrene after
injuries, particularly when the parts are crushed or
lacerated and soiled with earth, and is pathogenic to
man, horses, pigs, sheep, guinea-pigs, rabbits, rats, mice,
and some birds. The readiest plan for isolation is to
inoculate subcutaneously a guinea-pig with garden earth.
On death, which may occur in twenty-four to forty-eight
hours, the bacillus is found in the cedematous fluid, but
, not, like anthrax, in the blood, except later, when it has
multiplied after death, when it may form filaments 15 to
40 jui in length.

The gas manifested in the frothy exudation when an
animal is inoculated with garden earth is absent, or nearly
so, when the inoculation is made from a pure culture,
and is therefore probably due to other organisms.

SPORE-FORMING PATHOGENIC ORGANISMS 83

Bacillus (Clostridium) Ckauvcei.

Black-leg, quarter-evil, or symptomatic anthrax is un-
known in man, but in sheep and oxen a fatal termination
is to be apprehended one or two days after infection.
Localised swellings appear on the neck, shoulders, or
thighs, and the affected muscles become discoloured.
Puncture of infected muscle produces a frothy, sanguineous
fluid, which contains the organism. The organism is a
rod about 4 JLI long, and is actively motile. The large
spores give it club- and spindle-shaped appearances. It
is a strict anaerobe, and is usually Gram-negative. It
grows in agar and gelatin cultures with the production
of a foul gas. Gelatin is slowly liquefied. Kitt says the
odour is due, not to B. Chauvwi, but to contamination
with ' cadaver bacilli.' By drying an affected muscle at
35° C. and then heating to 85° or 90° C., attenuation in
virulence is effected, and the materials injected for the
production of immunity.

Red Braxy in sheep is associated with an organism
similar to B. Chauvcei, which, however, differs in its action
on other animals. The bacilli are very numerous in the
intestine and in the reddish-brown blood-stained mucus
that collects in the last and true digestive stomach.
After death, decomposition is very rapid, and the crimson
colour of the fourth stomach is sometimes mistaken for
a symptom of irritant poisoning. Hamilton found that
during July and August the serum of healthy sheep did
not allow multiplication of braxy bacilli. Very satis-
factory immunity is obtained by dosing lambs by the
mouth when their blood is most refractory to the organism.

Bacillus Botulinus.

Morphology. — A large bacillus (4 ft to 10 /LI long), with
rounded ends, and a tendency to form short chains.
It is an obligatory anaerobe, exhibits a slight motility,
and forms spores. The spores are often destroyed by a
temperature of 80° C. The position of the spores is
generally terminal, and the flagella number from four to
eight. The organism is Gram-positive.

Cultural Characters. — Cultures must be grown in the
dark. Milk is not curdled; glucose is fermented with
production of acid and gas; saccharose and lactose are

84 AIDS TO BACTERIOLOGY

not fermented. While Stockman mentions that cultures
of this organism have no putrefactive odour, other ob-
servers describe a rancid odour due to butyric acid. A
stab culture in glucose gelatin grows white along the stab
with lateral offshoots, liquefaction and disruption of the
medium with gas. On glucose-gelatin plates the young
colonies are translucent, of a yellowish-brown colour, and
surrounded by a liquefied zone. Growth is abundant at
ordinary temperatures, but scanty at 37° C., at which
temperature involution forms of long twisted filaments
develop. Sporulation does not occur at blood-heat.

Pathogenesis. — The organism has been found in liver
and blood sausages, pickled and smoked meat, tinned
meat, preserved foods, and ham that have caused ' botul-
ism ' or ' sausage poisoning.' Where symptoms show
immediately after ingestion of the infected food they
are those of gastro-intestinal disorder. It is not till after
a period of incubation of twelve or more hours that the
prominent symptoms described by van Ermengem show,
these being disordered secretion in the nose, dryness of
mouth and throat, dilated pupil, ptosis, paralysis of
accommodation, double vision, dysphagia, aphonia, and
retention of the urine. Fever is absent, and no impairment
of intellect occurs. While marked constipation has been
recorded, there may be slight and transitory vomiting
and diarrhoea. Death, from bulbar-paralysis, follows in
about a quarter of the cases.

Except for an instance where a bacillus indistinguishable
from B. botulinus was isolated by Kempner from the
intestine of a pig, there is no information as to the origin
of the organism. From the scanty growth obtained at
blood-heat it is fairly certain that little or no multiplica-
tion of the bacilli takes place in the living animal, so that
a sufferer is not a source of danger to his associates, and
it is pretty evident that infection of food must have
occurred after death of the animal, the infection coming
from sources outside the meat. Savage in reviewing the
reported cases of botulism found that in none of the out-
breaks had the foods incriminated been eaten in the fresh
state, all had been stored. There is often no external
sign of putrefaction in the infected meat, but it may
have a rancid odour.

An extracellular toxin of extremely high potency is

SPORE-FORMING PATHOGENIC ORGANISMS 85

formed by this organism, and the intoxication is due to
its formation in meat before ingestion. The toxin is
destroyed by exposure to 80° C. for half an hour, and
food causing the outbreaks has been either raw or im-
perfectly cooked. As growth of the bacillus is checked
by a 6 per cent, solution of salt, van Ermengem has
recommended that in salting a brine containing at least
15 per cent, of sodium chloride should be used.

The disease is now rare on the Continent, and Savage
was unable to trace the occurrence of a single outbreak
in this country.

An antitoxin prepared by Kempner is said to be of
value even some hours after ingestion of infected food.

Bacillus Welchii.

An organism commonly found in emphysematous gan-
grene was discovered by Welch and Nuttall (1892), and
named B. aerogenes capsulatus. Frankel (1893) inde-
pendently described B. plilegmones emphysematosce, which
is identical with Welch and Nuttall' s organism. Klein
attributed an outbreak of diarrhoea in St. Bartholomew's
Hospital (1895) to an organism (B. enteritidis sporogenes)
found by him in the dejecta of patients. Oftentimes,
regardless of all the attributes of Klein's bacillus, this
name is commonly applied to a normal inhabitant of the
digestive tract which is described here as B. Welchii.
Other observers have independently discovered organisms
either identical or very similar. Chester has renamed this
organism, or class of organisms, B. Welchii.

Morphology.— £. Welchii is a plump, long bacillus
(3^ to 6/i long), with almost square ends, occurring singly,
in chains, and in clumps. The length varies greatly on
culture, when different media are used. It frequently has
a capsule, and forms oval spores, generally situated near
one end, but only in serum cultures. It is strictly anaero-
bic, is Gram-positive and non-motile. B. enteritidis
sporogenes is described by Klein as multi-flagellate and
motile, and as sporing in gelatin.

Cultural Characters. — Gelatin is slowly liquefied. Gas
(two-thirds or three-quarters of which is hydrogen) is
produced in dextrose, lactose, and saccharose media.
•Since its growth is not suppressed by bile-salt it gives a
po bitive reaction in MacConkey's bile-salt media like the

86 AIDS TO BACTERIOLOGY

colon bacillus does. A characteristic fermentation is
produced in milk in forty-eight hours: a firm, white,
honeycombed curd and a clear, watery whey are produced
with abundant gas-formation. The culture is strongly
acid, and has a faint, sour smell, like that of butyric acid.
Most varieties liberate haemoglobin when grown in broth
containing blood (Jordan). When grown anaerobically
in undiluted human blood serum with staphylococcus,
very copious sporing occurs in two days (Douglas).
Fleming recommends neutral-red egg medium for surface
growths: in twenty-four hours bright pink spots, 2 milli-
metres in size, appear, having slightly raised margins
and characteristic prominences in the centres.

Occurrence. — B. Welchii is found in excrernentitious
matter and road dust. Its presence in water, milk, shell-
fish, etc., is to be regarded as an indication of pollution,
not necessarily recent, with faecal matter.

Pathogenesis. — Subcutaneous inoculation of guinea-pigs
with recent milk cultures produces in some cases intense
spreading hsemorrhagic oedema and necrosis, and death
in twenty-four to forty-eight hours. In other cases it
is not pathogenic.

Rabbits are almost immune, but if one be inoculated in-
travenously, killed, and the carcass incubated at 37° C. for
twenty-four hours, characteristic production of gas follows,
especially in the liver (Welch-Nuttall test). B. Chauvoei
produces a similar result, but forms spores, while (according
to Hewlett) B. Welchii does not under such conditions.

While the B. Welchii is either absent or rare in the
faeces of the young, Kendall has found it in the stools
of children with summer diarrhoea. Hewlett thinks it is
probably capable of producing necrotic changes in the
intestinal mucous membrane, and, because of its abund-
ance in the intestine in some cases of primary anaemia,
suggests it may have some relation to the condition. It
occurs in various pathological conditions, in septicaemic
and pyeemic infections of the gastro-intestinal and genito-
urinary tracts, and is often responsible for the gas seen
in ' foamy organs ' at autopsies.

In cases of gaseous gangrene occurring among our
troops in France B. aerogenes capsulatus (i.e., B. Welchii
as originally described by Welch and Nuttall) is dominant
in every case. Quinine hydrochloride is found to reduce

SFORE-FORMING PATHOGENIC ORGANISMS 87

the mortality considerably in experimental animals
(Kenneth Taylor, Lancet, September 4, 1915). The same
organism has been found in the expectorated matter
from cases of pulmonary gangrene supervening after in-
halation of the irritant gases employed by the Germans (De
la Riviere and Leclercq, Medical Press, October 20, 1915).
Detection in Water. — B. Welchii is regarded as of less
importance as an index of pollution in water than for-
merly. Large quantities of water must be examined, and
it was previously the custom to pass half a litre through
a Pasteur- Chamberland candle, suspend the deposit in
5 c.c. of sterile water, and inoculate three milk tubes with
3 c.c., 1 c.c., and 1 c.c. respectively of the concentrated
water. The milk tubes were then treated as described
below. Hewlett's method of conducting the test, though
cumbersome, is more satisfactory. Ten large boiling
tubes, each containing 50 c.c. of sterile milk, are inoculated
each with 50 c.c. of the water, melted vaseline is poured
on the surface of the mixture to exclude air, and each
tube is covered with a double layer of sterile filter-paper,
kept in position by a rubber band. The tubes are heated
in a bath of water to 80° C. for twenty minutes, and
incubated at 37° C. for two days. When smaller amounts,
such as 10 c.c. of liquid, are to be examined, an exposure
to 80° C. of ten minutes is sufficient. The gas produced
in the fermentation (vide supra) collects as a bubble under
the vaseline plug. The Closlridium butyricum (which is
principally responsible for the butyric fermentation of
milk, and which, when present, hastens the production
of tyrotoxicon therein) gives a similar reaction in milk,
but is not pathogenic for guinea-pigs. The only way to
distinguish B. Welchii from Clostridium butyricum, which
is regarded by some as a non- pathogenic form of the
former, is to inject 2 c.c. of the whey subcutaneously
into a guinea-pig of about 200 grammes weight, when
B. Welchii will kill the animal in forty-eight hours.

To isolate B. Welchii from pus, Alexander Fleming
(Lancet, August 21, 1915) recommends the following pro-
cedure: A tube of sterile milk is boiled and immediately,
while yet at a temperature close to 100° C., is planted
with some of the pus. The scum of cream suffices to
exclude air. If other spores get in, anaerobic plate
cultures on glucose agar are made.

88 AIDS TO BACTERIOLOGY

CHAPTER VII
THE COLON-TYPHOID GROUP

THIS group of bacilli is classed by Loffler as one family,
the TypJiaceoB. The characters of individual members
are not always clearly defined. As a general rule the
organism is short, plump, with rounded ends, often with
a tendency to develop long forms. No spores are formed
and the organisms are Gram-negative. None liquefies
gelatin except B. cloacce and a few coliform organisms
described by MacConkey (B. levans, B. oxytocus perniciosus,
and his No. 73). On a gelatin plate, they form thin,
irregular, notched colonies. All are aerobes and faculta-
tive anaerobes, none are chromogenic, and all grow well
on ordinary media.

The Colon Bacillus.

Bacillus coli (B. coli communis) inhabits the intestinal
tracts of man, the lower animals, birds, and, less fre-
quently, fish. The dispersion of the dejecta causes this
organism to be widely distributed. While a very large
proportion of the organisms when isolated are found to
conform to the salient characteristics of the recognised
type, ' atypical ' organisms are met with which, in one
or more respects, differ from the true type. Such are
described as ' coliform.'

Morphology. — The colon bacillus is a short rod, 2 JLI to
4 ]u long, with rounded ends, but may form much longer
rods, or be so short as to be oval in shape. It possesses
three or four flagella on an average, and although generally
feebly motile, non-motile forms are not uncommon. It
forms no spores, and is not stained by Gram's method.

B. coli is slightly more resistant to heat, to disinfectants
and other destructive agents than is B. typhosus. While
B. coli does not usually survive an exposure to 60° C. for
ten minutes, one variety, B. Griinlhal, produced a toxin
that withstood ten minutes at 80° C. According to
Ayers and Johnson 54-6 per cent, of cultures survived
thirty minutes at 60° C., and 6-9 per cent, survived
62-8° C. for thirty minutes.

THE COLON-TYPHOID GROUP 89

Cultural Characters. — On gelatin plates colonies appear
in one or two days, the growth being thin and lilmy,
irregular in shape, translucent at the margins, and moist
in appearance. Jn gelatin stab or shake cultures bubbles
of gas are produced. The gelatin is not liquefied.

On serum and on agar greyish-white, moist, shining
growths form. On potato, if acid, a yellowish growth is
obtained, but the growth may be colourless if the potato
is not fresh. Milk is rendered acid, and curdled in from
one to three days at 37° C. without subsequent digestion
of the casein and without subsequent production of alka-
linity. When grown in peptone-water containing one
of the following substances — glucose, laevulose, maltose,
galactose, arabinose, raffinose (?), lactose, mannite,
sorbite, dulcite or dextrin (cane-sugar, sometimes)— acid
and gas are produced. The gas production varies in
amount. From the fact that an organism may lose its
power of forming gas from sugars while retaining its
power of forming gas from alcohols, Penfold concludes
that two enzymes are concerned (see also p. 28). The
gas produced from dextrose consists of hydrogen 2 volumes
and carbon dioxide 1 volume. Present American opinion
places no reliance on this formula. B. coli produces indole
in broth or peptone-water cultures, generally within two
days, but sometimes a week is necessary before it can be
demonstrated. It reduces nitrates to nitrites. In
neutral-red lactose media the acid formed produces a
rose-red colour. On solid media no further change of
colour takes place, but in neutral-red lactose broth, the
dye is reduced with production of a greenish-yellow
fluorescence.

In the Voges-Proskauer reaction the organism to be
tested is grown for three days in 2 per cent, glucose broth.
Two or 3 c.c. of strong caustic potash are added, and a
pink eosin-like colour developing on exposure to air for
twenty-four hours constitutes a positive reaction. The
colon bacillus does not give this reaction. Rivas devised
a quick method of performing this test: 1-4 c.c. of a forty-
eight-hour culture is boiled with 5 c.c. of 10 per cent, sodium
hydrate solution, the reaction being hastened by blowing
air through the liquid or by shaking.

By means of fermentation and other reactions over a
hundred types of colon bacilli can be distinguished. It

90 AIDS TO BACTERIOLOGY

becomes, therefore, necessary to enunciate definitely
those attributes which, if possessed by an organism, will
entitle it to be regarded as a colon bacillus. Of course
when a pathogenic organism of this class is isolated from
a patient, the name it is called by in the present state of
knowledge matters little so long as it prove corpus delicti.
But when estimating the organism to ascertain existence
of fsecal contamination a reasonable degree of definition
is necessary. Different bacteriologists select different
characters to which they require an organism to conform
before identifying it as typical B. coli. Houston described
an organism as ' flaginac ' if it produced a fluorescent
greenish yellow in neutral red glucose peptone-water (fl),
acid and gas in lactose (ag), indole in peptone-water (in],
and acid and curd in milk (ac). MacConkey suggests the
omission of tests for the growth on gelatin, action on milk,
glucose, and neutral red, and the indole test. In the place
of them he recommends the action of the organism on (1)
dulcite, (2) adonite, (3) inulin, and (4) the Voges and
Proskauer reaction.

A negative indole reaction becomes more significant
when the para-dimethyl-amido-benzaldehyde test has
been used instead of nitrite and acid.

It is possible, but by no means certain, that certain
varieties of the colon bacillus are of greater significance
as indicators of excretal contamination than others. The
presence or absence of power to ferment saccharose is
particularly thought worthy of emphasis in this connection,
and Houston denominates those fermenting saccharose,
producing acid and gas in lactose peptone water, and
giving an indole reaction as ' sagin ' organisms. Those
having no action on saccharose, but giving the other two
tests, are similarly christened ' agin ' bacilli. The bacillus
producing acid and gas from cane-sugar (saccharose) has
also been called B. coli communior by Durham, and this
variety is supposed to be of more recent intestinal ancestry
than another not fermenting saccharose.

The American Committee mention the following
attributes as defining the colon group : ' Fermentation
of dextrose and lactose with gas production, short bacillus
with rounded ends, non- spore-forming, facultative anae-
robe, gives positive test with esculin, grows at 20° on
gelatin and at 37° on agar, non-liquefying in fourteen days

THE COLON-TYPHOID GROUP 91

on gelatin. Gram-staining negative.' Prescott and Wins-
low doubt there to be ' sufficient evidence to warrant
making the esculin reaction a general criterion of the
colon group,' and they desire to reduce the test to positive
reactions in a lactose fermentation medium, growth on
an aerobic agar streak, and microscopic examination.

Clemesha and Houston have independently shown that
with fresh faecal pollution in water most of the organisms
fermenting dextrose ferment lactose as well, while in
stored or filtered water dextrose -fermenting lactose-
negative forms relatively increase.

To obtain presumptive evidence of the presence of the
colon bacillus a medium containing a suitable carbohydrate
and some substance to inhibit the growth of other organ-,
isms is used, such as MacConkey's litmus lactose bile-
salt peptone-water, which is fermented with the pro-
duction, of acid and gas. This medium is used for further
work (see p. 230).

If the organism be present in large numbers and vigor-
ous condition, acid and gas production will be evident
in less than twenty-four hours, but attenuated organisms
may require three days' incubation at blood-heat or at
a slightly higher temperature. Bile-salt has the dis-
advantage of suppressing some of the weak coliform
organisms. In examinations for faecal contamination this
is not a matter for worry, as feeble vigour cateris paribus
denotes remoter faecal ancestry, and the attribution of
slighter significance to their presence and numbers. (It
may be mentioned here that the best ' pick-me-up '
media for rejuvenating sveak bacteria are liver broth, and
gelatin at blood-heat.)

Pathogenesis. — While in the intestine, the colon bacillus
is supposed by some to suppress the growth of less desir-
able organisms, and thus to protect the body. This
surmise is based on the diminution in number or absence
of the colon bacillus from the faeces in some bacterial
diseases affecting the intestines. Although chiefly of
importance as an ' indicator ' of pollution with excrement,
the colon bacillus is also capable of causing disease. The
organism is pyogenic, is commonly the cause of cystitis,
and may affect the gall-bladder and bile ducts and other
organs. Sir Almroth Wright suggests it may sometimes
be responsible for mucous colitis. Arnold Lawson

92 AIDS TO BACTERIOLOGY

describes cases of metastatic ocular inflammation as
associated with B. coli toxaemia. It is generally the
causal agent of calf diarrhoea (Bibby's ' Bovine Tuber-
culosis,' p. 434). It is possible that B. coli may be re-
sponsible for some outbreaks of bacterial food poisoning.
Gordon R. Ward thinks a variant of B. coli produces
pernicious anoemia.

Bacilli coli as pathogenic organisms are supposed not
to be identical with, but relations of the original B. coli ;
to use Metchnikoff s phrase, they are ' wild races.' Some
bacteriologists drop the term B. coli when speaking of
an infective organism of the species and speak of it as a
coliform organism.

Vaccines. — Antisera have not proved of value. Cystitis
and other affections of a chronic nature are successfully
treated with autogenous vaccines. Greater improvement
takes place when the vaccine is freshly prepared every
month.

By cultural characters alone, over a hundred types of
B. coli have been described. As organisms culturally
identical give widely different agglutination reactions it
is quite evident that more types still undescribed exist.
Even allowing for many types that are incapable of pro-
ducing disease there must remain an enormous number
of potentially pathogenic colon bacilli. In coli infections
it remains largely a matter of chance whether a stock
polyvalent vaccine will be antagonistic to an individual
pathogenic type.

Coli-like Organisms.

In addition to the atypical B. coli mentioned, other
organisms have been regarded as variants of the colon
bacillus. Some of these are mentioned among the capsu-
lated bacilli (vide infra).

B. neapolitanus, isolated by Emmerich from the dejecta
of cases of cholera, ferments saccharose and dulcite, and
produces indole, but is non-motile.

B. cloacce, a motile organism, liquefies gelatin quickly
or slowly, ferments saccharose but not dulcitol, and gives
the Voges-Proskauer reaction. It occurs in sewage, grave-
yards, and slaughter-house drains.

B. bifidus and B. acidophilus are coli-like, but Gram-
positive organisms, which occur in the large intestine of

THE COLON-TYPHOID GROUP 93

human infants. They arc described with B. bulgaricus,
with which Rodella thinks them identical (see p. 189).

B. coli anaerogenes, an organism of the coli-type, but
producing no gas in the fermentation reactions. Is
distinguished from B. typliosus by its production of acid
in lactose media and by agglutination reactions.

B. acidi lactici (pp. 188 and 108) is occasionally patho-
genic.

The Capsulated Bacilli.

B. pneumonice, or Friedlander's pneumo-bacillus (see
p. 143).

B. lactis aerogems, found in the bowels of nurslings
and sometimes in souring milk, is non-motile, does not
ferment dulcitol, and gives the Voges-Proskauer reaction.
In gelatin a projecting ' nail-head ' growth is formed, and
milk is usually curdled more rapidly than with B. coli,
with the formation of capsules. It is sometimes patho-
genic.

A short capsulated bacillus has been described in rhino-
scleroma, which is Gram-negative, does not liquefy
gelatin, and does not curdle milk.

Several organisms have been described in ozsena (foetid
atrophic rhinitis), one of which — Abel's B. ozcence — is
capsulated, and causes the atrophy of the mucous mem-
brane.

Bacillus Enteritidis (Gartner).

Morphology. — An actively motile bacillus, with shape
and size similar to those of the typhoid bacillus, carries
several flagella, forms no spores, and is Gram-negative.

Cultural Characters. — Ferments glucose, laevulose, mal-
tose, galactose, arabinose, raffinose, mannite, sorbite,
dulcite and dextrin, with production of acid and gas.
Has no action on saccharose, salicin (as a rule), nor
glycerin (as a rule). It is generally regarded as without
action on lactose, but some strains attack it. Neutral
red is reduced with production of yellowish fluorescence.
Milk is not curdled. In litmus milk in the first day a
slight acidity develops which is replaced by a permanent
alkalinity. The bacillus gives very little or no indole,
and does not give the Voges-Proskauer reaction. Identi-
fication as the cause of disease may be shown as in the

94 AIDS TO BACTERIOLOGY

case of the paratyphoid bacilli by a satisfactory agglutina-
tion with the patient's serum.

Pathogen esis. — Gartner's bacillus is the most frequent
cause of epidemics of meat poisoning. It has been dis-
covered in meat from pigs, cattle, horses, and fish.
In the only case of infection in mutton, the meat was
supposed to have been infected from contaminated ox
tongue (the organism found being B. suipestifer). In
many cases meat has been derived from animals that had
enteritis, in other cases all the evidence points to infection
of the meat post mortem. It is known that a piece of
infected meat will infect a sound piece if in juxtaposition.

It has been suggested that Gartner bacilli are more or
less natural inhabitants of the animal intestine. Savage
has shown that, although prepared meat foods are very
often subject to extensive contamination of excretal
origin, Gartner bacilli are not often to be found. He is
therefore of opinion that the Gartner Group organisms
(or other special bacilli) are ' derived from animals which
are either at the time suffering from disease due to Gartner
Group bacilli, or acting as carriers of these bacilli.'

Infected meat has usually been normal in appearance,
taste, and smell, but salty or other peculiar flavours have
been sometimes noticed, and turbidity of jelly was men-
tioned in one case.

Gartner's bacillus is easily killed by heat, not surviving
thirty minutes at 60° C., but it produces a heat-resistant
toxin that, as it is not destroyed in thirty minutes at
100° C., would survive most cooking processes.

Symptoms are due to action of the toxins, their onset is
generally sudden and may include vomiting, diarrhoea,
pains in abdomen and head (sometimes in back and limbs
as well), prostration, collapse, cold sweats, rigors, cramps,
rashes, furred tongue, offensive breath and moderate
pyrexia.

If the affected food contain toxins preformed in the
food, onset of symptoms may take place almost immedi-
ately after ingestion. In most cases, however, the food
contains toxins and bacteria, and symptoms may com-
mence any time within thirty-six hours. Bainbridge
suggests the rat's intestine to be the true home of Gartner's
bacillus (see also p. 97).

THE COLON -TYPHOID GROUP 95

Organisms Resembling Gartner's Bacillus.

Para-Gartner Bacilli (or pseudo-Gartner bacilli). —

Savage has described a number of organisms in the healthy
human and animal feces, that can only be distinguished
from true Gartner bacilli by extended schemes of fer-
mentation reactions and by their failure to agglutinate
with sera from animals immunised to Gartner bacilli.

Bacillus Paratyphosus p. — The paratyphoid bacillus /3
is similar to B. enteriiidis (Gartner) in cultural reactions,
and, like the latter, produces a toxin that withstands
100° C. for thirty minutes. While generally producing
symptoms like those of a mild typhoid infection, it has
been held responsible for some meat-poisoning outbreaks.
Savage, while not disputing the possibility of this organism
causing food-poisoning outbreaks, showed that where
B. paratyphosus /3 has caused acute gastro- enteritis
simulating a food-poisoning outbreak, the cases were
connected, not by a common food supply, but by con-
tact with a source of infection probably human. In the
Cholet outbreak, Chantemesse found the bacilli in the
confectioner's cream supplied for the wedding breakfast,
and ascertained that the confectioner was a paratyphoid
carrier.

Bacillus Paratyphosus a. — Like the /3 bacillus, this
organism produces symptoms resembling mild typhoid,
but it is much less frequent than the /3 bacillus.

In their reactions the paratyphoid bacillus a stands
nearer the typhoid bacillus than does the paratyphoid
bacillus /3. Group a produces less gas in glucose media
than group 13. Milk remains permanently acid with
group a; with group /3 it becomes alkaline after an initial
acidity. Though group a changes neutral-red to yellow,
the red colour returns after three weeks or so, while with
group /3 the yellow colour is permanent.

In paratyphoid fever, whether caused by a or /3 bacilli,
the organism may be found in the blood and fseces. The
'mortality is low (less than 3 per cent.), and in the autopsy
the characteristic typhoid ulcerations of the Peyer's
patches are absent. Milk and meat are supposed to have
conveyed the infection, and the organisms have been
isolated from water.

<)6 AIDS TO BACTERIOLOGY

In identification of the disease and of any suspicions
organisms isolated, agglutination tests are important.
Paratyphoid serum either fails to clump B. typhosus or
only does so in low dilution, but agglutinates the
appropriate paratyphoid bacillus in dilutions of over
1 in 100. Examination of the fseces is performed as for
typhoid.

At the time of writing paratyphoid appears to be more
frequent in the services than typhoid, often occurring
as a chronic dysenteric condition. The diseases are fre-
quently as severe as typhoid. Anti-enteric vaccine is
frequently a mixed preparation of typhoid, paratyphoid
a and paratyphoid /3 bacilli. This gives protection
against any or all of the three diseases. A triple infection
with the three diseases has been described (Castellani).

Bacillus Psittacosis. — Imported parrots are liable to an
infectious enteritis with septicaemia (psittacosis) which is
communicable to man, producing a fatal broncho-
pneumonia. The psittacosis bacillus belongs to the
Gartner group, and is partly clumped by typhoid serum,
some bacilli between the clumps retaining motility.

Bacillus Suipestifer (B. Suicholerae). — This organism was
formerly regarded as the cause of swine fever. The disease
is now known to be due to an ultra-microscopic organism,
but B. suipestifer always seems to invade the tissues of
animals with swine fever (hog cholera), and is credited
with pathogenic properties for that animal. Its presence
in meat has caused food poisoning. B. suicJiolerce is
supposed to be identical with Sanarelli's B. icteroides and
with B. Aertryclc. While German authorities consider
B. suipestifer to be identical with B. paratyphosus /3,
Englishmen maintain them to be distinct organisms.

Rat and Mouse Viruses. — The Danysz bacillus, cultures
of which (' Danysz virus ') are sold for the extermination
of rats, belongs to this group, as do the B. typhi murium,
used as a mouse virus, and organisms found in other makes.

Bainbridge showed that the viruses owe their potency
to bacilli indistinguishable from B. Aertryck and B.
enteritidis. Their destructive power on rats was incon-
stant, the death rate varying from 20 to 50 per cent.
Some of the rats recover and become immune, while
others do not even suffer at all. The use of rat viruses
appears to be sometimes attended with dangerous results.

THE COLON-TYPHOID GROUP 97

They are one and all certified to be harmless to human
beings, and in some cases this is based on actual ex-
periment. As Bainb ridge says, the entire innocence of
the bacterial viruses for man is a statement which needs
justifying.

Collingridge attributed an outbreak of illness lasting
forty-eight hours — and characterised by headache, giddi-
ness, and cramp — to a virus used for killing mice in a
London business establishment. Nine girls at an Irish
convent school died, and McWeeney attributed the out-
break to B. enteritidis in beef, which, he said, was possibly
infected by sick mice in the larder.

Savage and Read found true (Gartner) B. enteritidis
in the spleen and liver of apparently healthy rats and
regarded its presence as the result of an old infection
from which the rats had recovered. Savage thinks that
there is a strong probability that rats surviving an epi-
zootic of this nature will act as carrier cases for some
time.

Morgan's No. 1 Bacillus. — While this, or a very similar
organism, is to be found in the normal stools of children
occasionally, it is much more frequent in the motions of
summer diarrhoea of children, and is accepted as being
frequently the cause of this condition. It produces acid
and a little gas in dextrose, but does not ferment lactose,
saccharose, nor mannite. It does not liquefy gelatin nor
give the Voges-Proskauer reaction, but it produces indole
and slowly turns milk alkaline with no primary acidity.
It has been found on flies in houses with cases of summer
diarrhoea.

Diseases of Calves. — Gartner group bacilli are responsible
for or present in some cases of septicaemia, dysentery,
pneumonia, and other septic diseases of calves. Jensen's
paracolon bacillus of calf dysentery belongs to the group.

The Typhoid Bacillus.

Morphology. — B. typlwsus (the Eberth-Gaffky bacillus)
is 2 /UL to 4 //, long by 0-5 JLI thick, with rounded ends, but
shorter, longer, and, occasionally, filamentous forms are
seen in cultures. No spores are formed, but granules
and vacuoles may be seen. Involution forms 10 /LI to
30 fj, long are obtained on repeated subculture, and are
somewhat characteristic. The organism is aerobic and

7

98 AIDS TO BACTERIOLOGY

facultatively anaerobic and Gram-negative It is not
killed by drying ; its thermal death-point is 55° C. Diffused
daylight prevents its development, and direct sunlight is
fatal in five hours.

The typhoid bacillus usually carries eight to twelve
flagella arranged round its sides and end. It is actively
motile, and in hanging-drop preparations some of the
organisms are seen to progress at a surprisingly rapid rate-
while others rotate rapidly. While growth is best at
blood-heat, the bacillus develops well at room tempera-
ture, though less quickly than does the colon bacillus.

Cultural Characters. — On agar the typhoid bacillus
produces a thick, greyish, creamy layer. On gelatin it
gives a white, thin growth, usually almost confined to the
needle track, and without liquefaction. In a gelatin shake
culture the growth forni^> a diffuse haziness, without any
gas-bubbles. The surface colonies in gelatin plates are
small (1 millimetre), semi-transparent, bluish- white in
colour, with an irregular outline; the deep colonies are
roundish points with sharp margins, finely granular, and
yellowish- brown in colour. In broth a general turbidity
is produced, with some deposit, but no pellicle. Milk is
rendered slightly and permanently acid, but there is no
curdling. Occasionally alkalinity may succeed acidity if
culture in milk be prolonged. On slightly acid potato
a thin, moist, greyish, almost invisible layer is formed.
Glucose and mannite are fermented with the production
of acid only, no gas, but lactose and saccharose are un-
attacked. It forms no indole, does not give the Voges-
Proskauer reaction, and neutral red is unaltered.

Strains of typhoid bacilli showing slight divergence in
some cultural character from typical attributes are met
with. We found a carrier strain to slowly ferment lactose
with production of acid (in three days) (see also p. 28).

Strains grown on lactose, dulcite, or arabinose acquire
the power of fermenting the respective carbohydrate or
alcohol to which they have been ' educated,' but many
educated strains quickly revert to the non-fermenting
typical organism.

Channels of Infection — Water. — Although water con-
taminated with sewage may be drunk with impunity,
should the dejecta of a case of typhoid contribute to the
pollution, an epidemic of typhoid may be expected. In

THE COLON-TYPHOID GROUP gg

many cases infection has been traced to water which
chemical analysis showed to be of considerable organic
purity. Even bacteriological examination in which colon
bacilli and streptococci have been estimated will not
necessarily reveal danger from typhoid. Where a pre-
viously pure water is contaminated with urine from a
typhoid carrier, typhoid bacilli will be present without
colon bacilli and streptococci. Therefore, while as a rule
the ' bacterial indicators of pollution ' (normal intestinal
inhabitants) are to be found in a typhoid- bearing water,
it does not follow that because they are absent, typhoid
bacilli are also absent.

Bacteria are not evenly distributed through the bulk
in unfiltered water, many being aggregated in masses,
so that where typhoid infection is present some of the
bacilli will be discrete, but very many will clump together.
Consumers of this water only imbibing separated bacilli
are not likely, or are less likely, to acquire the disease.
But the consumer who receives a clump of typhoid bacilli
is the less likely to resist infection (spotted distribution).
Storage and filtration, Houston believes, remove all likely
danger from aggregated microbes, and tend to render
separated microbes fairly equally distributed throughout
a water supply.

There is no" ground for the suggestion that B. coli may
become transformed into the typhoid bacillus. The
persistence of the typhoid bacillus in water is largely
governed by the chemical constitution and bacteriological
characters of the medium (see also p. 225).

Typhoid bacilli persist longer in bacterially pure waters
than in waters containing large numbers of bacteria,
and Houston has shown that raw river water infected
with ' uncultivated ' typhoid bacilli from a case of typhoid
bacilluria cleared itself of 99-99 per cent, of typhoid bacilli
after a week, and apparently the organism had completely
disappeared in ten days (cf. pp. 202, 211, 225).

When they are present, infusoria and crustacean
organisms devour typhoid bacilli. Whipple and Mayer
found that in water kept under anaerobic conditions
(atmosphere of hydrogen) typhoid bacilli quickly died,
although under similar conditions it may thrive in nutrient
media. Houston found that between 0° and 18° C., the
higher the temperature the quicker was the disappearance

ioo AIDS TO BACTERIOLOGY

of typhoid bacilli in water. Provided it be sterile, organic-
ally polluted water such as sterile sewage does not clear
itself any quicker than a pure sterile water. Hewlett
says that in aerated water (CO2), B. typhosus does not
survive a fortnight.

Very different periods of survival have been found by
different workers, even a year in unsterilised tap water
having been insufficient for it to die out.

Shellfish. — Oysters, winkles, mussels, cockles, etc.,
from contaminated water may be a source of infection
(see p. 207). Klein has shown that oysters readily take
up B. typhosus, but clear themselves of the ingested
typhoid bacilli if they are kept in clean water which is
frequently changed. The process is slower if they
are kept in a ' dry ' state — i.e., out of the sea-
water. Oysters from a polluted locality clear them-
selves of the ingested bacilli to a less extent and
at a slower rate, even if kept in clean sea-water,
than oysters clean at starting. Oysters from a polluted
locality containing a large number of the B. coli very
rapidly clear themselves of this microbe, whether kept in
or out of the water. This shows that B. coli is foreign
to the oyster, and is rapidly destroyed by it. When,
therefore, it is present in the oyster, it must have been
derived from the surroundings. However largely infected
with typhoid bacilli, the oysters remain in all parts of
normal aspect. Cockles and mussels similarly take up
the typhoid bacillus, but clear themselves much more
slowly, particularly in the case of cockles, than do oysters.

The Board of Agriculture and Fisheries in their Report
for 1912 suggested a period of quarantine in pure water
for shellfish before sale.

Dust and Air. — Infection through the air for any dis-
tance, though possible, is unlikely. In India, where
excreta are buried in the ground, and dust-storms are
frequent, water-supplies and food are sometimes con-
taminated by faecal-loaded debris. Where latrines are
employed, as in military camps, it has been found that
the spot from which an epidemic started was that nearest
to, and directly to leeward of, the filth trenches; and the
direction in which it spread was always down the wind.

Flies. — After feeding on excreta, and other infected
material, bacilli adhere to the legs and wings of these

THE COLON-TYPHOID GROUP 101

insects, and should any be swallowed they may pass
through the fly unhurt. Living bacilli have been found
in or on the bodies of flies twenty-three days after infec-
tion. Dutton infected two healthy persons by exposing
them to bed-bugs that had previously bitten a typhoid
case.

Vegetables. — Watercress grown in sewage-polluted
streams, and vegetables from sewage farms, are proved
to have been the means for the conveyance of the disease.
Creel ascertained that plants from seed sown in infected
soil carried typhoid bacilli on their leaves for over thirty
days when shade was provided. In full sunlight the
t3^phoid bacilli lived ten days at the outside.

Soil. — Whereas in peat the bacillus dies within twenty-
four hours, in moist earth it may exist for two or three
months, but shows no tendency to increase in numbers.

Milk. — Through being handled by infected persons,
through the use of polluted water for rinsing out utensils
or for purposes of dilution, typhoid bacilli can obtain
access to milk, and it is probable that multiplication may
take place. The organism will survive for at least twenty
days, even after the milk has turned sour and curdled.
Butter made from infected milk retains the power of
infection for a time, and it is possible that the organism
may exist in curd cheeses. A series of cases on the round
of a specific milkman is the usual clue to the origin of
a milk epidemic. There is no evidence to warrant a
suggestion that cows may suffer from and transmit the
disease.

Filters. — A filter such as the domestic carbon type,
which only arrests a portion of the micro-organisms, if
contaminated by the passage of an infected water, will
continue to infect water passing through it, even if this
be pure.

Contact. — Direct infection of a healthy person by a
typhoid case is not uncommon. The ' typhoid carrier '
(vide infra] is especially dangerous on account of his
condition being probably unrecognised.

Pathogenesis. — The bacillus can nearly always be found
in the blood during the fever period, the number being
greatest during the early stages of the fever. The
number decreases with the temperature, and disappears
when the latter reaches normal. Inflammation and

102 AIDS TO BACTERIOLOGY

necrosis of the Peyer's patches is set up, forming ulcers,
which may become so deep as to lead to perforation. The
bacillus may rarely be detected in the faeces and urine
during the early stages of the disease, but appears in the
former after the eighth or ninth day, and from urine
enormous numbers can sometimes be found after the
third week. The organism may be found in the spleen,
sweat, mesenteric glands, liver, kidneys, bone-marrow,
and in some cases of pneumo-typhoid in the sputum.
From its presence in the kidneys, rose spots, and urine,
Wright and Semple consider the disease to be a septi-
caemia. The dorsi-lumbar region is not a very uncommon
site for the late activity of the bacillus (' typhoid spine ').
Such complications as cystitis (from infection by urine)
and inflammations of the gall-bladder are common.
Typhoid bacilli persist in the gall-bladder for a long time
and are suggested to be a frequent nucleus of gallstone.
Typhoid pleurisy is rare and typhoid meningitis rarer
still. Osteomyelitis may develop six or seven years
after recovery from typhoid (Jordan). Injected into
animals, the typhoid bacillus produces a general septi-
caemia. Animals, with the exception of chimpanzees and
young suckling rabbits, are immune to the bacillus
administered per os. In young rabbits a febrile affection
is produced, with diarrhoea and inflammation and ulcera-
tion of the lymphoid (Peyer's) patches and solitary
glands. The organism is most readily demonstrated in,
and isolated from, the spleen of a cadaver, in which it is
found in the form of small aggregates or colonies.

The view that the first lesion necessarily takes place
in the ileum is giving way before the idea that an entrance
into the blood-stream is effected through the tonsil or
other alimentary lymphoid area.

Typhoid Carriers. — After cessation of the fever, the
bacillus disappears from the faeces and urine fairly soon
as a rule, but a certain proportion (Semplo gives it as
11*6 per cent.) of cases are infectious for more than six
weeks after infection (chronic carriers). It is estimated
that three or four persons in every thousand are carriers.
Cammidge records a case where the bacilli wore still being
excreted in the faeces ten years after recovery from the
disease. The gall-bladder and the urinary passages are
the sources from which the faeces and urine are infected.

THE COLON -TYPHOID GROUP 103

Mild typos of the disease arc as likely to give rise to bacilli
carriers as the severest forms. Typhoid carriers may also
be recruited from those who have not passed through an
attack of the disease in a clinically recognisable form and
from those who have been in contact with typhoid cases.
The handling of food by such persons is a great source of
danger, and outbreaks of the disease have been definitely
traced to them.

Serum-Therapy. — Chantemesse grows the typhoid
bacillus in a maceration of spleen, bone-marrow, and
defibrinated human blood. This, on the injection of an
animal, produces a serum which, Wright suggests, perhaps
owes its value to the presence of toxins and not of anti-
bodies. Macfadyen and Hewlett, by injecting horses
with the cell- juice of typhoid bacilli, prepared by grinding
the bacilli in the presence of liquid air, obtained a serum
of promise.

Anti-Typhoid Vaccine (Wright). — Typhoid bacilli (the
virulence being kept by intraperitoneal passages through
guinea-pigs) are grown in nutrient broth in flasks, at
37° C., for a maximum period of thirty-six hours, and not
that of ten to fourteen days as formerly. A temperature
of 60° C. as originally employed for killing the bacilli
seriously affected the keeping property of the vaccine.
Now, after the contents of several flasks have been mixed
(to get a uniform product), the culture is killed by exposure
to 53° C. for an hour. Portions for testing sterility by
both aerobic and anaerobic methods are removed and the
bulk is allowed to cool, when 4 per cent, of lysol is added.
(Addition of lysol to the hot vaccine destroys the immun-
ising properties.) This killed culture forms the vaccine,
which is standardised by estimating the content of bacilli
(see p. 24). A dose of 500 million is injected subcutane-
ously, and is followed by a febrile reaction, which soon
passes off. To obtain more complete protection a second
injection of twice the original dose may be given after an
interval of ten to fourteen days. The success of this
vaccine is remarkable both as a prophylactic and as a
curative measure, and an expectation that it will clear
the organism from carriers appears reasonable. In the
Expeditionary Force in France the ratio of attacks is
fourteen times and of deaths forty-two times greater
among the uninoculated men. The vaccine loses its

104 AIDS TO BACTERIOLOGY

properties by degrees, and should never be used if it is
older than three months.

Antityphoid vaccine does not protect against the para-
typhoids (q.v.). Inoculation has proved to be quite a
harmless procedure, the great majority of men inoculated
experiencing nothing more than ' inoculation fever.'
Very occasionally it starts pyogenic organisms, already
lurking in the body, into activity.

Bacteriological Diagnosis — Blood Culture. — Two to five
c.c. of blood are withdrawn from a superficial vein with
a sterile syringe, and small flasks of broth (25 to 50 c.c.)
are inseminated, each with 1 to 2 c.c. of the blood, and
incubated. This method serves to differentiate cases of
paratyphoid from true typhoid by the differences in the
bacilli isolated. It is useful for diagnosis during the first
week.

Widal's Reaction (see p. 19). — The lobe of an ear is
Washed with ether or some other suitable liquid. With
a straight, spear-pointed surgical needle a prick is made
into the lobe and the blood collected in a capillary tube,
preferably sterile. An ordinary vaccine tube does very
well. The blood should form a continuous column in
the tube. The ends of the tube should be cautiously
sealed in the flame, dry end first, care being taken that
the blood is so far away from the ends as not to become
heated. It is better if the tube can be centrifuged, so
as to obtain a clear serum; if this cannot be done, it
will be found that after a few hours, on breaking off the
ends of the tube, a string of clot and some serum can be
blown out on to a glass slide, and the serum with some
corpuscles collected in another capillary tube, or in the
diluting pipette to be described immediately. If the
tube has been centrifuged, it is broken off at the junction
of serum and corpuscles, and the clear serum blown out.

A haemocytometer pipette may be used for diluting, or
a pipette may be made by drawing out a piece of glass
tubing in the blowpipe flame. A little of the serum is
drawn up into it, a mark made on the glass at the upper
limit of the serum, and the serum blown out into a watch
glass. Then any desired number of similar volumes of
diluting fluid (O7 per cent, salt solution) are added to the
serum, and the whole mixed.

Alternatively, Delepine's method may be adopted:

THE COLON -TYPHOID GROUP 105

Fifteen drops of broth or salt solution are successively
taken up in a loop of platinum wire, and placed on a glass
slide; close to them is placed one drop of the serum, and
the sixteen drops are then thoroughly mixed. A small
drop of this diluted serum is mixed on a cover-glass with
an equal-sized drop of a broth culture of the typhoid
bacillus. This gives a total dilution of the serum of
rather over 1 in 30.

The culture should preferably be from a specimen of
low virulence, but it is not an essential point; some
cultures agglutinate much better than others. It is also
better (but again not essential, if free from clumps) for
the culture not to be over twenty-four hours old. If no
broth culture is available, an agar culture rubbed up in
broth or salt solution will do as well, but should be filtered
through filter-paper to remove clumps before use. Dead
broth cultures (killed by heating to 65° C. for an hour)
may also be used. A control hanging-drop preparation
of the culture should always be made, and examined to
see that no clumps of bacilli are present in it.

The mixed drop of diluted serum and of culture is
examined as a hanging-drop (p. 47) and should have very
little depth, as the * clumps ' have a tendency to sink
and get beyond the focal distance of the lens. At first
the bacilli may be active, but if the case is one of enteric
fever they soon slow down or stop, then gradually groups
of two or three form; these groups soon aggregate until
nearly all are collected into various crowds or ' clumps,'
with very few isolated bacilli left. If the reaction is com-
plete within thirty minutes, the case is certainly one of
enteric fever. Generally it is not complete, and there may
be groups of only ten or twenty, but the occurrence of
grouping and loss of movement are in themselves decisive.

The dilution of the serum is necessary, because the
serum of normal individuals will often, undiluted, evince
an agglutinative power, but not, so far as experience goes,
in a total dilution of more than 1 in 16. More than one
dilution should always be examined, and it is advisable
that one should be high, such as 1 to 80 or 1 to 100. The
lower dilutions may conveniently be 1 in 30 and 1 in 50.

The time-limit is necessary since many normal sera
will act, even when diluted, if left an indefinite time;
with a dilution of 1 in 30 to 1 in 50 an hour should be

io6 AIDS TO BACTERIOLOGY

sufficient. Time and dilutions should always be recorded.
A positive reaction with a 1 in 80 dilution would in almost
all cases indicate the bacillus to be either specific for the
serum or very nearly so. It sometimes happens that
agglutination is obtained with a certain dilution but not
with a lower one, e.g., the 1 in 50 may be positive and
one of 1 in 16 may be negative. Such ' zone reactions'
are very rare.

The agglutination method is very satisfactory. It is
not obtained until about the seventh day of fever, is
rarely intermittent, and a negative result should not be
accepted unless repeated three or four times at intervals
of a few days; it is rarely absent throughout the course of
the disease, and in such cases the disease is frequently
severe. The reaction persists for years after an attack,
and, therefore, before applying the test a previous attack
should, if possible, be excluded. Inoculation with
Wright's vaccine also causes the blood of the inoculated
to acquire agglutinative properties. Pyrexia leads to a
diminution or disappearance of inoculation agglutinins,
and Tidy says that a positive Widal reaction after the
sixth day of pyrexia is as definite a proof of B. typhosus
infection in an inoculated as in an uninoculated man.
Delepine isolated a strain of typhoid bacillus, designated
as 7120, which clumped with the serum of an active case
of typhoid, but not with the serum of an artificially
inoculated person. The blood in paratyphoid infections
may sometimes agglutinate the typhoid bacillus, but
usually only in low dilution. Agglutometers for perform-
ing the test by macroscopical observation with dead
typhoid bacilli are sold.

Ophthalmo-Diagnosis. — Chantemesse applies typhoid
toxins to the conjunctiva — a reaction analogous to Cal-
mette's tuberculin conjunctival reaction.

Cutaneous Reaction. — Deehan applies a drop of fluid
containing the standardised toxin to the skin, and then
makes a slight abrasion with a lancet under the drop.
The reaction, which does not cause the patients any
discomfort, is supposed to be specific.

Cultivation from Faeces and Urine. — Surface-plate
cultures (p. 237) on Conradi-Drigalski agar, MacConkey's
lactose bile-salt agar or malachite-green agar are made
with the material, which should be as fresh as possible.

THE COLON -TYPHOID GROUP 107

The faeces are first diluted by emulsifying 1 or 2
grammes of fcecal matter with about 700 c.c. of sterile
normal salt solution; when the emulsion has stood for
about one hour, so as to allow the coarser particles to
subside, a small quantity of the supernatant fluid is
pipetted off for plating. It is necessary to use three large
Petri dishes for each case; about four drops of the fluid
emulsion is transferred to plate 1, and carefully spread
over the surface with a glass rod spreader bent at right
angles. The same spreader, but without being reinfected,
is then carefully rubbed in succession over the surface of
plates 2 and 3; all three plates are then incubated at a
temperature of 37° C. for twenty-four hours.

Colonies resembling typhoid or paratyphoid are worked
up.

To inhibit B. coli, some workers make a primary
inoculation of fseces into peptone water every 5 c.c. of
which contains 0-2 c.c. of a 1 in 10,000 solution of brilliant
green, incubate for one or two days and then plate out.
The simple plating is, however, quite satisfactory.

With urine it is only necessary to spread a small quan-
tity, varying from a few drops to 1 c.c. or thereabouts,
over the surface of a plate containing one of the media
mentioned, and then to proceed as above.

The Dysentery Bacillus.

The several varieties of dysentery are due to different
etiological agents. In one form — the tropical or amoebic
— an amoeba is believed to be the causative organism
(see p. 174); in a second form, the so-called epidemic or
bacillary, a bacillus (B. dysentericc:} is the infecting agent.
The paratyphoid bacilli and B. pyocyaneus may also
cause a dysenteric condition, and possibly other organisms
— e.g., the B. coli and Proteus vulgaris — and certain
parasites— e. g., the bilharzia— produce a pseudo-dysen-
tery.

As many as fifteen types of dysentery bacilli are said
to exist, the Shiga-Kruse and the Flexner groups being
best known.

Morphology. — The dysentery bacilli form no spores
and are Cram-negative. They are generally said to be
non-motile, and most workers have failed to demonstrate
flagella.

io8

AIDS TO BACTERIOLOGY

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THE COLON-TYPHOID GROUP 109

Culture. — The bacilli are aerobic and facultatively
anaerobic. The growths on surface agar and gelatin
are much like those of typhoid; there is no liquefaction of
gelatin. In broth and peptone-water there is a general
turbidity without pellicle; indole may or may not be
formed. On potato the growth is usually thin and colour-
less, sometimes yellowish or brownish. In milk it grows
well without clotting, the reaction being first acid, and
generally after a few days becoming alkaline. It ferments
glucose with the production of acid, but no gas; lactose
is not attacked, and neutral red is unchanged.

The varieties are identified by agglutination reactions
and action on mannitol. The Flexner type ferments
mannitol with production of acid, but no gas, while the
Shiga-Kruse bacillus has no action on this polyhydric
alcohol. Some varieties ferment maltose and saccharose
as well.

The thermal death-point is about 60° C. Soluble
thermostable toxins are formed in alkaline media, and
Todd has been able to produce an antitoxin by treating
an animal with the toxin.

Agglutinating Reaction. — Provided the type of organ-
ism causing the disease be employed, the blood-serum of
patients with bacillary dysentery gives an agglutination
reaction, sometimes in high dilutions. Whereas agglutina-
tion can be obtained on the second day in some cases, in
others it is not found till the twelfth day.

Pathogenesis. — The liver abscesses generally found in
amoebic dysentery do not occur in the bacillary disease,
and the organism is not found in the urine or blood, but
it is abundant in the bowel discharges. The incubation
period, after the bacilli have been swallowed, is from one
to two days.

The organism is met with in the so-called ulcerative
colitis, or ' asylums dysentery,' and in a considerable
proportion of cases of epidemic (summer) diarrhoea of
infants.

Direct or indirect contact with infected bowel dis-
charges is recognised as a principal means of infection.
After apparent recovery the convalescent may still
excrete the bacilli. Polluted water has been held re-
sponsible for infection, but Forster has shown that the
Shiga bacillus has but a short life outside the body, and

no AIDS TO BACTERIOLOGY

Buchanan (Medical Press, June 30, 1909) is of opinion
that water is not so often to blame as is generally imagined.
MacGonkey's lactose agar or Conradi-Drigalski agar
may conveniently be used for isolation of the bacilli
from faeces. On the former white colonies, and on the
latter small clear blue colonies, are worked up.

CHAPTER VIII
THE DIPHTHERIA BACILLUS

Morphology. — The Klebs-Loffler bacillus (B. diphtherice)
is a delicate, slender, non-motile, and non-sporing organ-
ism, having rounded ends. Its length is variable, long,
medium, and short varieties being described, the usual
length seen being 3 ^ to 5 //. Both in the membrane and
in cultures a remarkable parallelism in arrangement is
seen, and club-shaped forms are frequent.

It is not killed by drying; dust containing the bacillus
will retain its virulence for months under certain condi-
tions. The organism is aerobic and facultatively anaerobic,
and its thermal death-point is 58° C.

Staining. — When stained with Loffler's alkaline methy-
lene blue, the staining is frequently irregular, darker and
lighter stained portions alternating — the so-called ' seg-
mentation ' ; sometimes the poles are deeply stained,
appearing as dark dots at the ends — ' polar staining ' ;
and occasionally there is a change of tint to a pinkish
here and there — ' metachromatism.'

Wesbrook divides the bacillus into three types: The
' solid ' type stains uniformly ; the ' barred ' form shows
intervening segments, which either stain but slightly or
not at all; and the granular type (which predominates
in clinically characteristic affections) has deeply staining
granules. The bacillus is Gram-positive, and as con-
firmatory stains Neisser's or Pugh's stains are generally
used,

Neisser's diagnostic stain: A preparation for about
thirty seconds is treated with the following solution:
One gramme of methylene blue dissolved in 20 c.c. of
alcohol, and mixed with 950 c.c. of distilled water and
•50 c.c. of glacial acetic acid. It is rinsed in water, treated

THE DIPHTHERIA BACILLUS in

with Gram's iodine solution, and then counter- stained
in Bismarck brown (see p. 50) for about a minute, washed,
dried, and mounted.

The Klebs-Loffler bacillus treated by this method
appears a? a delicate rod, stained pale brown, and contain-
ing two inky- blue dots, one at each end. Sometimes a
dot is also seen in the centre. Most other organisms
simply stain brown, without any dots. (In the original
method the two staining solutions were alone employed;
Tanner introduced the use of Gram's iodine. Both
solutions should be filtered before use.) In some instances
it is possible to obtain characteristic stained preparations
from the cultures after five or six hours' incubation, long
before there is any visible growth.

Pugh's stain consists of 1 gramme of toluidine blue
dissolved in 20 c.c. of absolute alcohol, and made up to
a litre with distilled water. Fifty c.c. of glacial acetic
acid are added, and the stain filtered. Cover-glass
preparations are stained for five minutes, and then washed
in water. This stain shows up the granules very dis-
tinctly and saves the necessity for a double stain.

Cultural Characters. — On gelatin the growth is slow,
without liquefaction. On agar at blood-heat growth is
more rapid; on potato, unless first moistened with beef
broth, the growth is scarcely perceptible. On blood-serum
and glycerin agar growth is rapid, but Loftier' s medium
(p. 39) is most used, as the growth which appears as a
cream-coloured streak along the line of inoculation is so
rapid as to allow the bacillus generally to outstrip other
organisms that may have been present in the throat, and
is visible in twelve hours. A number of small isolated
dots near to, but not touching, the actual streak is
suggestive of the organism.

The bacillus exhibits heemolytic property, a yellowish
area encircling a colony on blood-agar. Hofniann and
xerosis bacilli are said to be devoid of this power. Milk
is turned acid without coagulation. Acid, but no gas,
is produced in glucose and lactose media.

In peptone-water, after a few days, an indole reaction
can be obtained with sulphuric acid; this is not due to
indole, but to skatole-carboxylic acid (Hewlett).

The bacillus (and some staphylococci) grows red with
a bluish-pink tint diffusing through the medium on a

112 AIDS TO BACTERIOLOGY

glucose neutral red sheep-blood serum containing 1
per cent, potassium sulphocyanate (Myer Coplans).
B. Hofmanni grows yellow!

Bacteriological Diagnosis. — The diphtheria bacillus
can occasionally be identified by direct examination of a
teased-up fragment of membrane, or in expert hands it
may be recognised in a smear made from a swabbing.
But generally a cultivation is necessary. The throat may
be touched with a platinum wire, and successive streaks
made along the surface of a sloped tube of Loftier' s serum.
Or an ' outfit,' consisting of a sterile cotton-wool swab
in a sterile tube, may be used. The swab is rubbed
over the suspected portion of the throat, replaced in the
tube, and sent to the laboratory. The use of antiseptic
gargles or tablets shortly before swabbing must not be
allowed, nor should swabbing be performed soon after a
meal, since particles of food containing bacteria may
render the examination more difficult. The early morn-
ing is a most suitable time for routine swabbings. In
contacts and convalescents, where no obviously diseased
patches are to be seen, the swab should be rubbed on
both tonsils. The swab is rubbed over the surfaces of
two serum-tubes in turn thoroughly, so that all parts
of the swab come in contact with the serum, but lightly,
so as to avoid ploughing up the surface, and the tubes
are incubated at blood-heat for twelve to twenty-four
hours. Cover-glass preparations are then made from the
most likely colonies. If no growth is visible, a scraping
of the whole surface should be taken, and should a micro-
scopical examination of this preparation be negative,
the tubes should be incubated for a further twenty-four
hours.

Millard, while regarding the ' long granular ' organism
as the B. diphtherice par excellence, expects solid stained
bacilli to show granules if further incubated. For
diagnostic purposes the barred forms are less satisfactory,
as B. coryzce segmentosus, commonly found in the nose in
a common cold, B. xerosis (vide infra), and B. auris and
B. ceruminis, found in the ear, are very similar.

Diphtheroids. — In many healthy throats or throats
diseased but not with diphtheria, are to be found bacilli
that closely resemble the Klebs-Loffler bacillus in every
particular except that of virulence, and it can hardly be

THE DIPHTHERIA BACILLUS 113

doubted that they are really diphtheria bacilli which have
lost their virulence. The Hofmann bacillus (' pseudo-
diphtheria bacillus ') differs from the Klebs-Loffler
bacillus in several particulars. It is a shorter, plumper,
somewhat spindle-shaped rod, staining pretty evenly,
only exceptionally presenting involution forms, and not
giving the Neisser staining reaction. Like the true
diphtheria bacillus, it stains well by Gram, and has the
same parallelism in arrangement. Its most marked
distinguishing feature is that it produces alkali and not
acid in milk and glucose media. It is virulent to many
small birds, but not to guinea-pigs, and Salter has claimed
that, though not forming toxin, it does form toxoids,
which are capable of combining with diphtheria antitoxin,
but his results have not been confirmed by Hewlett no£
by Petrie. Some have believed that the Hofmann
bacillus is a modified and non-virulent Klebs-Loffler
bacillus. Most observers have failed to transform the
one bacillus into the other, though success has been claimed
in a few instances. The Hofmann bacillus seems to
replace the diphtheria bacillus in the fauces during
convalescence, and this has suggested the transformation
of the latter into the former. But as Cobbett pointed
out, diphtheria bacilli being soon found in the acute
stage, Hofmann bacilli might pass unnoticed, but would
be found when scarcity or absence of Klebs-Loffler
bacilli necessitated a longer microscopical examination.
Present opinion inclines to regard the Hofmann bacillus
as a species distinct from the Klebs-Loffler bacillus. In
view of this uncertainty, cases of ' angina,' in which the
Hofmann bacillus only can be isolated, are often treated
as infective. Van Eiemsdyk (Lancet, 1915, ii. 766)
says that true diphtheria bacilli inhabit the throat for
choice and not the nose; pseudo-diphtheria bacilli,
contrariwise, are most often found in the nose and rarely
in the throat. The Hofmann bacillus has been found
in the nasal mucus of as many as 71 per cent, of dwellers
in large towns.

Ford Robertson believes the specific organism of general
paralysis to be a diphtheroid organism occurring in the cere-
bro-spinal fluid (B. paralyticans), and reports remissions in
patients by the use of a vaccine.

The xerosis bacillus, an organism met with on the

H4 AIDS TO BACTERIOLOGY

conjunctiva, is extremely like the diphtheria bacillus in
morphology and staining reaction, but it grows more
slowly, and the cultures are thinner, drier, and more
granular than those of the diphtheria bacillus, and it is
non-virulent.

These organisms can be differentiated by fermentation
reactions. Buchanan obtained excellent results by
coagulating ox-serum in an equal quantity of water,
filtering the mixture, adding 1 per cent, of glucose to one-
half and 1 per cent, of saccharose to the other half, and
' tubing,' neutral red being used as an indicator. In
twenty-four hours a marked acid reaction was produced
in the glucose tube by B. diphtheria, and in both tubes
by B. xerosis, while no change was produced in either
tube by the bacillus of Hofmann.

Hiss's serum-water medium is also used for the fer-
mentation reactions: Serum 1 part, water 3 parts, with 1
per cent, of a carbohydrate or glycerin, tinged with litmus.

The virulence of the bacillus varies considerably. The
average amount of a forty-eight hours' broth culture
required to kill a 250 to 300 gramme guinea-pig within
one to two days is 0-5 to TO c.c.

Channels of Infection. — Diphtheria is particularly a
disease of the young between the ages of two and ten;
the mortality is greatest at ages below five, and during
the last quarter, and is lowest during the summer months.
Its distribution is world-wide, but it is most prevalent
in cold and temperate climes. The ' school influence ' is
an undoubted factor in disseminating the disease, there
being a decrease in the number of cases during holidays.
The disease may be acquired by direct infection, as in
kissing, or by less direct means. The organism retains
its virulence for a long time in particles of dried mem-
brane, sputum, and discharges. As a rule, the bacilli
disappear from the throat within three or four weeks of
the beginning of the attack, but they often persist for
longer periods. Millard found bacilli present thirty days
after admission in 25 per cent, of his cases, and a case of
Hewlett's repeatedly gave the organism for twenty-two
weeks after the commencement of convalescence. A case
should not be pronounced as being free from infection
until at least two, preferably three, examinations have
shown that the bacilli are no longer present.

THE DIPHTHERIA BACILLUS 115

While non- virulent diphtheria-like bacilli are frequently
present in milk and its products, virulent Klebs-Loffler
bacilli have several times been isolated from milk. Klein
stated that cows inoculated in the shoulder with the
bacillus are attacked with an eruptive disease of the
udder, and that the diphtheria bacillus could be isolated
from their milk. Abbott and Hitter, repeating Klein's
experiments, failed to find the bacilli in the milk. Dean
and Todd investigated a small outbreak of diphtheria,
in which the Klebs-Loffler bacillus was isolated from
the milk and from an eruption on the udders of two
cows supplying it. They seem to have proved, however,
that the eruptive disease was not due to the diphtheria
bacillus, but was an eruption which had become infected
with the bacillus, and they suggest that both the lesions
on the cows and the milk had become infected from some
outside source, possibly the milker. Milk epidemics are
generally attributed to a human rather than a bovine
origin. Experiments show that the bacillus may survive
for some weeks in spring waters of little organic impurity,
although during this time the virulence is gradually
attenuated. If, however, at any time previous to its
ultimate disappearance the organism be transplanted
into a suitable culture medium, it can reacquire its full
initial virulence. There is, however, no well- authenticated
instance in which water has been proved to be the source
of infection.

Faulty sanitary conditions may also assist in the spread
of this disease by preparing the throat for the bacillus,
and may in this way apparently give rise to cases which
would never have arisen had it not been for the existence
of such conditions.

It is not unusual for an epidemic of diphtheria to be
preceded by a prevalence of ' sore throat,' which seems
to gather in intensity till cases arise of undoubtedly
true diphtheria.

Pathogenesis. — The incubation period varies from two
to seven days, but is usually from about two to four days.
The mortality is about 0-20 per cent, of the total death-
rate.

Mucous surfaces are most prone to infection. The
pharynx is most generally the area attacked, but infection
of the larynx (membranous croup) and of the nose

u6 AIDS TO BACTERIOLOGY

(membranous rhinitis) are common. The middle ear and
the mucous surfaces of the genitals may also be sites of
infection. Traumatic diphtheria may arise through
contact of an abraded surface with the organism. Con-
junctival infections have been caused by patients coughing
or sneezing in the eyes of attendants. In the skin an
eruption indistinguishable from eczema is produced.
The tonsil plays an important part in the defence of the
body and in pre-antitoxin days the mildness and low
mortality rate of tonsillar, as compared with other
diphtherias, was attributed to this defence.

Occasionally the organism may produce a septicaemia,
but generally only the toxins circulate, the organism
remaining more or less localised at the seat of infection
(toxaemia).

In a typical case a white wash-leather-like membranous
coating, consisting of a fibrinous exudation, is present,
and on detachment leaves a bleeding patch. Absorption
of the toxin produces lesions in the heart, nerves, and
kidneys, and paralytic sequelae may follow recovery
from an attack. Hewlett says that paralytic sequelae are
not found when infection of a non-diphtheritic nature is
concerned.

Kanthack and Stephens found that in fatal casein
diphtheria bacilli can, almost without exception, be de-
tected in the lungs, generally in the cervical and bronchial
glands and spleen, and sometimes in the kidney.

The term ' haemorrhagic diphtheria ' is applied to those
cases in which, in addition to other signs of malignancy,
haemorrhages appear in the skin at an early stage of the
disease, with or without haemorrhages from the mucous
membranes (Rolleston, Medical Press, 1909, 390). The
mortality is over 80 per cent., reaction to antitoxin
is delayed, and all the cases which recover suffer from
extensive paralysis.

A person may have the bacillus in the throat without
contracting the disease. It may be found in 20 per cent,
or more of contacts. Once lodged deep in the lacunae
of the tonsil, the bacillus remains there and the patient
becomes a ' carrier.' Pybus records a case that had
three attacks of diphtheria in as many years and none
after removal of the tonsils. A. G. Macdonald says that
the length of carrier-life of the bacillus appears to have no

THE DIPHTHERIA BACILLUS 117

effect upon its virulence, since the organism has been
proved to be virulent after four and eight months in the
ear and nose.

The guinea-pig, rabbit, dog, cat, horse, and cow, are
all susceptible to infection with the diphtheria bacillus.
Rats and mice are refractory. A disease of cats appears
to be identical with human diphtheria. They may also
harbour diphtheria bacilli without symptoms of the dis-
ease.

Although both are susceptible to infection with the
Klebs-Lo filer bacillus, the diseases known respectively
as diphtheria of poultry and calves (p. 121) are of a different
nature, and non-communicable to man.

Mixed Infections. — In the false membrane the Klebs-
Loffler bacillus is often associated with such organisms as
Streptococcus pyogenes, staphylococci, and pneumococci.
In such cases a high .temperature and foatid throat may
be expected, and probably the pathologic process is more
serious. W. J. Wilson says that staphylococci may be
antagonistic to diphtheria bacilli.

Diphtheria Toxin and Antitoxin. — The nature of the
extracellular toxin is not known, but it probably consists
of protein. When injected into an animal in doses,
sublethal at first and gradually increasing, an antitoxin
is formed.

In addition to the toxin, a diphtheria broth culture
contains other substances called toxones and toxoids that
combine with antitoxin. Toxoids are non-toxic deriva-
tives of toxin. Toxones are primary secretory bodies
that produce induration, necrosis, and paralysis.

Preparation of the Toxin. — A virulent culture (grown
in slightly alkaline beef broth at 37° C., with free access
of air, for a week) is filtered through a Pasteur-Chamber-
land filter. The toxicity of the filtrate should be such that
not more than O'Ol c.c. will kill a 250-gramme guinea-
pig in forty-eight hours.

Immunisation of the Horse. — A small dose of toxin is
injected into the withers, together with a dose of anti-
toxin. A slight swelling appears, and after a few days
subsides, when the operation is repeated, using a larger
quantity of toxin. A swelling again appears; when this
has in turn subsided, further injections are made, till
it is possible to inject 500 c.c. of toxin without injury

nS AIDS TO BACTERIOLOGY

to the animal. When sufficiently immune, a sterile
cannula is inserted in the jugular vein, and the blood
drawn off into sterile bottles. The bottles are placed
in an ice chamber to allow the clot to separate. The
clear serum is separated for use. A small quantity of
trikresol is added. For use in hot climates the serum
may be evaporated to dry ness in vacuo, and then forms
amber-coloured scales or granules, which, for use, may be
dissolved in five to ten parts of sterile water (one part
of dry serum = about ten parts of the fluid serum).

There is gradual diminution of antitoxic power in the
serum yielded by horses, even though they continue to
receive toxin, so after some months fresh animals have to
be employed.

The preparation of a horse to give serum of very high
antitoxic value by the above method involves treat-
ment extending to six months or more. Cartwright
Wood grows the diphtheria bacillus in ordinary peptone
broth containing serum for three or four weeks at 37° C.,
and, after filtration, heats for an hour at 65° C. It is
thereby claimed that powerful antitoxic serum can be
produced in a short time.

During immunisation a small quantity of blood is with-
drawn from time to time, and its antitoxic power tested.

Standardisation of the Serum. — The serum is tested
against a certain amount of the toxin, the result being
recorded in units. The methods of Behring and of Roux
were formerly employed for this purpose.

According to Behring's standard, a serum that contains
one normal antitoxin unit per c.c. is of such a strength
that ^o °f a c-c- completely neutralises the action of ten
lethal doses of toxin.

The lethal dose of toxin is the amount required to kill a
given weight of guinea-pig. Of this toxin, ten times the
amount required to kill a guinea-pig of about 250 grammes
weight is injected, together with the antitoxin to be tested.
By noting the absence or presence of local reaction and
the increase or loss of weight, it is stated that an opinion
may often be formed after twenty-four hours, but that
after forty-eight hours a decision can always be given.
When the toxin is completely neutralised, as it should be,
the animal should not only live, but there should be no
trace of local reaction (oedematous swelling). This

THE DIPHTHERIA BACILLUS 119

swelling may not be apparent in the first twenty-four
hours, but a rapid fall in weight will at that time fre-
quently indicate its probable occurrence within the next
twenty-four hours. In this connection it should be borne
in mind that guinea-pigs, taken from stock and put into
small cages, usually rise in weight when not injured by
the action of the toxin.

Roux's method defines the proportion of serum in
relation to the weight of the animal which would protect
a guinea-pig against a lethal dose of toxin.

Ehrlich's method of standardisation, now universally
adopted, eliminates errors due to toxoids and toxones.
A standard antitoxin is employed, as this is more stable
than toxin if dried and stored in vacuo. For use, this is
diluted so that one unit is contained in 1 c.c. The
laboratory toxin is then standardised with this standard
antitoxin, and the exact amount of the toxin is ascertained,
which, when mixed with one unit of antitoxin, just
suffices to cause the death on the fourth or fifth day of a
250-gramme guinea-pig. This amount of the toxin is
termed the L+ dose (L = limes = boundary — i.e., be-
tween life and death, the neutral point — L+ meaning
that there is one lethal guinea-pig dose of the toxin left
unneutralised by the unit of antitoxin). The L+ dose
of the laboratory toxin having been ascertained, this
amount of the toxin is mixed with varying amounts of
the antitoxic serum to be tested, and each mixture is
injected into a 250-gramme guinea-pig. The amount
of the serum which just suffices to completely protect
the guinea-pig from the toxic effects of the L+ dose of
toxin is then known to contain one unit of antitoxin. The
standardised toxin is preserved by the addition of toluol,
and is kept in a cool, dark place; but even then its toxicity
gradually diminishes, and it has to be restandardised
every three to six weeks.

As Sudmersen and Glenny found that the active
immunity of the doe guinea-pig is transferred passively to
her offspring, animals from parents used previously in
the test cannot be used.

The unit of antitoxin corresponds to 105 to 115 minimal
lethal doses of a toxin for the guinea-pig, or, roughly, to
100 minimal lethal doses.

The most suitable place for injection of antitoxic

120 AIDS TO BACTERIOLOGY

serum is the subcutaneous tissue of the flank. The
dose given varies from 2,000 to 4,000 units up to 30,000
or more. A child may require as much as an adult.

Injections of serum are often followed by the appearance
of various rashes, sometimes erythematous. at other times
urticarial, and in a few cases not unlike the rash of scarlet
fever or of measles. These rashes usually come on in
from seven to ten days after the injection; sometimes the
rash is accompanied by more or less pyrexia, and in a
small number of cases by pains, and even effusion into
some of the joints. Such rashes may appear after
injection of normal horse-serum (see Serum Disease,
p. 26).

Diphtheritic paralysis seems to occur more frequently
than formerly, the explanation being that more cases,
and especially severe ones, recover. For cases treated
on the first day of the disease the mortality is nil, but
rises with delay in treatment, until in cases not treated
until after the fourth day the mortality is nearly as great
as in pre- antitoxin times. In many cases the injection
of the serum is followed by a speedy reduction in the
severity of the symptoms, and a rapid separation of the
membrane in cases where it was causing obstruction of
the air-passages, thus diminishing the number of cases
which would otherwise require tracheotomy.

Prophylactic Use of Diphtheria Antitoxin. — To protect
contacts the prophylactic dose should be about 500
units. The immunity so obtained does not last longer
than three weeks.

Vincent's Angina.

This affection of the throat often closely simulates
diphtheria, but the diphtheria bacillus is absent. In
the lesions Vincent describes the presence of two sym-
biotic organisms — one a bacillus, with pointed ends 6-8 //
to 10-12,0. in length and 1-1-5^ in diameter, sometimes
motile, not staining by Gram, and cultivable anaerobic-
ally on the ordinary media with the addition of human
serum (B. fusiformis). With this is usually associated
a long, delicate, motile spirillum (Spirochceta Vincenti),
which has not been cultivated, and which is supposed
to be the fusiform bacillus in another stage.

THE DIPHTHERIA BACILLUS 121

Diphtheria of Birds.

' Pigeon diphtheria ' appears to be generally due to an
organism resembling the bacillus of hsemorrhagio septi-
caemia, but the 1907 epidemic in wood-pigeons and
some other epidemics were presumably due to Loffler's
B. diphtheria? columbarium, a short, non-motile organism,
which is Gram-negative and forms no spores.

Diphtheritic roup of poultry is supposed to be due to a
protozoon. Macfadyen and Hewlett isolated a Klebs-
Loffler-like organism from the throats of healthy birds,
but the organism was non- virulent to guinea-pigs. Jordan
states that an organism usually present in roup differs
essentially from the Klebs-Loffler organism; diphtheria
antitoxin is without effect upon the progress of roup. A
tough, yellow, false membrane is found on the conjunctivas
and the mucous membranes of the mouth, pharynx, and
breathing passages of the birds, and, apart from the ques-
tion of transmissibility of the disease to man, the affected
birds are emaciated to a degree that renders the flesh
unfit for food.

Calf Diphtheria (malignant or ulcerative stomatitis) is
said by Hewlett to be produced by an anaerobic strepto-
thrix, while Stockman attributes it to Bang's necrosis
bacillus.

CHAPTER IX
THE BACILLI OF H^MORRHAGIC SEPTICAEMIA

THIS class of organisms comprises the infecting agents
of bubonic plague, rabbit septicaemia, swine plague, fowl
cholera, and septic pleuro-pneumonia of cattle. These
affections are characterised by the presence of hsemor-
rhagic areas under the skin and throughout the internal
organs. The organisms themselves all show bipolar
staining (i.e., the ends of the organisms stain deeply,
whereas the central portion hardly stains at all). The
bacilli are Gram-negative, are short and non-motile, and
do not form spores.

Bacillus Pestis.

Morphology. — In the body it occurs as a short, almost
ovoid, rod, measuring on the average about 2'3 ft by

122 AIDS TO BACTERIOLOGY

1-7 /JL; but longer forms are seen, measuring 5 JLI. As
convalescence approaches, round and ovoid involution
forms, totally unlike the bacillus, appear. In cultivation
the young bacilli are so short as to be almost coccoid or
slightly oval, but in older cultures rod, thread, and involu-
tion forms occur. In broth culture the organism forms
chains (streptobacillus).

In a film made from an agar culture the bacilli are
swollen and yeast-like, but it is questioned whether a
capsule exists.

Staining Reactions. — B. pestis does not stain by Gram's
method, but with ordinary dyes shows marked bipolar
staining.

Cultural Characters. — B. pestis is an aerobe and a
facultative anaerobe. Growth is slow at 18° to 20° C.,
rapid at 37° C., but 30° C. is the optimum tempera-
ture.

In broth a characteristic flocculent, wavy deposit is
formed, which settles to the bottom, leaving a clear
medium above.

In broth containing a little butter or coco-nut oil, and
kept absolutely at rest, flocculent, tapering masses of
growth depend from the droplets of oil floating on the
surface (Haffkine's stalactite growth).

On the surface of gelatin it forms a thin, whitish,
punctate growth, which is confined to the inoculation
streak ; the medium is not liquefied. On the surface
of agar and of serum it forms a thick, cream-coloured,
very sticky growth.

On agar containing 2-5 to 3-5 per cent, of common salt
pear-shaped and spherical involution forms are so common
that Hankin recommends this salt agar as a diagnostic
medium. It grows in milk without coagulation. On
potato little or no growth takes place. It grows well in
bile-salt media. While the virulence of some strains
is retained in cultures for a long while, that of other
races quickly diminishes.

Resistance. — The organism is very easily killed by
disinfectants. The thermal death -point appears to be
between 58° and 70° C. While cold has little effect,
complete desiccation kills the bacillus. In sterilised
water the bacillus has remained alive for fifteen days at
room - temperature .

B A CILLI OF HJEMORRHA GIC SEPTIC&MIA 123

Pathogenesis. — Three types of the disease are common
— bubonic, septicsemic, and pneumonic. The bacillus is
found in the buboes (sometimes with streptococci and
staphylococci) in the bubonic form, in the blood-stained
(' rusty ') sputum in the pneumonic form, and in the blood
in the septicsGmie form. It is also found in the blood in
the other forms on the approach of death. The period of
incubation appears to be usually from three to six days,
but may extend to nine days.

Pathogenesis for Lower Animals. — The injection of
cultures into mice, guinea-pigs, and rats, produces plague
symptoms, and the animals die in two to seven days.
A mere scratch with a needle dipped in an emulsion of
a recent culture of plague will generally kill a white
mouse of ordinary size in one to three days. Rats and
mice can be infected per os. The bacilli are found in
the spleen and lymphatic glands of inoculated animals
but arfe not very numerous in the blood.

Calves and poultry may contract the disease in a
chronic form, as the result of feeding on plague-infected
offal (Simpson). Plague also attacks cats, dogs, ferrets,
bats, squirrels, pigs, hares, rabbits, and monkeys.

Transmission, — The plague bacillus probably obtains
entrance through wounds in the skin, or through the
mucous membrane of the respiratory or (more rarely)
alimentary tracts. The sputa of patients with the pneu-
monic disease, when discharged in droplets during cough-
ing, etc., probably constitutes the main way in which
disease spreads in these cases.

The lower animals often convey the disease. An
epizootic among rats is almost constantly seen before
the disease becomes epidemic among men, and ground
squirrels have been held responsible for epidemics in
California. Rats may become infected by ingestion of
carcasses of men and animals dead of the disease. When
infection is experimentally produced in this way mes-
enteric buboes are most frequent. But Jordan says that
an examination of 5,000 naturally-infected rats showed
no instance of mesenteric bubo, cervical buboes being
most often found. Different species of rats vary greatly
in regard to their susceptibility to the disease. The
white rat is the most susceptible, the brown ship rat or
brown dock rat coming next, then the black rat and a

i24 AIDS TO BACTERIOLOGY

Norwegian species, and finally, the least susceptible of
all, our own common sewer rat.* In the Bombay district,
a new outbreak first attacks M us decumanus (the grey
sewer rat), passing to M us rattus (the black house rat),
and then on to man. The disease is largely spread by
fleas. On the death of an animal the fleas desert it for a
living host. The common rat-flea found in the tropics
(Xenopsylla cheopis) readily attacks man, and laboratory
experiments show that, provided a healthy animal can be
protected against the fleas of one that is plague-stricken,
the former is not likely to develop the disease, in spite of
the proximity of the respective cages. (X. cheopis has
only been found once on English rats.)

Sambon insists that in epidemic plague transmission
from man to man is more frequent than transmission
from rat to man. He believes that during a true epidemic
the rat strain of B. pestis is replaced in many cases by a
human strain, and the rat fleas are replaced by the fleas
of man, and by those of the cat and dog, which attack
man as well. Jordan states that cervical buboes are
most common when flea infection has occurred.

Serum Treatment. — A horse is inoculated intravenously
with a suspension of dead bacilli weekly for three months.
During the next three months living organisms are given.
Various reports have been given of the efficacy of serum
treatment.

Anti-Plague Vaccine. — The ' Haffkine prophylactic '
is prepared by growing a virulent bacillus in nutrient
broth containing butter-fat. After a week the stalactite
growth (see p. 122) is detached by shaking, and the broth
reinoculated with the organism. By the time the medium
has been treated thus four or five times development
becomes slow and scanty. The culture is sterilised by
heating to 65° C. for one hour, and 0'5 per cent, of carbolic
acid added. The dose employed is about 2 -5 c.c., which
is injected subcutaneously. A second injection, given
a week after the first, increases the immunity. By the
use of the Haffkine prophylactic the incidence of the
disease is much reduced, and among the vaccinated who
contract the disease the mortality is much less than
among the unvaccinated.

* ' The Bacteriology and Etiology of Oriental Plague' (Klein).

BACILLI OF HJEMORRHAGIC SEPTICSEMIA 125

Bacillus Pseudo-Tuberculosis Rodentium*
A disease occurring spontaneously in guinea-pigs and
rabbits, accompanied by wasting, and progressing to a
fatal issue in about three weeks, is caused by the
B, pseudo-tuberculosis of Pfeiffer. This bacillus grows
readily and rapidly, forms a creamy growth on agar, and
a whitish growth on gelatin without liquefaction, not
unlike that of the colon bacillus. It also grows well in
bile-salt media (MacConkey). It is not acid-fast, and does
not stain by Gram. It has been met with in milk and
sewage. MacConkey has shown its fermentation attri-
butes to be similar to those of B. pestis. In the Suffolk
plague epidemic of 1910, a few rats whose appearances
were suggestive of plague, were found to be infected with
B. pseudo-tubercul

Swine Plague.

In the lung lesions of pigs affected with swine plague
(contagious pneumonia of swine), an organism very similar
to the bacillus of chicken cholera is found, but is not
regarded as the cause of the disease. Stockman is of
opinion that this disease comprises cases of swine fever
with pneumonia as a complication, and confirms
McFadyean's opinion that these organisms are normal
inhabitants of the mouth and air-passages in pigs, which,
owing to the weakened powers of resistance, have been
able to invade and multiply in the lung tissue.

Chicken Cholera.

This disease as a rule runs a very rapid course, pro-
minent symptoms being profuse diarrhoea, drooping wings,
ruffled feathers, and somnolency. It is said to be some-
times introduced into this country through foreign maize.
In an epidemic investigated by the authors, a fresh con-
signment of maize had certainly been fed to the birds
shortly before the outbreak. The specific bacillus is
a small oval bacillus, which exhibits polar staining so
markedly as to look like a diplococcus in stained prepara-
tions. The organism can be readily found in the blood,
but Stockman points out that the presence of such an
organism is, from his experience, insufficient to prove
cause of death, as bacilli of a similar type, which may in

126 AIDS TO BACTERIOLOGY

no way be connected with death, are not infrequently
present in the heart- blood of fowls. There seems little
doubt that the organism is identical with the bacillus
of rabbit septicaemia, and probably with the so-called
bacillus of swine plague. It differs from the similar
bacilli isolated from the following diseases in that the
latter are not communicable to the fowl: ' grouse disease,'
epizootic pneumo-pericarditis in turkeys (McFadyean), and
cholera in ducks. It also differs slightly from B. septicus
agrigenus, an organism isolated by Nicolaier from manured
soil, and from an organism found by Klein in fowl enteritis.

B. cholerce gallinarum has been described as an infect-
ing agent in some cases of gunshot wounds in the head at
Alexandria (Bartlett).

The bacilli of fowl cholera and its allies show very little,
if any, multiplication in bile-salt media (MacConkey).

The Committee of Inquiry on Grouse Disease decided
that lesions thought to be due to acute and infective
pneumonia in grouse were in many cases nothing more than
normal post-mortem changes and therefore dismissed the
bacteria isolated from further consideration.

The undermentioned organisms differ from those
previously described in this chapter in retaining the stain
by Gram's method.

The Bacillus of Swine Erysipelas.

Many cases of swine erysipelas (commonly known as
nettle-rash) amount to little more than a passing in-
disposition. There is a rise of temperature, more or less
shivering and loss of appetite, followed by a patchy red
eruption on the skin about the base of the ears, thighs, and
body. In severe cases there is vomiting and great
prostration, the pigs stagger about, breathe rapidly,
and perhaps die in about forty-eight hours. The specific
organism is a non-motile, Gram-positive bacillus, with
rounded ends, about 2jn long. It is found in the spleen,
bone-marrow, and lymphatic glands.

A vaccine is used, together with an anti-serum for
prophylaxis, producing an active immunity lasting about
a year. This is largely a seasonal disease, and animals
should be immunised before the season of prevalence —
not later than May. Swine erysipelas is said to be
communicable to man, though rarely.

SUPPURATION AND SEPTIC DISEASES 127

Bacillus Murisepticus.

The bacillus found in mouse septicaemia is similar to if
not identical with the bacillus of swine erysipelas. In
the tissues it is an extremely minute bacillus, I /u in length,
but in cultures filamentous forms occur. It stains well by
Gram's method. It is aerobic and facultatively anaerobic.
On surface agar it forms minute delicate colonies, or a
thin, delicate, greyish film, not unlike that of the strepto-
coccus; in stab gelatin it grows well without liquefaction,
forming a well-defined but delicate cloud-like growth
radiating from the stab.

CHAPTER X

MICRO-ORGANISMS OF SUPPURATION AND
SEPTIC DISEASES

THE action of organisms is not an indispensable condition
for the formation of pus. Abscesses may be produced by
the injection of turpentine, sodium cinnamate, and other
substances. Such an abscess intentionally produced (Fixa-
tion Abscess) is used by French surgeons for relief of certain
bacterial infections, toxaemias, and even for cases of metallic
poisoning (Brelet, Medical Press, 1915, i., 440). Pus
may also be produced by the introduction into the tissues
of sterilised bacteria of several kinds, with or without
the soluble products of their growth, so that the exciting
cause may be either the intracellular contents of the organ-
isms, or, possibly, the mechanical effect combined with
the positive ' chemiotaxis ' that most bacteria and their
products exhibit to the leucocytes.

Aseptic pus (sterile pus) is, however, rarely encoun-
tered, and pus formation (suppuration) is almost always
due to microbic agency. An organism capable of pro-
ducing pus is termed ' pyogenic.' An abscess is a local
and well-defined collection of pus, and the activities of
these so-called septic organisms may produce boils,
carbuncles, erysipelas, cellulitis, septicaemia, sapraemia,
and pyaemia (for definitions, see p. 14). According to the
presence of factors other than the organisms, different

128 AIDS TO BACTERIOLOGY

manifestations may be brought about by the same species.
By the injection of a broth culture of Staphylococcus
pyogenes aureus into the blood-stream of a rabbit a
septicaemia alone is produced, except perhaps in the
kidneys, where abscesses may be formed. If, however,
before injection the culture is rubbed up with finely-
ground potato, abscesses will be produced in the heart
and a pyeemic condition be induced, owing to the inability
of the potato particles, with the bacteria adherent thereto,
to pass through the capillaries. The washing out of a
loccilised abscess causing a saprsemia results in an im-
provement of the condition of the patient, owing to the
removal of organisms producing the offending toxins.

In addition to the organisms dealt with in this chapter
the following are also pyogenic: the tubercle, typhoid,
colon, and glanders bacilli, the pneumococcus, the
Actinomyces, and certain of the Blastomycetes and
Hyphomycetes (q.v.).

Staphylococcus Pyogenes Aureus.

Morphology. — This organism is a spherical coccus,
about 0'75^a to l/u, in diameter, and occurs as a diplococcus,
or, more commonly, in grape cluster-like masses. It is
non-motile, forms no spores, and is Gram-positive.

Cultural Characters. — The coccus is an ae'robe and a
facultative anaerobe. It grows well at room-temperature
and at blood-heat. The virulence of cultures persists for
many months. In broth a general turbidity forms within
eighteen hours. Gelatin begins to liquefy as soon as there
is any visible growth, liquefaction occurring in stab culture
all along the stab, an orange-yellow sediment being pro-
duced. On agar and blood-serum a thick streak develops,
which is at first pale, but later becomes golden -yellow;
exposure to diffused daylight favours chromogenesis.

Resistance. — The thermal death - point is 58° C.
(Sternberg), provided the organism is in a moist con-
dition; if desiccated, much greater heat is required. It
also seems that certain of the cocci in a culture are more
resistant than the majority to destruction by heat and
antiseptics.

Habitat. — The surface of the body appears to be the
normal habitat; it has been found in dust, earth, and
water, but its presence in these is probably accidental.

SUPPURATION AND SEPTIC DISEASES 129

Pathogenesis. — Different strains exhibit considerable
variation in virulence. Injected subcutaneously, it
forms a local abscess; into the circulation, a septicaemia;
into the peritoneum, a purulent peritonitis; and rubbed
into the skin, local inflammation, with small pustules
(impetigo). Eczema is now generally regarded as being
due to the irritative action of the chemical products of
8. pyogenes aureus. The organism has been found in
ulcerative endocarditis, furunculosis, osteomyelitis, em-
pyema, boils, carbuncles, and abscesses, in acne pustules,
and occasionally in septicaemia and pyaemia.

Little or no toxin is formed by 8. pyogenes aureus, and
attempts to prepare an antiserum have proved un-
successful. Vaccine treatment of staphylococcal infec-
tions is, however, quite popular.

Other Pathogenic Staphylococci.

Staphylococcus pyogenes albus and 8. pyogenes citreus
are not to be distinguished from the aureus variety, except
by the colours of the growths on agar or potato. The
former produces white and the latter lemon-yellow
growths. Andrewes and Gordon regard these three
organisms as a single species, owing to their ability to
produce intermediate varieties of colour on cultivation.
The albus is said to be frequently found in styes. A
feebly virulent variety of the S. pyogenes albus is fre-
quently present on the skin — S. epidermidis albus. Welch
finds it to be so deeply buried in the epidermis as to
render it difficult to destroy by means of disinfectants.
It is the most frequent cause of stitch abscess. While
S. pyogenes aureus ferments mannite, with production of
acid, this organism has no action.

Staphylococcus cereus albus forms a greyish-white,
wax-like growth, and 8. cereus flavus a wax-like growth,
first white, and then yellow, on gelatin. Neither liquefies
gelatin. These organisms are specially met with in
localised inflammatory conditions, and negative results
follow inoculation experiments.

Micrococcus salivarius, occurring in the saliva, and a
micrococcus found in scurf from the scalp, give white
growths on agar, and are non-pathogenic for animals.

136 AIDS TO BACTERIOLOGY

Pathogenic Tetracocci.

Micrococcus tetragenus occurs in phthisical cavities,
and sometimes in the pus of acute and chronic abscesses.
The organism occurs in pairs or fours, often seen sur-
rounded with an ill-defined capsule. It is Gram -positive,
slowly develops on gelatin as a thick white growth without
liquefaction, and on injection into white mice produces
general septicaemia.

Micrococcus catarrhalis is present in pairs or tetrads
(often within the polymorphonuclear leucocytes) in the
discharges of the ' influenza cold ' and other forms of
nasal catarrh and bronchitis. It is sometimes concerned in
producing pyorrhoea and other buccal abscesses. It
grows at 22° C. It is Gram-negative, and produces
opaque colonies of so tough consistence on serum agar
that they often come away intact on a platinum
loop.

Sarcina ventriculi occurs in the stomach, particularly
in cases of dilated stomach.

Bacillus Pyocyaneus.

The B. pyocyaneus is a small bacillus found in blue
and green pus, very actively motile, non-sporing, giving a
creamy growth on agar, to which it imparts a greenish
fluorescence and rapidly liquefying gelatin, the fluid being
similarly coloured. It may not produce blue pigment
for some days, and perhaps not at all until it has been
passed through animals (Lartigau). It does not stain
by Gram. The pigment can be extracted with chloro-
form, and consists of two pigmented bodies — pyocyanin
and pyoxanthose. The organism occurs in various condi-
tions, accompanied by debility, wasting, and diarrhoea —
the so-called marasmus — and occasionally its presence
in water has caused dysentery. It has also been re-
sponsible for septicaemia. There are probably several
varieties of the organism, varying in virulence and pig-
ment production, of which the B. fluorescens liquefaciens,
an organism common in water, may be one. A body of
the nature of a ferment, ' pyocyanase,' when extracted
from cultures, is used for the prophylaxis and cure of
anthrax and diphtheria.

SUPPURATION AND SEPTIC DISEASES 131

The Acne Bacillus.

Unna's B. acnes occurs in many conditions of the skin,
such as acne vulgaris. It is Gram-positive and resembles
Hofmann's bacillus. Sabouraud regards it as the cause
of seborrhcea, but proof of this is thought to be wanting
by Cooke and Dold. The organism, which is a facultative
aerobe, is most easily grown anasrobically on glucose
agar. Raised greyish-white opaque colonies appear in
three to five days. Sabouraud' s original medium con-
sisted of agar, 15 grammes; peptone, 20 grammes; glycerin,
20 grammes; distilled water, 1 litre; and concentrated
acetic acid, 5 drops. Fleming's medium (Lancet, April
10, 1909) consists of oleic acid (1 to 5 per cent.) in nutrient
agar. Western (British Journ. Dermat., January, 1910)
and others are of opinion that infection of the sebaceous
material with the acne bacillus is a secondary event, and
leads, by the irritation caused by its presence, to prolifera-
tion of the adjacent epithelium and formation of the
comedo. Autogenous acne bacillus vaccine is recom-
mended for treatment, but this has apparently failed in
seborrhcea (Practitioner, 1910, 526). Cases with much
induration are most frequently due to a mixed infection
of the acne bacillus and Staphylococcus albus, sometimes
with 8. aureus or citreus as well.

Streptococcus Pyogenes.

Morphology. — In pus a chain may contain up to fifteen
elements, but much longer ones of thirty or forty elements
are met with in broth cultures, of which the individual
cocci may vary very much in size, both large and small
cocci being found in one chain. It also sometimes
happens that a new chain starts away from one of the
cocci in a chain, thus producing branching. The variation
in the size of the cocci is also noticed in cultures on other
media. The organism is Gram-positive.

Cultural Characters. — The organism grows well in the
presence or absence of oxygen. When grown in broth
at 37° C., the medium becomes turbid in twenty-four
hours; after three or four days multiplication ceases,
owing to the production of an inhibitory metabolic
substance, but living organisms have been found after
ninety days.

132 AIDS TO BACTERIOLOGY

Growth on gelatin is slow and without liquefaction.
The colonies are generally very small and discrete, while
on agar at 37° C. the colonies are also small and seldom
coalesce with neighbouring colonies that almost touch.
This tendency of streptococcal colonies to remain small
is of great assistance in picking them out from plates
containing a variety of bacteria. In stab or shake culture
in gelatin the colonies appear as small whitish spheres.
In milk, acid is produced without a clot. It ferments
lactose, saccharose, and salicin. Growth on blood agar
shows it to be strongly haemolytic. The thermal death-
point lies between 52° and 54° C. (Sternberg).

Pathogenesis. — Streptococci of the pyogenes type are
more frequently found to be concerned in pathogenic
processes than are those of the short chain varieties. It
is found in ulcerative endocarditis, occasionally in
osteomyelitis and frequently in pyaemia, mammary
abscess, cellulitis, lymphangitis, and other suppurative
conditions. S. pyogenes is generally supposed to be
identical with S. erysipelatis, an organism found at the
periphery of the zone of redness in the lymph channels
of the skin in cases of erysipelas. Some doubt exists
whether the infective agent in puerperal fever is S. pyo-
genes or an allied organism (see S. puerperalis, p. 133).
There appears to be little doubt that several species of
streptococci have been described under the name of
S. pyogenes.

Serum Treatment. — The virulence of a culture of
S. pyogenes is ' exalted ' by passage through a series of
rabbits. A culture medium of 1 part of human or ass's
serum with 2 or 3 parts of broth is then inoculated, and
a horse is immunised, first with killed and then with living
cultures. A serum prepared with one variety of strepto-
coccus may not immunise against another, so the horse is
inoculated with several strains of streptococci (' poly-
valent ' serum). This serum is antimicrobic Vaccines
of killed cultures of streptococci are also used.

Coley's Fluid. — An attack of erysipelas supervening
upon a malignant growth has sometimes caused the dis-
appearance of the latter, and sterilised cultures of the
$. pyogenes have been used in the treatment of inoperable
tumours with varying success. Coley grows S. pyogenes
and B. prodigiosus together for two or three weeks, and

SUPPURATION AND SEPTIC DISEASES 133

the cultures are sterilised by heating to 65° C. Injection
is followed by a marked temperature reaction. The
treatment appears to be more successful in sarcoma than
in carcinoma.

Streptococcus Puerperalis.

While puerperal fever may be caused by B. coli and
Staphylococcus pyogenes aureus, in most cases it is a
streptococcus infection, and this is popularly supposed
to be S. pyogenes. While this may be the case, a strepto-
coccus found in the uterine discharge and in any secondary
pus, pleuritic fluid, or sputum by Mackey and Furneau
Jordan is considered by them to be probably the most
frequent cause. S. puerperalis grows freely upon agar,
producing opaque colonies which are much larger than
any other streptococci, producing chains of moderate
length. It produces acid and clot in milk, acid in lactose,
glucose, saccharose, and salicin, but no change in raffinosc,
mannite, and inulin. Furneau Jordan suggests that this
streptococcus, like 8. fcpcalis, is present in the contents
of the bowel, and that the puerperal woman is very
susceptible to its action.

Diplococcus Rheumaticus.

Poyriton and Paine's organism is usually a diplococcus
measuring 0*5 ^ in diameter, but forms masses on solid,
and chains in liquid, media. It stains feebly by Gram,
grows slowly on gelatin without liquefaction, and forms
in broth a flocculent deposit with clear supernatant
fluid. On agar it forms minute, white, discrete, slightly
opaque colonies, on potato hardly grows, and in litmus-
milk forms much acid and a firm clot. It is found in
the arthritic and valvular lesions in rheumatic fever, and
on inoculation into rabbits may produce arthritis and
endocarditis. Poynton and Paine's organism has been
found in affected tonsils in the tonsillitis preceding
rheumatic fever (Pybus).

Varieties of Streptococci.

Attempts at classification have hitherto proved unsatis-
factory. Lingelsheim attempted a distinction between
the long-chain (S. longus) and short-chain (S. brevis)
streptococci, the former being considered more virulent

134 AIDS TO BACTERIOLOGY

than the latter. But virulent short-chain streptococci
are sometimes met with, and it is possible to transform one
variety into the other by culture. Penfold describes
the variations obtained as ' haphazard.' Strains that
correspond to haemolytic streptococci have been converted
into typical pneumococci and vice versa.

Gordon divides the streptococci into four groups:
(i.) S. longus (from the mouth). Very long and compara-
tively straight chains. Broth remains clear with floccu-
lent deposit. Acid but no curd in milk, (ii.) S. medius
includes most pyogenic streptococci, which as a rule form
fair-sized curling chains; corresponds to Lingelsheim's
longus type. Broth acquires a flocculent deposit but
supernatant liquid remains clear. Milk becomes slightly
acid without curd, (iii.) 8. brevis. Short chains. Broth
uniformly turbid. Slight acid and usually curd in milk.
Includes pneumococcus. (iv.) S. scarlatina1 or conglo-
meratus. Masses of chains. Deposit in broth but upper
liquid keeps clear. Acid and curd in milk. Later,
Gordon introduced fermentation tests as a basis of differ-
entiation and using these Andre wes and Horder dis-
.tinguish (1) S. pyogenes, already described. (2) S.
salivarius (found in saliva), a brevis type that clots milk,
is not haemolytic nor pathogenic for mice. (3) S. angino-
sus (inflamed fauces, scarlatinal throats, and rheumatism),
a longus type, with no action on salicin, is haemolytic
and pathogenic for mice. (4) S. fcecalis, a faecal organism
sometimes found in cystitis, meningitis, and pus. A brevis
type that clots milk, is not hsemolytic nor pathogenic
for mice, the only class to ferment mannitol. Perhaps
the Diplococcus rheumaticus is identical with this organism.
(5) The pneumococcus (q.v.). (6) S. equinus (from
horse dung), a brevis type, not clotting milk and the only
class that does not ferment lactose.

Too much stress must not be placed on these differ-
ences, experience showing the characters mentioned are
anything but absolute.

•Salivary streptococci do not usually ferment salicin,
and Savage thinks the discovery of salicin-fermenting
streptococci in throats during an epidemic of sore throat
would point to a milk infection.

Streptococcus mastitidis, found in streptococcic in-
flammation of the mammae of cows (mastitis), and S. an-

SUPPURATION AND SEPTIC DISEASES 135

ginosus, found in inflamed and scarlatiniform throats,
are said to be indistinguishable morphologically and
culturally (Local Government Board Reports, 1907-08,
'Garget in Cows'). S. mastitidis only produces a local
abscess in animals, however, while S. anginosus produces
general symptoms and death. The former produces
mastitis in goats, while the latter does not.

Jordan thinks most of the streptococci in milk are
probably descended from saprophytic, not from patho-
genic, ancestors (see pp. 221, 222).

Wright (Lancet, October 30, 1915) describes the most
frequent organism in wounds received in action as a
streptococcus that, in film preparations of pus, nearly
always shows up as a diplococcus. As obtained from agar
and broth cultures, the elements of the diplococcus are
lancet-shaped and the pair form an angle resembling a
circumflex accent. In broth, a few short chains are
also formed. Colonies on agar show up very faintly grey-
green and when planted closely tend to run together.
Growth is much more rapid than with ordinary S. pyogenes,
luxuriant cultures being obtained at 37° C. on broth
or agar in four or five hours. In normal serum, and on
agar when transplanted in blood, it grows out. Wright
finds this organism to correspond to the enterococcus of
the French authors, and he also regards it as the ordinary
streptococcus of faeces (see p. 265).

Mutch says that in diabetes the duodenum is infested
by the S. brevis and that the only other condition in which
this abounds in the duodenum is rheumatoid arthritis.
Rosenow considers it reasonable to suppose that in man
gastric ulcer may be caused by streptococci. Houston
describes a case in which phthisis was simulated by a
streptococcic infection and in a case of duodenal ulcer
he found in the contents of the stomach streptococci and
staphylococci, from which a vaccine was made with
promising results. Streptococcic infections occur in
measles leading to septicaemia, which may prove fatal
in the middle or end of the second week. Streptococci
are frequent in alveolar abscesses and infected root canals :
Gilmer and Moody found many varieties, including a
haemolytic streptococcus with a wide zone of haemolysis,
S. mucosus (see p. 141) and S. viridans. (S. viridans
resembles 8. salivarius, but grows green on bloocl-agar.)

136 AIDS TO BACTERIOLOGY

Strangles, an equine disease, is supposed to be due to
streptococci, and they have been found in scarlet fever
(p. 195), variola (p. 200), and other diseases.

The Gonococcus.

Morphology. — The gonococcus is a small organism
measuring about 0'7 /a by 0'5 ja, tending to be somewhat
like a coffee-bean in shape, usually grouped in pairs, the
flattened sides of the two organisms being adjacent,
occasionally single or in tetrads. It is killed in ten minutes
at 60° C.

Cultural Characters.— The gonococcus is aerobic. Not
growing on ordinary media, it can be cultivated on
Wertheim's medium, a mixture of equal parts of nutrient
agar and human blood-serum, blood-smeared agar, or
the medium of Christmas — rabbit's blood-serum coagu-
lated by heat. A simple method of cultivation is to
deposit drops of blood obtained with aseptic precautions
from the finger on the surface of an agar plate, then to
add a drop of gonorrhceal pus, smear over the plate, and
incubate at blood-heat. A pure culture of the gonococcus
assumes a raised appearance, similar to a mulberry, and
is of a greyish-white colour. It is necessary to sub-
culture every few days, or the vitality is lost.

Whitehouse uses ordinary agar, with the addition of
human blood-serum and a few drops of human urine
(Practitioner, 1910, 489).

Pathogenesis. — The human urethra is generally the
site of attack, producing an inflammation, which may be
followed by posterior urethritis and stricture. In gonor-
rhoea the pus usually contains gonococci in pure culture
during the first few days, but later on staphylococci and
streptococci will often be found. After the acute stage
of gonorrhoea has passed, and there is no longer any con-
siderable flow of pus, the gleet that follows still contains
the gonococcus.

After the discharge has ceased, an examination of the
centrif uged deposit from the urine may reveal the organism
in large numbers. The organism may persist in the
genitals for years after apparent recovery, and the
patient still be capable of infecting others. In the female
the infection often spreads to the Fallopian tubes,
ovaries, and peritoneum. Gonorrhoea! ophthalmia of the

SUPPURATION AND SEPTIC DISEASES 137

new-born is due to maternal infection (see Stephenson's
* Ophthalmia Neonatorum '). In the male infection may
produce epididymitis and prostatitis. The circulatory
system may be invaded and produce arthritis (see Murrell,
Medical Press, 1910, 87) or endocarditis. Infection may
also be transferred by the use of infected towels, sponges,
etc. Entrance of the pus into the eye may result in its
loss unless properly treated.

Christmas, by growing the gonococcus in a medium
containing fluid rabbit's serum, obtained a feeble toxin,
with which he immunised rabbits, the serum of which
was feebly antitoxic. Vaccines are sometimes used.

Microscopical Examination of Pus. — In the female an
examination is best made directly after a menstrual
period, as in some cases, especially in chronic and sub-
acute conditions, the gonococci are then, and then only,
to be found. Films are prepared from the cervix uteri,
Bartholin's ducts, and Skene's tubules in the female, or
from the discharge as it exudes from the urethra in the
male. The films should be fixed by immersion in alcohol
and ether (equal parts) for fifteen minutes. Some films
are stained with Loffler's methylene blue, and the others
by Gram's method. In using the latter method for this
organism it is of the utmost importance that the technique
be most carefully and religiously observed. Bismarck
brown is a suitable counter-stain (Neisser's 6. stain,
acting for two minutes.) Identification of the organism
is only permissible when all the following characters are
shown. The organism must completely lose its stain
when treated by Gram's method, should have the coffee-
bean form, and occur in the pus cells. In the early stage
of infection the organisms may be found outside the cells.
Abundance of other organisms may lead to the gonococci
being missed, and in all cases a negative result should
be received with great caution.

The characters described will usually suffice to dis-
tinguish the gonococcus, but in cases of importance
cultures should be made. Organisms similar to the
gonococcus are found in the genitals, but these grow on
gelatin, with liquefaction of the medium, and on nutrient
agar. Some of these urethral diplococci are Gram-positive,
and there is no doubt that they are sometimes responsible
for the urethritis or whatever form the infection takes

138 AIDS TO BACTERIOLOGY

Eyre and Stewart state that the B. xerosis, which is
frequently present in the healthy and seldom absent from
the diseased, genital tract, forms colonies on blood- agar
which are indistinguishable from those of the gonococcus
to the naked eye.

Seeligman considers pruritus vulvse to be due to a diplo-
coccus, which resembles the gonococcus in appearance,
but is Gram-positive.

The Meningococcus.

Morphology. — In epidemic cerebro- spinal meningitis
(' spotted fever ') the causative organism is the Diplococcus
intracellularis meningitidis of Weichselbaum (meningo-
coccus).

While found in the polymorphonuclear leucocytes of
or lying free in the cerebro-spinal fluid as a diplococcus,
on culture it is a markedly pleomorphic organism, and
involution forms appear on media early. Lundie, Thomas,
and Fleming (Lancet, September 25, 1915) state that
associated with the meningococcus in the naso-pharynx
and brain, there is nearly always a Gram-positive ' strepto-
coccus,' tending on culture to become Gram-negative
and producing involution forms — in the blood they are
often Gram-negative. Donaldson (Lancet, June 26, 1915)
suggests the true cause of cerebro-spinal fever to be a
diphtheroid bacillus of which the meningococcus is only
a phase. Lundie, Thomas, and Fleming have also de-
scribed pseuclo-diphtheroids and they state the clots
do not stain by Neisser like the Klebs-Loffler bacillus.
These authors (loc. cit.) describe the ' caterpillar,'
' Zeppelin,' and ' bomb ' forms also met with.

Like the gonococcus, which it resembles in appearance
and arrangement, the meningococcus is Gram-negative.
It is very susceptible to cold, and infected swabs or other
material intended for culture experiments must be kept
as near body-heat as possible, if an interval between
sampling and plating-out is unavoidable. Halliday
Sutherland (Lancet, October 16, 1915) says the meningo-
coccus soon dies at 72° F. and is killed in thirty minutes
at 62°.

Culture. — For primary cultures from suspected material,
blood-smeared agar may be used, but Wassermann's
nutro.se ascitic agar ('Nasgar') is the popular medium.

SUPPURATION AND SEPTIC DISEASES 139

Nasgar is prepared as follows: 6 grammes of nutrose and
90 c.c. of ascitic fluid are added to 210 c.c. of distilled
water. This is heated with constant agitation to boiling,
when the nutrose dissolves. This solution is added to
600 c.c. of nutrient agar (previously melted) and the mixed
liquid is heated in the steam steriliser for half an hour,
filtered and tubed and sterilised as for nutrient agar.
Cultures are incubated at 37° C. and colonies appear in
twenty-four to forty-eight hours. The colonies are
characteristic. They are pearl-grey, clear, smooth, and
translucent with a firm, regular, oval or round outline.
They are larger than the colonies of pneumococci and
streptococci which are often found in material from the
naso-pharynx. The meningococcus ferments maltose and
glucose with the production of acid (distinction from
Micrococcus catarrhalis) and fails to ferment saccharose.
Muir and Ritchie point out that these fermentation re-
actions are most satisfactorily carried out on solid serum
media containing one per cent, of the sugar to be
tested.

Mclntosh and Bullock (Lancet, November 27, 1915) use
a medium of nutrient 3 per cent, agar, 3 parts; unheated
horse- serum, 1 part. The nutrient agar should be neutral
to phenolphthalein and made from beef-broth. These
authors mention that bile seems to inhibit growth of the
meningococcus.

Agglutination reactions may sometimes be obtained,
but negative results are worthless. Morgan (Lancet,
1909, ii., 156) found that the serum of patients may
agglutinate t}^phoid bacilli in a dilution of 1 in 50 — a
phenomenon which would be looked upon as a positive
reaction to Widal's test.

A recently suggested diagnostic reaction is performed
by adding a drop or two of anti-meningococcus serum to
a tube of fresh cerebro-spinal fluid obtained by lumbar
puncture and cleared by centrifuging. The tube and a
control are incubated at 52° C. for a few hours. If the
meningococcus is the cause of the meningitis, a precipitate
is said to develop in the tube to which the serum was
added, but not in the control.

Channels of Infection and Pathogenesis (see also p. 265).
— Cerebro-spinal meningitis is described in the horse,
cattle, sheep, and dog, but it is not known if these arise

140 AIDS TO BACTERIOLOGY

from the meningococcus, and the probability of the
disease in man being derived from animal sources does
not appear to be likely. Cerebro-spinal meningitis more
often affects country districts than cities (Osier). It
arises under cold atmospheric conditions and disappears
with the advent of warm weather. Overcrowding, bad
sanitation, and privation are cited as predisposing
causes.

In the body the meningococcus has primary residence
in the naso-pharynx. While it is there the host is a
carrier case, often without developing the disease himself.
Though carriers are usually free from the meningococcus
in two or three weeks, it persists in a small percentage
for two or even seven months. Mayer reported a case
probably existing two years. Carriers may develop
meningitis after two or three weeks. There is consider-
able variation in the number of carriers among contacts
in different epidemics. Among 300 soldiers who were
contacts Arkwright only found four carriers. In other
epidemics as many as 23 per cent, to 37 per cent, have
been reported.

During the stay in the naso-pharynx, whether the host
be a healthy carrier or in the incubation stage of the disease,
the small drops of secretion expelled during sneezing,
coughing, or speaking serve to carry and disseminate the
meningococcus. Halliday Sutherland says that infection
is carried by currents of warm moving air. Kissing is
obviously dangerous. In the developed disease the
meningococcus is found in the cerebro- spinal fluid and
very often in the blood.

Where a case is definitely attacked, the cerebro-spinal
fluid obtained by lumbar puncture shows distinctive
features: an increase of pressure, increase in albumin
content, and a polymorphonuclear leucocytosis frequently
so marked as to render the fluid quite turbid. An examin-
ation of the centrifuged deposit shows the meningococci
which are often within the leucocytes. If no diplococci
are found, recourse must be had to culture, but even then
negative results are sometimes obtained when all the
other evidence suggests meningococcal activity. Carrier
cases are detected by bacteriological examination of the
naso-pharyngeal secretion. As several non- or feebly-
pathogenic micro-organisms closely resembling the

SUPPURATION AND SEPTIC DISEASES 141

meningococcus are present in the healthy mouth, contami-
nation of the swab with saliva must, as far as possible, be
avoided. West's swab is recommended. The swab from
the naso-pharynx is lightly brought in contact with one
portion only of ready set nasgar in a Petri. The material
from the infected area is spread over the medium by means
of a sterile glass spreader, and, without reinfection, this
spreader is rubbed over the surface of a second plate.
The plates are then incubated at blood-heat. It is
imperative that the culture should be made immediately
after the swabbing of the naso-pharynx or else the in-
fected swab should be kept warm until it can be used.
When the colonies are up, a film is stained by Gram's
method, meningococci being Gram-negative. Sub-
cultures are made and incubated at 37° C. and 23° C.
respectively. The vast majority of the Gram-negative
cocci of the normal mouth grow readily at 23° C., while
the meningococcus does not.

Gaskell (Jour. R.A.M.C., September, 1915) says that
when the puncture fluid is allowed to stand for twelve to
eighteen hours in the blood-heat incubator and the
sedimented pus sown on blood- agar slopes, success is
more frequent than with generous sowings of fresh fluid.

Organisms that may possibly be mistaken for the
meningococcus are —

(1) Gonococcus. For practical purposes this may be
excluded.

(2) Micrococcus catarrhalis. Distinguished by fermen-
tation tests (vide supra) and by its growth at 23° C.

(3) Diplococcus pharyngis siccus. Grows at 23° C.
Colonies are very tough, those of the meningococcus
being easily removed and readily emulsified with water.

(4) Diplococcus mucosus. Gives slimy colonies. Grows
at 23° C.

(5) Diplococcus crassus. Gram-positive.

(6) Chromogenic cocci are not likely to lead astray,
unless the colour does not develop. Under this circum-
stance Micrococcus flavus gives colonies that closely
resemble those of the meningococcus.

(7) Pneuinococcus. Gram-positive.

(8) Pseudo-meningococcus.

(9) Streptococcus mucosus. Gram-positive; on serum -
agar gives a colony as clear as water.

142 AIDS TO BACTERIOLOGY

Flexner's curative serum has been used with success
(Lancet, October 30, 1909), and has considerably dimin-
ished the mortality, but it is important to obtain a serum
which is polyvalent to as many strains as possible. A
serum prepared from one strain may be entirely ineffective
when used to combat infection with a meningococcus
morphologically and culturally identical.

The judicious use of polyvalent vaccines is said to have
met with frequent success. Colebrook (Medical Press,
May 12, 1915) states that the vaccine did not help to
eradicate the meningococcus from the naso-pharynx
of carriers with any regularity.

CHAPTER XI

The Diplococcus (Streptococcus) Pneumonise.

Morphology. — D. pneumonice is found in large numbers
in the affected lung and in the rusty expectoration of
acute croupous or lobar pneumonia, and occasionally in
the blood. It is seen usually as a diplococcus, sometimes
in chains of four elements. The cocci are oval or lance-
head shaped, measure 0'5 JLL by TO [JL, and are surrounded
with marked gelatinous capsules. The organism is
Gram-positive. Maynard (Med. Press, November 4,
1914) thinks it probable that the pneumococcus can
assume the form of a bacillus of diphtheroid type.

Cultural Characters (see also Streptococci, pp. 134,
15). — The pneumococcus is an aerobe and facultative
anaerobe. It grows well on blood-serum and glycerin
agar at 37° C., but the dewdrop growth is only visible on
close examination. On gelatin at room-temperature it
does not develop. Under cultivation the capsule is lost,
except in media containing blood-serum, and it forms
short chains of a few cocci; hence it is probably a strepto-
coccus. It rapidly (five to six days) loses its vitality on
agar, but can be preserved alive for a considerable time
on agar smeared with blood or in gelatin kept in the
blood-heat incubator. It does not- develop on potato.
It grows in milk, with the production of acid, and usually
with curdling.

THE DIPLOCOCCUS PNEUMONIA 143

Clinical Examination. — With sputum the microscopical
examination and the inoculation of a drop into the
peritoneal cavity of a mouse give more reliable results
than culture methods, since other species of organisms
are frequently present; but with pus and exudations pure
cultures can generally be obtained.

Pathogenesis. — D. pneumonia? is pyogenic and may be
found in broncho - pneumonias, pleurisy and empyema,
endocarditis and pericarditis, meningitis (one- third of
the cases), peritonitis, arthritis, osteomyelitis, and con-
junctivitis. It can apparently also produce inflammation
of the throat, with the formation of a false membrane,
and is sometimes met with in this situation in association
with the diphtheria bacillus. It frequently occurs in the
healthy mouth. As it occurs naturally, its virulence is
subject to great variation. In its clinical features pneu-
monia presents strong resemblances to the specific fevers,
and though isolated cases are most common, epidemics
do occasionally occur. The diplococcus is very fatal to
mice on subcutaneous or intraperitoneal inoculation,
less so to rabbits and guinea-pigs, while pigeons and fowls
are immune. Antipneumonic serum has not given very
satisfactory results.

Vaccine Treatment. — Willcox and Morgan commence
treatment with a stock vaccine, while an autogenous
vaccine is being prepared by culture: (1) from sputum;
(2) from blood; or (3) by aspiration of the pleural cavity
or superficial part of the consolidated lung by a small
syringe with a fine needle. Vaccine treatment has
sometimes been found beneficial.

Resistance.— When protected by an albuminous coat-
ing the pneumococcus may retain its vitality for three or
four months, and has been found in the dust of a room
occupied by pneumonic patients and in the dust of
hospital wards.

Mice may play an active part in the dissemination of
pneumonia, particularly the epidemic variety (Gamaleia),

The Pneumo-Bacillus of Friedlander.

This organism, in the sputum, occurs as a short rod

1 fji to 2 /a in length, with rounded ends, though longer

forms are seen. In the exudations it is encapsuled, and

frequently occurs in pairs. It is non-motile and non-

I44 AIDS TO BACTERIOLOGY

sporing, and is aerobic and facultatively anaerobic. It
is Gram-negative.

Cultural Characters. — The pneumo- bacillus grows well
on all the ordinary media, both at room-temperature and
at blood-heat, but loses its capsule under cultivation. On
agar and blood-serum it forms abundant moist, thick,
cream-coloured growths. On surface gelatin it forms a
whitish spreading layer, and in stab gelatin a nail-shaped
growth — the growth being heaped upon the surface like
the head of a nail, and tapering from above downwards in
the line of the puncture; the gelatin is not liquefied. On
potato a well-marked white sticky layer develops. Milk
is usually slowly coagulated. There is an abundant
growth in broth, with a considerable deposit. It ferments
glucose, saccharose, mannite, and lactose energetically,
with the formation of gas and acid. Variations in its
power of fermenting occur.

It is sometimes met with in the sputum, particularly
in bronchitis, and occasionally in association with the
Diplococcus pneumonia?. It is also found in stomatitis
and rhinitis, ulceration of the cornea, affections of the
throat (sometimes with a false membrane), and in broncho-
pneumonia.

Mice are susceptible to infection, the guinea-pig is less
so, and rabbits are infected with difficulty.

Other Pneumonic Conditions.

There are also pneumonic conditions frequently com-
plicating other diseases, and most frequently seen in
young children in the course of measles and whooping-
cough, in influenza, in typhoid fever, in plague, and
after operations about the mouth and throat (' septic '
pneumonia). Although acute croupous pneumonia may
complicate these diseases, the pneumonic process is usually
of a different type, starting in a number of scattered
patches, which, however, may coalesce and so involve
large areas.

Micrococcus Melitensis.

The causative agent of Malta or Mediterranean fever is
the M. melitensis, a small coccus occurring singly, in
pairs, or in short chains, with an active Brownian move-
ment. (It is doubtful if it is a true motility, though

THE INFLUENZA BACILLUS 145

flagella have been described by Gordon.) Bacillary
forms have been observed in old cultures, and frequently
occur in even young cultures. Maynard (Medical Press,
November 4, 1914) says this bacillary form grows more
rapidly and luxuriantly than the typical micrococcus.

M. melitensis is of slow growth, especially in the primary
cultures from the spleen; on agar in three to four days it
forms small semi-transparent droplets, which later become
opaque and yellowish -orange in colour. It develops
slowly on gelatin as a limited dirty white streak without
liquefaction. It does not stain by Gram's method.
Inoculated into monkeys, it produces a febrile condition,
with enlarged spleen, simulating the human disease;
but it is non-virulent to guinea-pigs and rabbits, except
on intracerebral inoculation.

The disease is diagnosed by an agglutination reaction,
but dilutions up to 1 in 100 should be prepared, as Hewlett
points out that old laboratory strains agglutinate with
normal serum in dilution of 1 in 20 or 30. The organism
occurs in the blood and milk of goats, and the latter
constitutes the main source of infection. Since tli3
prohibition of goat's milk to the garrison at Malta (1906)
the disease has practically disappeared. The organism
is sometimes found in the urine, and less often in the
ffeces and milk of patients. It is not apparently a
water-borne disease.

CHAPTER XII
The Influenza Bacillus.

Morphology. — Pfeiffer's B. influenza is found in the
sputum and nasal secretion during the febrile period of
influenza. It is a very small rod, not exceeding 1-5 JJL
in length and 0-3 JLC in thickness. It has rounded ends,
and is generally found in pairs, but on cultivation grows
out into strings. It is Gram-negative. When stained
with dilute carbol-fuchsin there is a tendency to bipolar
staining. Resistance to outside influences is very
slight.

Cultural Characters. — The influenza bacillus is a strict
aiTobe. No growth occurs below 25° C., the optimum

10

146 AIDS TO BACTERIOLOGY

temperature being 37° C. On the surface of blood-
smeared agar it forms small transparent colonies, which
are perceptible with difficulty. Growth on ordinary agar
medium is slight and uncertain, but it grows better in
broth containing grape-sugar and glycerin. The organism
must be subcultured every eight days on blood- agar, or
its vitality will be lost.

Pathogenesis. — 'The period of incubation is twelve to
twenty-four hours. The disease may occur in an un-
complicated form, or may be accompanied or followed by
respiratory or gastro-intestinal lesions or neuroses, but no
case involving both respiratory and gastro-intestinal lesions
has been recorded. The simple form lasts from three to
five days, and the complicated from eight to ten, except
those affecting the nervous system, when the patient is
often months, or even years, in shaking off the effects,
and there are a few cases where insanity or paralysis has
resulted. It is not uncommon for the same patient to
have two attacks in one year, and in each fresh epidemic
those who have had the disease once are far more liable
to be attacked than those who have previously escaped.

The bacillus varies greatly in virulence and in the type
of infection produced. The pneumococcus may be the
causative agent in many cases of so-called influenza
(Allen). In a small percentage symbiosis of the influenza
bacillus with the pneumococcus or Staphylococcus albus
occurs. Broncho-pneumonia of an epidemic type has
been caused by the influenza bacillus. Influenza is
transmissible to cats, and is the cause of ' pink eye ' in
horses.

The Bacillus of Ducrey.

The bacillus of soft sore ( Ducrey 's bacillus) is found
in the ulcers and buboes of soft chancre. It is minute
and generally arranged in groups or chains, mostly outside
the leucocytes. It does not stain by Gram, and is culti-
vated on blood -agar, producing small shining grey
colonies, or in guinea-pig's blood. It produces the disease
on inoculation of the human subject.

The Koch- Weeks Bacillus.

This small bacillus is the cause of an acute contagious
conjunctivitis Morphologically it resembles the influenza

THE GLANDERS BACILLUS 1/17

bacillus, and growth is difficult except on serum agar
(ininute transparent colonies) or ascitic fluid glycerin
agar. It is not pathogenic for animals.

The Bacillus of Whooping-Cough (Bordet and
Gengou).

B. pertussis has much the same characters as the influ-
enza bacillus. It is a strictly aerobic small cocco-bacillus,
non- motile, negative to Gram, and staining feebly with
methylene and toluidene blues. The best medium is
agar, with which has been mixed a large proportion of
blood drawn off aseptically. The serum of patients who
have recovered from the disease shows specific reactions
to this organism. The serum of patients suffering from
this disease agglutinates the bacillus sometimes in as much
as a sixty-four-fold dilution, and gives the complement
deviation method of Bordet-Gengou.

The Glanders Bacillus.

Morphology. — B. mallei is a straight or slightly curved
rod, 2]n to 5// long, with rounded ends. Stained prepara-
tions may show beading or bipolar staining. From the
production of long filaments with swollen ends and ex-
hibiting lateral branching, some regard it as belonging
to the Trichomycetes, It does not form spores, and is
non-motile, though an active Brownian movement is
present in broth cultures. Its thermal death-point is
55° C. (Loffler).

Cultural Characters. — The glanders bacillus grows
slowly on gelatin without liquefaction, and readily oit
glycerin agar as a creamy layer. On potato the growth,
which is apparent in three to four days, at first has the
appearance of drops of honey, but later on deepens in
colour and becomes thicker, and eventually darker, till
it approaches a chocolate colour. The potato itself
remains unstained.

Staining Characters.— B. mallei is Gram-negative, is
not acid-fast and does not readily stain with ordinary
dyes. Smears of glanders pus or material are best
stained, according to McFadyean, with methylene blue,
and then treated with 4 to 5 per cent, acetic acid for a few
seconds, which decolorises the nuclear detritus, but still

148 AIDS TO BACTERIOLOGY

leaves the bacilli well stained. In sections from the edge
of an ulcer, glanders bacilli are few in number and difficult
to recognise.

Pathogenesis. — The disease is communicable to man,
the horse, mule, ass, sheep, goat, field-mouse, and guinea-
pig. Cattle are entirely immune, and white mice and
rabbits partially so. Nocard considers the path of en-
trance to be often through the alimentary canal. This
cannot often be the case with human infections. In man
glanders occurs generally through the infective discharge
from a diseased horse coming into some traumatic injury,
and is a very serious affection. In the horse there are
a persistent nasal discharge, ulcers on the septum nasi,
and usually an enlarged submaxillary gland. In the
horse, when the disease affects the skin on the insides of
the legs it is popularly known as ' farcy,' but lesions are
invariably found in the lungs (McFadyean). The swellings
of superficial lymphatics and glands are known as ' Farcy
buds.' Suppuration usually follows.

The discharge, either from the nostrils or from ulcers
or pus, contains comparatively few bacilli, so that it is
not easy to demonstrate the bacillus by staining.

Straus's method of obtaining a pure culture consists in
the injection of the suspected discharge into the abdominal
cavity of an adult male guinea-pig. If B. mallei is present
the scrotum will be red and shining after three days,
and the testicles much enlarged and caseous, and the
caseous material will contain the bacillus in pure culture.
A similar orchitis may, however, be induced by other
organisms, or a fallacious result be obtained through the
animal not developing orchitis at all, or else dying
from general peritonitis before orchitis has time to
develop.

Addison and Hett give the incubation period for man
as from a few hours to a year, most generally four to seven
days. They also emphasise the necessity for making
the injection for Straus's reaction with a culture grown
on potato.

Mallein. — The organism is grown in glycerin broth
for about six weeks; the culture is sterilised by heat,
filtered through porous porcelain; the filtrate when
concentrated constitutes mallem. If about 1 e.c. of
maUcm be injected into a healthy animal, nothing, or

THE GLANDERS BACILLUS 149

only a slight febrile reaction, occurs, in the horse not
exceeding about 102°, the normal being about 100°; but
if glandered ever so little, the temperature runs up to
103° or even 106° in eight to sixteen hours. At the seat
of inoculation a large swelling appears, and any local
lesions, if present, become much enlarged. This swelling
is of more importance diagnostically than the rise of
temperature.

It was discovered at the Wellcome Research Labora-
tories that many non-glandered horses, if immunised
against other bacterial products such as diphtheria toxin,
react to mallein, but the local swelling rapidly disappears
and the rise in temperature persists for a shorter period.
With this exception, the mallein test as a diagnostic
agent is practically infallible. It seems to act but
feebly as a curative agent, although a few cases of ap-
parent cure after its use have been reported.

The Glanders and Farcy Order, 1894, sec. 17, compels
seizure and total destruction of every part of an animal
that had glanders at time of death.

Epidemic Abortion in Cattle.

Bang described a very small non-motile, oval or rod-
shaped, Gram-negative bacillus, found in the exudate
betAveen the foetal membranes and the uterine mucous
membrane, and also in the stomach and the blood of the
foetus. The bacillus was sometimes lying singly, but in
many cases was markedly clumped. This clumping is
attributed to the agglutinating action of the animals'
serum, and is especially noticeable when the bacillus is
grown on serum media. Bang described the bacillus as
growing in a shake culture from \ to 2 centimetres below
the surface only, but McFadyean's cultures grew best
either on or just underneath the surface. The bacillus
grew on all the ordinary media, the agar-gelatin serum
medium being the most favourable. On agar gro\vth
takes ten days or more to appear. On potato the growth
closely resembles that of glanders. The organism only
grows between 30° and 37° C. The thermal death-point
is 00° C.

A Departmental Committee appointed in 1905 issued
a Report in 1909, in which Sir John McFadyean and
Mr. Stewart Stockman found that, although vaginal

150 AIDS TO BACTERIOLOGY

infection may sometimes cause the disease, food contain-
ing virulent material is a very important factor in its
dissemination. Ewes, goats, bitches, and guinea-pigs can
be infected experimentally.

Non-pregnant cattle have been immunised by means
of large doses of the living cultures of the abortion bacillus,
and treatment of infected cattle by vaccines gave very
promising results. Stockman thinks abortions can be
reduced from the 30 per cent, that sometimes occur to
6 or 7 per cent, by prophylactic inoculation.

CHAPTER XIII
THE SPIRILLA

The Spirillum of Asiatic Cholera.

Morphology. — Spirillum cholerce Asiatics (Koch's ' comma'
bacillus), as it appears in the excreta, is a curved rod
('vibrio'), 2 //, by 0-3 JLL. On cultivation, especially in
liquid media, S-shaped and spirillar forms develop.
It is actively motile, with a single flagellum at one end
only, exceptionally more. No spores are formed. Desic-
cation and sunlight are rapidly fatal, and its thermal
death-point is about 50° C. The organism is Gram-
negative, but is readily stained with anilin dyes, especially
with dilute carbol-fuchsin.

Cultural Characters. — The spirillum grows readily on
most media. An alkaline reaction is essential, develop-
ment being hindered by small amounts of acid. Aerobic
conditions allow a much better growth than anaerobic
ones, and its resistance to disinfectants increases as a
saprophytic habit develops.

It slowly liquefies gelatin, giving in a stab culture in
forty-eight hours a bubble of liquefaction at the top of
the stab only. While growing on most media at 22° C.,
on potato there is no perceptible growth at this tempera-
ture. On potato at 37° C. there is a slow, light greyish-
brown growth.

The rapid formation of indole is very characteristic of
Koch's comma. It gives a creamy growth on agar and

THE SPIRILLA 151

a general turbidity, delicate pellicle and sulphuretted
hydrogen in broth cultures.

Bacteriological Diagnosis. — It is frequently possible to
report positively at once as to the nature of the disease
on the microscopical examination of one of the rice-like
flakes, whether from the contents of the ileum or in
a living patient from the stool. A minute fragment of
one of the flakes is suspended in sterile salt solution
(0-7 per cent.) and examined in a hanging drop, when
the spirillum of Koch is recognised by its characteristic
screw-like movement. If a portion of a flake be crushed
carefully between two cover-glasses, which are then drawn
apart and stained, the organisms lie with their long axes
in the same direction, and present the ' fish-in-stream '
appearance. (Isolated vibrios may be found in normal
dejecta, so no significance can be attached thereto.)

Such appearances are, however, only to be found in
perhaps half the cases, and it is generally necessary to
perform the following cultural experiments: A flake or
two are washed in two or three rinses of sterile salt
solution, and then broken up and thoroughly emulsified in
a little salt solution. Several gelatin tubes are melted,
and inoculated with loopfuls of this suspension, and
poured into plates, while at the same time six to twelve
flasks containing sterilised Dunham solution are similarly
inoculated. (This solution consists of peptone 1 per cent.,
salt 1 per cent., in distilled water.) The flasks should be
conical Erlenmeyer ones of about 120 c.c. capacity, and
containing 40 to 50 c.c. of the Dunham's solution. After
inoculation the flasks are capped with a loose cap of
sterile filter-paper and incubated at 37° C. The gelatin
plates are examined after twenty-four hours' incubation at
22° C. The colonies are then macroscopic, and appear
microscopically as granular discs, with faintly sinuous
margins. After forty-eight hours there are small funnel-
shaped depressions in the gelatin, having yellowish points
at their apex, while the gelatin begins to liquefy. Frag-
ments of colonies having these characters are picked out
with a platinum needle for microscopic examination, both
in the hanging-drop culture and in cover-glass specimens.
The Dunham solution flasks are incubated for twelve hours
only, and are then probably cloudy from the rapid growth
of the organisms, and the production of indole and

i52 AIDS TO BACTERIOLOGY

nitrites has proceeded sufficiently far to cause the appear-
ance of the indole reaction (a distinct rose-madder
tint) on the addition of a few drops of pure sulphuric
acid.

Many other organisms besides Koch's comma also
produce indole and nitrites in sufficient quantities to
yield the indole reaction, but not in this time (twelve
hours — it can often be obtained in five or six). The
commas tend to form a delicate film on the surface of
the medium. This should be examined, care being taken
not to shake the flasks, so that the film may be preserved.
In cases of true cholera the organism frequently cannot
be demonstrated in the stool when the patient is on the
way to recovery, so that the inability to demonstrate the
organism in cases three or four days from the commence-
ment of the attack must not be taken as evidence that
the disease was not true cholera.

An agglutination reaction may be performed with the
isolated vibrio. (Agglutination reactions with the patient's
serum on a pure strain of the vibrio are said to be of
doubtful value.)

Major Glen Liston (Kept. Bombay Bact. Lab., 1913)
says the distinction between cholera and ' cholera-like '
vibrios is based solely on their behaviour with a standard
agglutinating cholera serum, as no morphological or
cultural differentiation was discovered that could dis-
tinguish in a trustworthy way between the cholera and
the ' cholera-like ' strains.

Hccmolysis Test. — True cholera vibrios do not hacmolyse
apparently even after prolonged contact, while several
similar vibrios are ha3molytic. An emulsion of a young
agar culture in 5 c.c. of normal salt solution is. made,
O'l c.c. of which emulsion is mixed with 0-9 c.c. of normal
salt solution, and then a drop of a suspension of well-
washed rabbit corpuscles added. Haemolysis is gener-
ally apparent in twe e to twenty-four hours if the
organism produces a haemolysin, but some of the cholera-
like vibrios described by Ruffer require forty-eight
hours.

Saturation Test. — The saturation of a specific agglu-
tinating scrum with the homologous organism removes
most or all of the specific agglutinin. (a) Ten loopfuls of
a young agar culture of the isolated vibrio are mixed with

THE SPIRILLA 153

10 c.c. of a 5 per cent, solution of a highly agglutinating
serum. After standing for two or three hours at room-
temperature, the mixture is centrifuged and the clear
supernatant fluid decanted. (6) The agglutinating power
of the latter on the organism with which the serum was
prepared is ascertained. If the organism treated in (a) is
homologous with the organism with which the agglutin-
ating serum was prepared, the decanted fluid will have lost
most, or a considerable proportion, of its agglutinating
power for the latter (Hewlett).

Fixation Test.— (See p. 20).

Pfeiffer's reaction (p. 22) is particularly valuable in the
diagnosis of cholera.

Pathogenesis. — In Asiatic cholera urine is suppressed,
and the copious and watery stools have the characteristic
rice-water appearance due to flakes of detached epithelium.
The most prominent symptoms are subnormal tempera-
ture, distension of the abdomen, and ultimately profound
collapse.

The organisms are practically confined to the intestine,
and are not to be found in other organs nor in the
blood. Death may occur in twelve or even six hours
after infection, or three hours after the first symptoms
are noticed. The incubation period rarely exceeds two or
three days.

Unless some heroic measure, such as a device to
neutralise the acidity of the gastric juice, be employed,
experiments on animals fail to produce cholera. Intra-
peritoneal injections into guinea-pigs, though fatal, do
not produce cholera. Young suckling rabbits are an
exception, ingestion of the organism producing choleraic
diarrhoea.

Occurrence and Distribution. — The disease is endemic
in many parts of India, particularly the delta of the
Ganges. In other countries its course may be traced
along the ordinary lines of traffic, showing that it is
carried by travellers. There has been no epidemic of
cholera in the British Isles during recent years.

Cholera spreads most rapidly when the earth tempera-
ture is high; this happens chiefly when the ground-water
is low, which is in accord with the observation of Petten-
kofer that increase in cholera is often preceded by a fall
in the ground -water.

154 AIDS TO BACTERIOLOGY

Transmission of the disease may take place by means
of water (as at Hamburg), by milk (rare), uncooked
vegetables, or by fomites. The infection is confined to
the bowel and stomach discharges, and is not found in
the urine.

During outbreaks of cholera a number of persons show-
ing only slight or no symptoms get infected at the same
time as those who fall ill, and harbour the organism for
some time. Haffkine has proved that such * vibrio-
carriers ' can spread the disease.

As the vibrio offers little resistance to drying, it seems
unlikely to be disseminated by dust. At the same time,
it is readily capable of a saprophytic existence. Un-
cultivated vibrios die more speedily than cultivated
ones and the duration of their life is shorter in the hot
season than in the cold (Greig).

In some waters the cholera vibrio will live for consider-
able periods (see p. 225). Charcoal filters, once infected,
have been known to continuously pollute water otherwise
pure for many weeks, and cause grave epidemics.

The best-known instance of milk infection is that of the
outbreak of cholera in the Gaya Gaol, in which it was
surmised that flies carried the infection.

Under ordinary conditions, little or no toxin is found in
cultures, but a powerful toxin (presumed to be an endo-
toxin) has been obtained by disintegration of the vibrionic
structure.

Vaccine. — Haffkine's vaccine is prophylactic, not
curative. An attenuated vaccine (prepared from cholera
spirilla grown on agar at 38° or 39° C., over the surface
of which a current of moist sterile air is passed) is injected.
Five days later a stronger (' exalted ') vaccine is ad-
ministered, which does not exert its full power of immuni-
sation till five days after inoculation. The virulence of
the latter is obtained by passage through the peritoneal
cavities of guinea-pigs. Both inoculations are made
subcutaneously. Neither vaccine is sterilised or filtered,
both living bacilli and their products being injected.
The protection is of a very decided character.

Treatment by antisera has met with little success.
The cholera immune serum is bacteriolytic, not antitoxic.

THE SPIRILLA 155

Cholera-like Vibrios.

Finkler-Prior Spirillum. — ^Etiological significance un-
certain. Is likely to be confounded with Koch's comma.
It is occasionally found in the stools in English cholera
(cholera nostras), cholera infantum, etc. The vibrio is
rather thicker and longer than the Koch's comma, and
has the following cultural characters: In gelatin stab
culture liquefaction is rapid, extending in shape of a
funnel to the bottom of the stab within forty-eight hours.
On potato at 22° C. there is a slightly yellowish growth,
and at 37° C. a rapid, slimy, yellow growth. Grown in
peptone-water, a feeble indole reaction may be obtained
after three days. A vibrio having very similar characters
has been found in decaying teeth (Miller's Spirillum).

From cases of true cholera spirilla have been cultivated
that closely resemble Koch's comma, yet differ slightly
in cultural and other characters. Therefore ' Koch's
comma ' may be taken to be an organism with variable
attributes, or more probably a group of organisms all
capable of producing cholera, but not all exactly similar
in character.

Sanarelli has isolated no less than thirty-two vibrios
from water, morphologically distinct from each other, all
of which gave a distinct indole reaction. Four of these
organisms he found to be extremely pathogenic to
animals, producing symptoms in guinea-pigs indistin-
guishable from those given by the true cholera spirillum.

Spirillum Metchnikovi. — This organism, which is patho-
genic for fowls, pigeons, and guinea-pigs, but non-
pathogenic for mice, closely resembles the cholera spiril-
lum in morphological and cultural characters, even giving
the indole reaction on the addition of sulphuric acid
alone. The growth on gelatin affords a means of distinc-
tion from the cholera spirillum. On gelatin plates small
white colonies form, which produce cup-like depressions
on liquefaction in two or three days. In a gelatin stab
liquefaction is more rapid than with the cholera spirillum,
and takes place in the form of a funnel-like tube. It is
more pathogenic for guinea-pigs than the cholera spirillum,
and can be distinguished from the latter by the readiness
with which pigeons succumb to septicaemia after inocula-
tion, and by fatal results from feeding to fowls.

156 AIDS TO BACTERIOLOGY

Spirillum Tyrogenum.

(Syn., Deneke's cheese bacillus.) Forms no indole, is
but feebly pathogenic for laboratory animals, and does
not develop readily at blood-heat.

Spirillum Rubruin.

This organism, which is non-pathogenic, is found in
water and garden earth. In broth long threads, with up
to fifty twists, are formed. The shorter spirals are very
motile. Colonies on gelatin out of contact with air and
those on potato are red, and a red sediment is produced
in broth.

Other spiral organisms, sometimes classed as Spirilla,
are more correctly placed among the Protozoa, and will
be dealt with later.

CHAPTER XIV
THE TRICHOMYCETES

THE Trichomycetes are thread-forming organisms and
form a group intermediate between the Schizomycetes
and the Hyphomycetes. Much confusion exists as to the
terminology of the class, the same term being used in
different senses by authors. The following classification
is convenient:

Leptothrix: No branching.

Cladothrix: ' False ' branching.

Nocardia, or Streptothrix : True branching, with forma-
tion of rounded bodies, regarded as spores.

Aetinomycosis.

Morphology. — Actinomyces, or ray-fungus, is a strepto-
thrix occurring in three types in the colonies as they
grow in the tissues — namely, filaments, cocci, and clubs.
The filaments (seen better in cultures) are thin, measuring
about 0-5 /* across, and are often of great length. The
central protoplasm is enclosed in a sheath ; the filaments,
particularly in the centre of a colony, interlace, forming
a network. In oklor filaments the protoplasm may be
segmented, giving rise to a streptococcal appearance.
These bodies are regarded as gonidia. The clubs are

THE TRICHOMYCETES 157

involution forms, perhaps produced by resistance of the
tissues. The filaments are sometimes ' acid-fast.'

When pus, sections, or teased-up specimens from human
sources are stained by Gram's method, the filaments and
gonidia are Gram-positive, while the clubs situated around
the periphery and showing a radiating structure do not
usually stain Jby Gram's method. Carbol-fuchsin and
picric acid may be used, when the fungus stains red and
the tissue yellow. In the bovine organism the clubs
stain well by Gram and are well marked, clubs of the
hominis variety being stunted. The prominent central
filamentous network of the hominis variety is generally
replaced by a mass of debris in the bovine type.

Cultural Characters. — In artificial media clubs are not
found. The organism grows well, and for almost an
unlimited time, on glycerin-agar and potato. Fully
developed cultures on potato are peculiar and character-
istic: a dull raised and wrinkled growth, of considerable
thickness, of a bright sulphur-yellow or light chocolate
colour, somewhat similar to the lichen commonly seen
on apple-trees.

Occurrence and Distribution. — Actinomyces grows on
grasses and cereals, particularly on barley, and especially
on damp rich soils. Infection is due to the piercing of
a mucous surface by a portion of a cereal bearing the
fungus; possibly the fungus may also gain access to the
system by inspiration.

It does not seem likely that cattle are infected by
contact, and although some human cases have occurred
after eating grains of barley, Wright is of opinion that
actinomyces is normally present in the mouth, and that
irritation caused by the foreign particle merely facilitates
invasion of the tissues. The change of teeth in young
animals is regarded as a period when risk of infection is
greatest. Scirrhous cord, the fibrous tumour found on
the end of the spermatic cord of the ox, is stated by
Stockman to be caused by the entrance of the actinomyces
by the wound of castration.

Pathogenesis. — Cattle are the principal subjects of
the disease, but it also occurs in pigs, sheep, horses,
squirrels, and man. In animals the disease is usually
local and may arise in various parts of the body, the
head and neck, particularly the tongue, being most

1 58 AIDS TO BACTERIOLOGY

common sites. The? disease commences with a swelling
(' wen ') that in due course ulcerates with the discharge
of pus. In the tongue the growth of fibrous tissue makes
it painful, hard, and immobile ('wooden tongue'), the
tongue may protrude and ulcerate at its base. There is
constant dribbling. The lower jaw-bone and the neck
glands are commonly infected, the teeth often falling
out from the former. The disease in man corresponds
pretty closely to that observed in animals, but there is
less tendency to localisation, to the abundant formation of
connective tissue, and to the frequency of calcification.
The tendency of the disease in man is to become chronic,
and it is only by the implication of some vital organ or
by the exhaustion following prolonged suppuration that
the patient succumbs. The disease spreads by continuity,
and no tissue seems able to resist its invasion. Besides
this, secondary embolic foci may occur, perhaps the
commonest seat being the liver.

Griffith (Report to L.G.B., New Series, No. 107, 1915),
after examining a number of actinomycotic ox-tongues,
supports the view that under the term actinomycosis a
number of distinct conditions are included. Most of his
specimens showed an organism probably identical with
the actinobacillus of Lignieres and Spitz.

If a little of the pus be allowed to run gently down the
side of a test-tube, which is then held up to the light, the
small yellow grains which will be visible may be picked
out, placed on a slide, and pressed down with a cover-
glass. It will transmit to the finger a sensation similar to
that of squeezing a drop of solid fat, if the granule be
taken from man; while if from an animal, the granule
may be gritty, from calcareous infiltration. On examining
the slide with a low power, a number of ovoid kidney-
shaped masses are seen, which with a higher power show
the characteristic club-shaped structure.

Mycetoma, or Madura Disease.

The foot is generally attacked, occasionally the hand,
and rarely other parts. Three varieties are seen. The
most common is the white, the black being less frequent,
while the red variety is rare. The varieties are named
after the colour of the granules occurring in the cavities.

The white form is produced by the Streptothrix madur<r,

THE TRICHOMYCETES 150

distinguished from the actinomyces by its pathogenicity
and cultural characters. It is not infective for rabbits,
does not form yellow or black pigment in cultures, and
does not liquefy gelatin. The cause of the black variety
is uncertain, but it is credited to a hyphomycete.

Streptotrichosis.

In addition to actinomycosis and madura disea.se,
other affections in which tubercles and suppuration are
involved are caused by Streptotrichece. The manifesta-
tions of some of these organisms strikingly resemble
and are apt to be mistaken for tuberculosis. Some
species, at any rate, are acid-fast and Gram-positive
when the filaments are young, but older or degenerate
threads are Gram-negative and not acid-fast.

Streptotrichosis may affect the mouth, neck, lungs,
skin, kidneys, conjunctiva, appendix, and peritoneum.
Foulerton (Lancet, 1910, i., 551, 626, and 769) says that
cases show a preponderance of males, and the influence
of occupation and other circumstances involving special
exposure to the possibility of infection from a vegetable
source is very noticeable. Eppinger's streptothrix (St. Ep-
pingeri) belongs to Group II. of the Streptotrichecp, a
class possessing acid-fast properties, and showing more
active pathogenic action than the freely-growing species
of Group I. and the slow-growing species of Group III.
St. Eppingeri produces an infection which may be in-
distinguishable anatomically from a tuberculosis.

The occurrence of branching forms in B. tuberculosis,
sometimes in typical actinomyces form, has led to a
suggestion that it should be placed among the Trichomy-
cetes. To quote Foulerton: ' There can be no reasonable
doubt but that at one stage of its growth the bacillus of
Koch is represented by branching mycelial threads of
the streptothrix type. And this being so, the common
" bacillary " forms represent the persistent rod segments
of a streptothrix.' Streptotrichece are commonly found
on grasses, and among the reputed bacilli which display
acid-fast properties are those having the characteristics
of a streptothrix — Bacillus phlei I. and //. (Timothy-
grass bacilli), and the cow-dung bacillus (' Mistbacillus ')
All three are pathogenic for guinea-pigs but grow readily
on culture media.

i Go AIDS TO BACTERIOLOGY

Leptothrix.

Varieties of Leptothrix are common in the mouth and
slime of the teeth. L. buccalis is a term perhaps compris-
ing several species, some of which may cause dental
caries. The threads have been thought to penetrate
the tissue of the teeth, after the enamel has been acted
upon by the acids generated by bacteria, including the
fermentation of the food.

L. innominata (Miller), commonly found in the deposit
on teeth, does not give the blue granulose reaction when
treated with dilute sulphuric acid and iodine. This
distinguishes the organism from L. buccalis maxima which
gives the blue granulose reaction.

It is an open question whether all the Leptothrices
in the mouth are not either bits of the same or stages in
the life of the same.

Leptothrix epidermidis. — A non-pathogenic sporulating
organism found in variolous crusts and on the skin
between the toes.

Cladothrix Dichotoma.

This organism, which is common in natural waters,
consists of long motionless filaments, sometimes a milli-
metre in length, which may possess pseudo-branches.
The sheaths of the filaments are often coloured yellow,
red, olive-green, or brown by oxide of iron. The
C. diclwtoma withdraws iron from water, and thus fixes
it, often causing obstructions in iron pipes. On gelatin
plates it forms small yellowish dots surrounded by a
brown halo, and a depression due to the slow liquefaction
of the gelatin. On agar it grows at 35° C. as a thick
shining expansion, which adheres so closely to the medium
that it is impossible to remove it without carrying away
some of the agar. The growth has a tendency to form
concentric rings, and sometimes becomes covered with a
greyish efflorescence, which is dry and very brittle. The
agar becomes brown in colour. All the cultures have a
very strong mouldy smell.

Beggiatoa.

Beggiatoa arej found in both fresh and salt water;
in sulphur springs and brewery effluents they are

THE BLASTOMYCETES 161

particularly in evidence. They are sometimes considered
diagnostic of pollution with animal matter, but this is
not invariably true. The growth may be white, grey,
pink, red, or violet. The filaments contain sulphur
particles, seen as highly refracting granules (see p. 6).
They can decompose sodium sulphate in suitable organic
solutions.

Crenothrix Kuhniana.

This is found in water containing organic matter or
iron. It sometimes occurs in such great numbers that
the water is unusable owing to the unpleasant odour
and taste it produces. The organism produces a thick
vegetable mass in the water, either brown or greenish
in colour, frequently imparting a reddish or greenish tint
to the water (see p. 6).

CHAPTER XV
THE BLASTOMYCETES

Blastomycetes, or yeasts, are round or oval unicellular
organisms, consisting of granular ' cytoplasm ' (hyaline
in healthy, granular in old cells), surrounded by a wall
of cellulose. One or more colourless lacunae (vacuoles)
are sometimes seen, particularly in healthy cells. A
nucleus (or what is taken for one) may sometimes be
seen. The salient character of this group is the method
of reproduction by budding (gemmation) : a bud is
extruded from the parent cell, and, after enlarging,
separates. Torulce seem to be restricted to this method
of multiplication, but Saccharomycetes can, in addition,
reproduce by spore formation. Minute spherical bodies
occur within the vacuoles in active movement, but their
function is not known. In the absence of ample suit-
able nourishment, the contents of the cells become homo-
geneous, and then two, four, or, in the case of Schizo-
saccharomyces octosporus, eight, shining spots are seen,
surrounded with a thick membrane. In the course of
time these new cells (ascospores) become liberated by
dissolution of the mother cell. On introducing these
spores, which are 4 IJL to 5 JLI in diameter, into a saccharine

11

1 62 AIDS TO BACTERIOLOGY

liquid, they form mature cells, which multiply, as usual,
by gemmation. Dextrose, laevulose, mannose, and (some-
times) galactose, are the only hexoses fermentable by
the yeast cell. Maltose, cane-sugar, malto-dextrin, and
lactose are fermented, but different species vary in
fermentation ability.

Saccharomyces Cerevisise. — Of the typical brewery
yeast there are two main varieties, which are intercon-
vertible: the typical English brewery yeast — a ' top '
fermentation variety — and a yeast of the Continental
lager beer brewer — a ' bottom ' fermentation. The ' top '
and ' bottom ' varieties are known also as ' high ' and
' low ' yeasts respectively, as they ferment at high and
low temperatures respectively. The rounded or slightly
ellipsoidal cells are from 8 ^ to 9 //, in diameter, and occur
both singly and in short chains. Spores occur three or
four together in a mother cell, each being 4 /n to 5 /,<, in
diameter.

Fermentation with ' low ' yeasts, in the manufacture of
lager beer, takes place at 5° to 10° C., at which tem-
perature other forms of yeast are inert. The process
requires about fourteen days.

' High '-fermentation yeast consists of cells which are
rather larger and more globular, and have a greater
tendency to form branched chains than the ' low ' yeasts.
The temperature best suited for this fermentation is
between 15° and 18° C. The reaction in the fermenting
vats is much more violent than is the case with the
' low ' yeasts. The rapid emission of carbonic acid
brings the cells to the surface.

A soluble ferment (invertase) is present, which can be
extracted by precipitating with chloroform, and which
converts sucrose into invert sugar. Another enzyme
occurs within the cells (maltase), which is capable of
converting maltose into glucose.

Buchner, by grinding up yeast with kieselguhr and
filtering under pressure, obtained a liquid which caused
the evolution of carbonic acid and formation of alcohol
in solutions of glucose. This he termed zymase, and
regarded it as a solution of an intracellular ferment.
Von Lebedeff prepares zymase by macerating 1 part
of dried yeast with 2-5 — 3 parts of water, and filtering
through paper after being allowed to remain overnight.

THE BLASTOMYCETES 163

He claims for the solution greater activity and stability
than that prepared by the usual method.

Zymase shows extreme instability, apparently due to a
powerful proteolytic enzyme (endotryptase or endotrypsin),
always present in yeast- juice, which, when digesting the
coagulable protein, doubtless digests the zymase at the
same time. The activity of zymase seems to be absolutely
dependent on a co-enzyme, that withstands the tempera-
ture of 100° C. (thermostable) and is dialysable, but of
which the constitution and function are unknown.
Harden has shown the presence of phosphates is also
necessary.

The juice extracted from yeast also contains rennin,
a glycogen-hydrolysing enzyme, and a reducing enzyme
which can liberate sulphuretted hydrogen from sodium
thiosulphate and decolorise methylene blue. The yeast
cell possesses special ferments capable of destroying
zymase and its co-enzyme, also an anti-enzyme which
prevents such destruction. See also Harden' s ' Alcoholic
Fermentation ' (Messrs. Longmans' * Monographs of
Biochemistry ').

8. cerevisice ferments saccharose, maltose, and dextrose.
When the alcohol produced reaches 12 per cent., growth
stops, and with 14 per cent, fermentation ceases altogether.

Saccharomyces Ellipsoideus. — The yeasts classed under
this name are bottom fermentation forms, and are usually
rounded or ellipsoidal in shape, but sometimes assume
a sausage form. The cells average about 6 // in length,
are single or united in little branching chains. Two to
four spores are found in a mother cell, each 3 fj, to 3-5 /u,
in diameter (twenty- one to twenty- seven hours at 25° C.).

S. ellipsoideus I. is found on ripe grapes, and plays an
important part in the fermentation of grape- juice.

S. ellipsoideus II. is a dangerous ' disease ' yeast,
producing yeast turbidity, in bottom fermentation
breweries.

Saccharomyces Pastorianus. — This yeast — of which
three varieties, known as L, II., and III., have been
isolated by Hansen — is very polymorphic in shape. The
cells are oval or sausage-shaped, and also occur in elon-
gated, ellipsoidal, or pear-shaped forms. Two to four
spores are usually found in a mother cell (twenty-five to
twenty-eight hours at 25° C.). They take a part in many

i64 AIDS TO BACTERIOLOGY

spontaneous fermentations, and in the vinous fermenta-
tion usually succeed 8. apiculatus. They are classed as
' wild ' yeasts, the spores of which frequently occur in
the atmosphere of breweries.

8. pastorianus /. is a ' bottom ' form, and causes a
disagreeable smell and a strong, bitter taste in beer.

& pastorianus II. is a feeble ' top ' form, and of no
particular importance.

S. pastorianus III. is a top form, and is a dangerous
disease yeast, causing yeast turbidity.

Saccharomyces Apiculatus. — This very common yeast
can scarcely be termed a true saccharomyces, as no spore
formation has yet been demonstrated. It occurs in
wine fermentations and spontaneously fermented beer;
on sweet succulent fruits, such as grapes, cherries, plums,
gooseberries, etc. The cells, which are 6 /a to 8 ^ long
and 2 /j, to 3jbi broad, have a most characteristic citron
shap3 (hence the name), from the prominences at the end
of which the budding takes place. It invariably appears
at the onset of the vinous fermentation of grape-juice,
but soon gives way to the S. ellipsoideus and 8. pastorianus.
It is a bottom yeast form, only gives rise to a very feeble
alcoholic fermentation, and is incapable of fermenting
cane-sugar and maltose.

Saccharomyces Mycoderma (Myeoderma Cerevisise). —
The cells are oval, elliptical, or cylindrical, 6^ to 1 ja long
and 2/z to 3/bi thick, united in freely-branching chains. It
forms the skin, or ' mould,' on the surface of fermented
liquids, without, however, exciting fermentation. When
forced to grow submerged in a saccharine liquid, it gives
rise to a small quantity of alcohol, but development is
feeble. In liquids already containing alcohol it produces
ethyl acetate.

Saccharomyces Exiguus. — Conical or top-shaped cells,
5 fju long, and reaching 2*5 //. in thickness, in slightly
branching colonies. There are two or three spores in
a row in each mother cell, but spore formation is scanty,
and film formation does not occur. It is incapable of
fermenting maltose. This yeast is often present in the
after-fermentation of beer.

Saccharomyces Marxianus.— A yeast found on grapes.
The cells are small, oval, or sausage-shaped, and form more
or less kidney-shaped spores, the optimum development

THE BLASTOMYCETES 165

of which is between 22° and 25° C. It only produces
1-1-3 volumes per cent, of alcohol.

Saccharomyces Anomalus. — Found in impure brewery
yeast, on green malt, and on fruit. It is peculiar in that
the spores are hemispherical, with a projecting rim, and
are like a bowler hat. It forms but little alcohol.

Saccharomyces Pyriformis. — A yeast which, with a
symbiotic bacterium — B. vermiforme — was found to cause
the change in a solution of sugar and ginger produced by
the addition of the ' ginger- beer ' plant (Marshall Ward).

Schizosaccharomyces Octosporus. — Found on currants
and raisins. In the genus Schizosaccharomyces of Lindner
the cells are like yeast cells, but multiply by fission through
a septum formed across the middle of the cell, and not
by budding. Ascospores are also developed, in this
particular species being usually eight in number.

Examination of Yeasts.

Hanson's scheme involves the isolation of a pure
culture, and the observation of the time taken at various
temperatures to form ascospores and ' films.'

1. Microscopical Appearance. — The growths, after grow-
ing in sterilised wort for twenty-four hours, are examined.
When S. cerevisue and 8. pastorianus are mixed or other
varieties are present, little is to be learnt from a direct
microscopical examination.

2. Formation of Ascospores. — The formation of spores
in the saccharomycetes is regulated by the following
conditions: (a) The cells must be placed on a moist
surface, and have plenty of air. (6) Only young and
vigorous cells can exercise this function, (c) The most
favourable temperature for most species is about 25° C.
(d) A few saccharomycetes form spores when present in
fermenting nutrient liquids.

A small portion of a young and vigorous growth is
transferred to a moist gypsum block, prepared as follows:
To well- baked plaster of Paris distilled water is added
until the plaster is nearly liquid; this is poured into a
small mould of metal or paper. The blocks are allowed
to set, dried, and sterilised. They are then laid in a shallow
tray containing a little sterile water, the whole arrange-
ment being kept well covered by a bell- jar.

Two sets of cultures on plaster blocks are made, one

166 AIDS TO BACTERIOLOGY

set being incubated at 25° C., the other at 15° C. In a
pure brewery yeast no ascospores will be detected under
thirty hours and seventy-two hours respectively.

Hanson found that the formation of spores takes place
slowly at low temperatures, more rapidly as the tempera-
ture is raised to a certain point; when this point is passed
their development is again retarded, until finally a
temperature is reached at which it ceases altogether.
After a time, which varies with the species, roundish
plasma particles appear in the cells. These are the first
indications of spores. They become surrounded by a
wall, seen more or less distinctly in different species.
The spores may expand to such an extent that the pres-
sure which they exert on each other whilst still enclosed
in the mother cell develops the so-called partition walls.
Later complete union of the walls may take place, so
that a true partition wall results; the cell then becomes
a compound cell, divided into several chambers. During
germination the spores swell, the wall of the mother cell,
originally thick and elastic, stretches thinner, finally
ruptures, and then remains as a loose shrivelled skin,
partially covering the spores, or else dissolves.

3. The Formation of Films. — With the pure cultivation,
drop cultures are made into 4-ounce flasks, half filled
with sterile wort, and protected from falling particles
by a well-fitting cap. The films first appear as small
opaque points, which gradually increase in size and then
run together, forming irregular floating patches. As soon
as the film becomes apparent to the naked eye it is
examined. The film at length overspreads the whole
surface of the liquid, and becomes adherent to the walls
of the flask. Perfect rest of the liquid is a very necessary
condition for the formation of films.

Hansen's Pure Culture Method. — Bdttcher's moist chamber
consists of a short glass cylinder or ring, cemented to
the upper surface of a glass slip. A cover-glass rests on
top of the ring. A small drop of water is placed at the
bottom of the chamber. A mixture of yeast growth
and sterile water is mixed with a suitable amount of
wort gelatin, which has been previously liquefied at 30°
to 35° C. A very small quantity of the mixture is spread
in a thin layer on the sterile cover-glass and left under
a sterile bell -jar to set. The edge of the ring of the

THE BLASTOMYCETES 167

Bottcher chamber is -painted with sterile melted vaseline,
and on this the gelatin- coated cover-glass, infected
surface downwards, is placed and pressed down, so that
the chamber is completely sealed. The edge is painted
with a melted mixture of 1 part of wax and 2 parts of
vaseline, to prevent the cover-glass moving. The prepara-
tion is examined, the position of isolated cells is marked,
and the chamber incubated at room -temperature till the
colonies have become sufficiently large to be transferred
to liquid media. With a Bottcher chamber with a ring
of 30 millimetres diameter, twenty to thirty cells is a
convenient number to have in the gelatin mixture. The
method needs some practice to obtain results.

Torulse (vide supra). — The Torulce produce little or no
alcohol in saccharine liquids. Saccharomyces rosaceus,
niger, and albus are met with in air, and form coral pink,
sooty black, and white growths respectively on potato
and bread.

Yeasts in Sour Milk.— Herschell and Emerson agree
with the use of a yeast in the preparation of Bulgarian
soured milk for therapeutic purposes, not only to improve
the flavour and to attack the milk-sugar after lactic
acid has been formed, but also to inhibit the overgrowth
of pathogenic and putrefactive bacteria. It is not un-
usual for the yeast to get the upper hand, and carry on
the fermentation much too far.

Pathogenic Yeasts.— Blastomycetic dermatitis (blasto-
mycosis) is sometimes seen in man. The disease may
become generalised, and sometimes ends fatally. Yeasts
have also been obtained from tumours, and some con-
nection between yeasts and malignant tumours has been
suggested. An outbreak of sore throat at Lincoln was
thought by Klein to be caused by a yeast found in a
throat. The yeast produced swelling of the throat,
fauces, and larynx in rabbits.

CHAPTER XVI
THE HYPHOMYCETES

THE moulds or Hyphomycetes are multicellular organisms
composed of filaments (hyphae), which, when interlacing,
form a mycelium. A mycelium may form a hard woody

168 A IDS TO BACTERIOLOGY

mass (sclerotium). Lower members of the group have
both asexual and sexual methods of reproduction, while
among the high fungi sexual development is less evident.
According to the form of the seed-bearing organ, the
moulds are divided into:

1. Mucor inse. — The end of an aerial hypha swells into
a knob, known as a columella, around which a spherical
seed-capsule or sporangium forms. When ripe, the spores
burst the enclosing membrane, and thus become free.

2. Aspergillinse (Knob -Moulds). — The heads of aerial
hyphae are studded with spore-carriers, or sterigmata.
Each sterigma bears a chain of spores (gonidia).

3. Pemcilliacese. — Aerial hyphse (goniodophores) branch
at the apex forming basidia, on the terminals of which
are the sterigmata, from which the conidia, or spores,
are separated in the form of chains.

The most important members of these groups are the
undermentioned :

Mucor mucedo. — A mould frequently seen on food-
stuffs, particularly stale moist bread, and on animal
excreta. It grows well on an acid medium, forming a
white fur. It is not pathogenic.

Mucor rhizopodiformis forms a similar growth to the
above. A culture on bread has an aromatic odour.

Mucor corymbifer forms a dense white fur on bread,
resembling cotton-wool.

Mucor ramosus grows on bread and potato as a white
fur, which soon changes to greyish -brown.

These last three mucors are pathogenic. Intravenous
injection of their spores causes fatal disease in rabbits.

Aspergillus niger, A. albus, and A. glaucus grow upon
bread, candied fruit, etc. The last two organisms grow
best at blood-heat, when they soon overgrow the nutrient
medium.

Aspergillus flavescens and A. fumigatus. — The former
is distinguished by its well-marked fructifications and the
greenish colour of its culture, the latter by its fine fructifi-
cations and ash-grey fur. On gelatin plates the filaments
grow rapidly into the medium, causing liquefaction.
Both organisms grow at blood-heat. Both are pathogenic,
growing in various parts of the body — particularly the
ear, producing otomycosis. They have been also found
growing in the lungs and on the nasal mucous membrane.

THE HYPHOMYCETES 169

The spores cause the death of rabbits on intravenous
injection. Most cases of aspergillosis occur among bird
fanciers, especially those handling pigeons. In the birds
the disease takes a pulmonary form, and A. fumigatus
is most frequently found.

Penicillium glaucum, the commonest mould, is seen as
a pale bluish-green fur on jam and damp surfaces. It
produces a peculiar musty odour. The mycelium consists
of horizontally arranged straight or slightly undulating
jointed filaments, from which the spore-bearing hyphae
stand vertically up, dividing at their upper ends into
forks (basidia), from which fine processes branch off
(sterigmata] in the shape of a hair pencil, and are segmented
at their ends into rows of fine globular spores or conidia.
The mould grows well on bread-pap in the form of a
fur, white at first, but afterwards green. On gelatin
plates fine threads diverging from a point, and not giving
rise to sharply defined colonies, radiate over a consider-
able surface. The spore-bearing hyphae which rise above
the level of the gelatin are put in motion by air currents,
when the spores disperse. The earliest formation of
spores takes place in the centre of the colonies, and is
indicated by a green colour. The gelatin is liquefied.

The volatile arsenical compound, which is given off by
arsenical wall-papers to cause poisoning in the tenants of
the room, is almost certainly liberated from its combination
in the pigment by micro-organisms thriving in the paste.
Penicillium, Mucor, and Aspergillus are found to be
capable of so acting, most marked powers being given by
P. brevicaule. Gosio has elaborated a test whereby
P. brevicaule is used for the detection of minute amounts
of arsenic (see Glaister's ' Arsenic Gas Poisoning ').

Brown mould is brownish-yellow in colour, and is dis-
tinguished from P. glaucum, which it otherwise resembles,
by its closely-felted mycelium, the hyphae being scanty,
ramified, and segmented. It grows on gelatin, which it
quickly liquefies. According to Trelease, this mould is
identical witli the Cladothrix dichotoma (</.?;.).

Microscopical Examination of Moulds. — Moulds cannot
be easily moistened with water, owing to the presence on
their surface of a very thin layer of fat. Alcohol to which
a little ammonia has been added removes Ihe fat, after
which they can be mounted in glycerin or glycerin and

I yo AIDS TO BACTERIOLOGY

water. Loffler's methylene blue stains the filaments of
the mycelium and hyphaB, the spores remaining unstained.
For permanent preparations moulds are mounted in
glycerin jelly, the cover-glass being ringed with varnish.

Culture of Moulds. — The moulds grow on nutrient
gelatin and agar, on potato and bread-paste. The best
media are glucose agaf, wort gelatin, and wort agar.

Fermentation by Moulds. — Some species of Mucor,
when immersed in a fermentable saccharine liquid, such
as wort, very quickly change their appearance: the
submerged hyphse swell irregularly, and a large number
of transverse septa appear, which divide them into barrel-
shaped or irregular cells, filled with highly refractive
plasma; these cells seem to multiply by budding, like
true yeast. If, then, the above-mentioned cells are
brought to the surface of the liquid, or otherwise under
aerobic conditions, they are again able to develop the
typical mould form. The most active fermentative power
is possessed by Mucor erectus, which in ordinary beer-
wort can be made to yield up to 8 per cent, by volume of
alcohol. Another form, the Mucor (amylomyces] Eouxii,
obtained from a Chinese rice fermentation, is used com-
mercially for the conversion of starch into alcohol.

OVdiaeese.

The Oidiacece are generally classed among the Hypho-
mycetes. No special spore- bearing organs are present,
but the hyphse bud at the extremities, forming spores.

Oidium lactis, found in sour milk and butter as a white
fur, grows on gelatin without liquefaction, producing a
smell of sour milk. On agar it grows in the form of little
stars, which then overgrow the medium. In a stab
culture the fibres of the mycelia permeate the medium.
O. lactis grows very readily in milk, which it does not
change in any special way. It is not pathogenic to man
or animals.

Cndium albicans grows on the mucous membrane of
the mouth and pharynx of children, producing thrush. If
one of the white patches be removed and teased up, the
filaments and yeast-like cells can be distinguished. It
does not flourish in an alkaline medium, but can be grown
on milk, bread, gelatin with liquefaction, agar, and potato.
On all these media it produces a very copious growth,

THE HYPHOMYCETES 171

showing yeast-like cells, the growth on potato being a
whitish, thick, raised patch. After an intravenous
injection rabbits die in about thirty-six hours, with their
viscera full of the mycelium.

Gibbons says sometimes it causes pruritus and Bahr
(Medical Press, October 7, 1914) describes it or a similar
organism as occurring in the tongue lesions of sprue.

Oidium carnis (see p. 209).

Sporotrichon Beurmanni.

This organism may attack practically any of the tissues
of man. The disease (Sporotrichosis) simulates tuber-
culosis and syphilis and produces pus. It can seldom be
found in smears but is readily cultivated. Gougerot
(Medical Press, October 7, 1914) recommends the pus
from closed lesions being inoculated on to glucose gelatin
and kept at room-temperature. He says that colonies
of sporotrichum make their appearance between the fourth
and the twelfth day ; white at first, they soon become brown
and then chocolate colour. He likens the colour and the
wrinkling to mountains on a relief map or the cerebral
convolutions. A brownish halo surrounds the colonies.
Gougerot says infection takes place through scratches
with thorns or with knives used for peeling potatoes,
for example, and the ingestion of uncooked vegetables.
Animals may convey infection.

The organism produces filaments and spores, and is
thought to be a hyphomycete.

Ringworm.

American authorities include the forms of ringworm in
a class called the Kcratomyceles, owing to their power of
living on the keratinised products of skin, which they
break up and digest by means of keratolytic ferments. It
rarely affects sheep and pigs, and in cattle is usually
confined to the head and neck. In France it is frequent
among horses. Dogs and cats arc affected, and frequently
transmit the disease. In the human subject most forms
of ringworm disappear before the host reaches twenty
years of age.

Two varieties at least of the disease are to be dis-
tinguished :

172 AIDS TO BACTERIOLOGY

Microsporon Audouini is met with in young children.
Sequeira (School Hygiene, August, 1912) found it present
in 89 -8 per cent, of ringworms in children. Hewlett says
that it never attacks the scalp of adults, never affects
the beard or nails, is very intractable, and frequently
epidemic. It occurs as a whitish sheath round the stumps
of broken hairs. Microscopically the parasite appears as
round or ovoid spores measuring 3 JUL to 4 jbt, in diameter.

The second form comprises two varieties. In one,
which is exclusively of human origin, the spores are met
with chiefly in the interior of the hairs, and hence is
termed the endothrix variety. It occurs in late childhood
(10-2 per cent, of children's ringworms [Sequeira]). In
the ectothrix variety, which principally affects the beard
and nails, and is derived from animals, the spores lie on
the exterior of the hairs. In both the spores are large,
measuring 4 /u, to 12 /j, in diameter, and the parasite is
known as the Trichophyton megalosporon endothrix and
ectothrix respectively.

The fungi can be readily cultivated on all the ordinary
media, but beer- wort gelatin and agar are to be preferred,
and the best of all is a maltose agar (Blaxall's English
Proof Agar), composed of peptone 1 gramme, maltose
4 grammes, agar 1;3 grammes, and water 100 c.c. The
growths are white and fluffy, and rapidly liquefy gelatin,
the different varieties showing certain differences, and
microscopically they consist of a tangled mycelium,
with spores.

A pure culture may be often obtained by chopping up
an affected hair and strewing it over an agar plate.
After incubating for three to seven days at 24° or 30° C.,
colonies appear as little, fluffy, whitish spots. To
diagnose a case of ringworm, it is generally sufficient to
examine one of the suspected hairs under a low power
(£ inch), when it will be found to be covered with spores.
To facilitate examination, the hair may first be soaked
in alcohol and ether and then in 10 per cent, caustic
potash solution. If the patch itself is examined, spores
will be found on the surface, while a little below will be
seen a matted mass of mycelial branching. The organism
may be stained with fuchsin or by Gram's method, and
permanent preparations (unstained) may be mounted
in glycerin jelly.

THE HYPHOMYCETES 173

Gilbert Brooke states that M. Audouini and T. endo-
thrix are rare in the tropics, and thinks that dhobie
(washerman's) itch is caused by Tinea, ectothrix. The
p. access of X-ray treatment lies not in any power to destroy
the ringworm spores, but in a copious defluvium of hairs
from the part exposed, leaving a follicle free from disease.
Germicides are recommended as an accompanying treat-
ment.

Strickler and Kolmer (Journal Amer. Med. Assoc.,
July 17, 1915) found that on injection into the superficial
layer of the skin of the arm of children suffering from this
disease of 0-05 c.c. of a suspension of dead ringworm
fungus in salt solution, usually there occurs a local re-
action. These authors recommend a vaccine.

Microsporon Furfu:.

This organism, which is found in pityriasis versicolor,
probably belongs to the same family as the T. tonsurans.
It occurs as masses of short threads and clusters of oval
refractile spores in the scales of the skin, but has not yet
been artificially cultivated.

Achorion Schonleinii.

The yellow cup-shaped crusted masses of fungus in
favus are caused by A. Schonleinii. The disease affects
man, dogs, cats, caged rabbits, mice, and rats. In the
last two cases it is commonly fatal. In the fowl lesions
occur mostly on the comb and wattles as a grey crust
(' white comb '). Favus can be transmitted from animals
to man. A. Schonleinii in the earlier stages is indistin-
guishable from the T. tonsurans, but soon assumes the
honeycomb appearance. It grows on all ordinary media
except milk. Gelatin is liquefied. On agar the colonies
appear distinctly in forty-eight hours. They are sur-
rounded by a fine fringe of threads. On blood-serum
star-shaped colonies are formed, which radiate out from
the centre, producing a flower-like appearance. The
medium is not liquefied. It also grows well on bread and
potato.

174 AIDS TO BACTERIOLOGY

CHAPTER XVII
THE PATHOGENIC PROTOZOA

THE Protozoa are unicellular organisms belonging to the
animal kingdom. Reproduction generally takes the forms
of simple fission and spore formation, but complex life-
cycles, involving the alternation of sexual and asexual
phases, occur in some. A conjugation of two dissimilar
cells (gametes) may take place to form a zygote. Gametes
fulfilling a duty analogous to that of spermatozoa are
called microgametes, the other variety being macrogametes.

The following is one classification of the Protozoa:

Sarkodina. — The Amoebae, the only type of this division
parasitic for man, have no cell-wall, movement is slow,
and takes place by the projection of temporary limb -like
processes (pscudopodia). Multiplication is by fission or
spores.

Mastigophora. — Flagellated forms, including trypano-
somes and probably spirochsetes.

Sporozoa. — Passive and exclusively parasitic organisms,
reproducing by spore formation, including malarial para-
sites, Coccidium and Piroplasma.

Infusoria. — Actively motile ciliated protozoons, with
definite cell-walls, and small micronucleus participating
in conjugation, and a vegetative macronucleus. Includes
Balantidium.

Parasitic Amcebse.

Three Amrebse, known as Entamcebse, are parasitic for
man:

Entamoeba buccalis occurs in dental caries.

Entamoeba coli is an apparently harmless, and not
infrequent, inhabitant of the bowel.

Entamoeba histolytica is found in the faeces in the form
of chronic dysentery common in the tropics, and in the
pus of tropical abscess of the liver, a not infrequent
sequela of this form of dysentery. A. histolytica measures
about 15 /LI to 50// in diameter. The pseudopodia are short
and blunt, and vacuoles, a nucleus, and highly refractile
granules are to be seen in the endosarc, often together
with blood-corpuscles, pus cells, and bacilli. Amrabae can
be separated from ingested bacteria by a 20 per cent.

THE PATHOGENIC PROTOZOA 175

solution of sodium carbonate; but in order to obtain
growth,* it is necessary to use an agar medium, and supply
the organisms with living bacteria. In the faeces spore
formation takes place. The disease is supposed to be
spread by drinking water and raw foods. Jordan says
that sand filtration will not remove Amoebae from water.

The Trypanosomes.

The trypanosomes have a somewhat elongated eel-like
form, the average length being from 20 ft to 35 IJL. They
belong to the Flagellata class of the Mastigophora division
of the Protozoa. The description of T. Brucei given below
is with slight modifications accurate for other species.
Trypanosomes have no mouth, nutrition being effected
by the imbibition of soluble nutrient material. When
living they move rapidly about in the fluids of the body,
being propelled by a flagellum, by the undulations of
a fin-like structure — the undulating membrane — and by
alternate contractions and relaxations of the body proto-
plasm. They reproduce by a simple longitudinal division.
While some authorities aver that no true conjugation
has been discovered, others differentiate a bulky, granular,
slow-moving organism as the female, and a slender,
active one as the male. These sexual types are said to
conjugate in the Glossina or other invertebrate host and
a transitional form between the male and female varieties
is regarded as an indifferent form, or as a daughter cell
split off from a fertilised female.

Trypanosoma Gambiense is found in the glands, cerebro-
spinal fluid, and blood in sleeping sickness. The organism
is conveyed by a tsetse-fly * (Glossina palpalis), which
feeds on an infected being, and conveys the trypanosome
in its alimentary canal till it bites a healthy subject
(frequently on the back of the neck), and thus causes
infection. An exogenous cycle appears to take place
in the gut of the fly, involving differentiation into sexual
types and fertilisation of the female. The fly remains
infective after feeding on infected blood for ninety-six
days, and possibly for entire life. Glossina palpalis does

* Tsetse-flies belong to the same order as the house-fly (Musddce),
which they resemble in general aspect, but when at rest the wings fold
completely over each other, like the blades of a pair of scissors. The
flies are viviparous, extruding single annulated adult larvce.

176 AIDS TO BACTERIOLOGY

not feed on ordure, but frequents the brushwood near
water where birds and fishes congregate, and from these
it derives the blood it must have every two or three days.
It feeds on fishes lying on the surface, hippopotami, and
crocodiles, its proboscis being able to pierce even through
elephant-hide. Glossina, fusca and mosquitoes of the
genera Mansonia and Stegomyia are possibly important
auxiliaries in conveying the disease.

The organism may be found by direct examination of
a wet blood-film or in the juice of the enlarged glands.
The parasites are often difficult to find in the cerebro-
spinal fluid and blood. They may be absent from the
blood and present in the cerebro-spinal fluid, and vice
versa.

The inoculation of monkeys with the blood or cerebro-
spinal fluid may detect the disease when direct examina-
tion is negative. A count of the white cells of the cerebro-
spinal fluid shows an increase in the small lymphocytes
(Broden and Rodhain).

Two distinct and separate forms of sleeping sickness
are recognised: the Uganda form, which is violently
epidemic, and the variety prevalent in Nyasaland and
Rhodesia. The latter is almost certainly not epidemic,
and is caused by a different trypanosome, which is
carried by 0. morsitans, a tsetse-fly that appears to be
independent of water.

Yorke and Kinghorn found that a large proportion of
the wild game in Equatorial Africa, though apparently
quite healthy, are veritable reservoirs of trypanosomes
constantly infecting the tsetse- flies who feed upon them;
and the tsetses in turn infect man. In the case of the
Rhodesian or Nyasaland form, the Inter-Departmental
Committee on Sleeping-Sickness concluded that * the
evidence is conflicting as to whether the wild animals
which are a reservoir of the disease affecting domestic
stock are a danger to man.' And further, that in the
case of the Uganda form ' the part played by wild animals
is of minor importance, as compared with that played
by man himself,' as a reservoir from which the fly derives
the infection.

Sleeping-sickness may have a conjugal origin. Monkeys,
dogs, and rats are susceptible, but donkeys, oxen, goats,
and sheep, up to the present, have shown themselves

THE PATHOGENIC PROTOZOA 177

refractory. There is no evidence that a previous attack
of sleeping-sickness gives any immunity. Trypanosomes
surviving medication with arsenic compounds acquire
a tolerance of them.

Trypanosoma Brucei. — Domesticated horses, cattle, and
dogs entering a ' fly country ' are liable to nagana, or
' fly disease,' the aetiological agent of which is T. Brucei.
This parasite has a somewhat slender, spindle-shaped body,
about 15 ILL to 20 JLI in length, bluntly pointed posteriorly,
and prolonged into a long, delicate flagellum at the
anterior end. On one aspect of the organism is a delicate,
undulating membrane, commencing near the posterior
extremity at a micro- nucleus, or blepharoblast, and becom-
ing prolonged into the flagellum. Near the centre of the
organism is a macro-nucleus, and the protoplasm is refrac-
tile and finely granular, and contains a vacuole. The
appearance of this hsematozoon in the blood is signalised
by a rise in temperature; the incubation period is from
seven to twenty days, after which period the hsematozoa
may be found swimming actively among, and apparently
' worrying,' the corpuscles, red blood-corpuscles becoming
very largely reduced in numbers. G. morsitans transmits
the parasite. Wild game harbour the parasite without
showing any sign of the disease — in a happy expression
of Bruce, they act as ' reservoirs,' and removal of the big
wild herbivora from a district considerably lessens the
incidence of the disease.

Trypanosoma Evansi is found in surra, a disease of
horses and camels in India. The parasite is more active
than T. Brucei, but is regarded by some as identical
with it.

Trypanosoma equinum occurs in an equine disease
prevalent in South America, mal de Caderas, the prominent
symptom of which is paralysis of the hindquarters.

Trypanosoma dimorphon, or dimorphum, occurs in a
trypanosomiasis of horses in Senegambia. Two forms,
long ands hort, occur, and by the prolongation of the
protoplasm to the tip of the flagellum a free process is
eliminated.

Trypanosoma Theileri measures up to 65 //. in length,
and is found in South African cattle affected with galziekte.

Trypanosoma Transvaaliense measures up to 50^ long
and up to 6^ thick. It is found in South African cattle.

12

178 AIDS TO BACTERIOLOGY

Trypanosoma equiperdum is found in dourine (mal
du coit) in horses, and is transmitted by coitus.

Somewhat similar organisms are frequently found in
the sewer-rat (T. Lewisi), apparently doing little or no
harm, and in many other mammals, birds, reptiles, and
fishes. T. Lewisi is conveyed by a rat-flea. Noller
and also Wenyon say that it is not the act of biting which
infects the rat, but that infection is brought about by the
rat licking up the faeces passed by the flea while feeding.

The trypanosomes stain well by Leishman's method.
By cultivating on a blood agar (ordinary agar, with an
equal quantity of sterile defibrinated rabbit's blood) many
of the trypanosomes may be grown — T. Lewisi with ease,
T. Brucei with more difficulty.

Leishmania Donovani. — The spleen and liver of persons
suffering from kala-azar (tropical febrile splenomegaly,
dumdum fever) contain Leishman-Donovan bodies. They
are round or oval bodies, measuring 2-5^ to 3'5/^. Some
sort of cuticle is apparently present, and there are two
chromatin masses — a large one, staining pale red with
Leishman's stain, and forming part of the periphery of the
parasite; and the other a small one, staining deep red
with Leishman's stain, situate opposite the other, in the
short axis of the parasite. Donovan suspected a reduviid
bug (Conorhinus rubrofasciatus, de Geer) as being a
possible transmitter of the disease. Patton has de-
scribed the complete development of the parasite in
Indian and European bed-bugs. A parasite indistin-
guishable from Leishmania Donovani was found by Nicolle
in the splenic smears of Tunisian dogs, but Donovan
failed to find it in an enormous number of smears from
Madras dogs. Nicolle' s parasite is considered to be a
separate species and is called Leishmania infantum.

A goitre affection occurring in Brazil is said to be a
trypanosome infection. Donovan tritely remarks that
there appears ' to be no limit to the existence of the
parasitic flagellates in animal organisms, but what is
more astonishing and subversive of previously held views
is the occurrence of these parasites in the latex or milky
juice of plants.' Lafont found Herpetomonas (Lepto-
monas) in the latex of Euphorbia pilulifera in Mauritius,
and Donovan discovered these flagellates — small, narrow
forms — in the latex of the same plants growing in Madras.

THE PATHOGENIC PROTOZOA 179

The organisms differ from the known flagellates parasitic
on animals, and will doubtless be placed in a new genus,
for which the name of Phytomonas has been suggested
by Donovan.

The Spirochsetes.

Authorities are divided as to whether the members of
this group should be regarded as Bacteria or Protozoa.
The presence of a definite nucleus, blepharoblast, undu-
lating membrane, and multiplication by longitudinal
division suggest a protozoan nature, but some observers
have thought the flagellum to be of a bacterial type in
8. recurrentis, and Jordan states that this organism
multiplies by transverse fission.

Spirochseta recurrentis (Spirillum Obermeieri) is an
actively motile, delicate, spirillar filament, found in the
blood-plasma in relapsing fever during the febrile period.
It measures about 8 ^ long and about 0-3 /LL thick, and
is pointed at the ends. Monkeys can be inoculated, but,
with the exception of rats, other animals are immune.
Bed-bugs and pediculi are supposed to transmit the dis-
ease. Darling found granular forms in the tissues of
animals infected with the spirochsete of the relapsing
fever which occurs in the Isthmus of Panama, which
granules he suspects to be biogenetically connected with
the spirochaetes, and inoculation of such tissue, apparently
completely free from spirochsetes, was followed by in-
fection.

Spirochaeta Duttoni occurs in the body of the tick
(Ornithodoros moubata). Clumps of chromatin granules,
considered by Leishman to be biogenetically connected
with the spirochaete, are found in the Malpighian tubes of
the tick (Lancet, 1910, i. 12). These ticks, by feeding on
a man or monkey, produce tick-fever.

A fowl spirochsetosis occurs in the Soudan, and is trans-
mitted by Argas persicus. Spirochaeta anserina occurs
in geese. Fantham, Chalmers, and O'Farrell found^a
spirochaote in a form of bronchitis endemic in some^parts
of the Soudan.

Spirochseta pallida, or Treponemal pallidum. — This
actively motile spiral-shaped protozoonjs very thin and
varies from 4 {JL to 14^ in length, with an "average thickness
of 0*25 ji. The ends taper. Six to eight spirals are

i So AIDS TO BACTERIOLOGY

generally seen, though more (up to twenty) are often
found. The existence of a flagellum is uncertain. The
virus is destroyed by exposure to X rays or to a tempera-
ture of 48° C. for half an hour.

T. 'pallidum is now accepted as the cause of syphilis.
It is found in the primary sore and neighbouring lymphatic
glands, in the papular and roseolar eruptions and condylo-
mata of the secondary lesions. It is also found but with
some difficulty and only in small numbers in gummatous
lesions. Considerable numbers of T. pallidum are found
in the liver, lungs, pancreas, spleen, thymus, and adrenals
of syphilitic foetuses. Noguchi demonstrated the presence
of this organism in the brains of patients dying of general
paralysis; Stoddart holds the view that the treponeme of
general paralysis is not absolutely identical with that of
syphilis. Moss has suggested that there is a special general -
paratysis-producing variety of the syphilitic organism.

McDonagh thinks the protozoon enters the body while
in the spore phase, and that T. fallidum probably re-
presents the end phase of the life cycle and is a male.
McDonagh says that where extracellular development
(only described in chancres and condylomata) occurs,
the immature treponeme that is first formed resembles
the refringens type.

T. pallidum can be cultivated by Noguchi's method;
material for inoculation is obtained not from human
lesions, but from the artificially infected testicular tissue
of the rabbit. This is introduced into serum water
to which a piece of sterile rabbit tissue (preferably kidney
or testicle) has been added. The surface of the medium
is covered with a layer of sterile paraffin. Once primary
culture has been effected strict anaerobiosis is not essential ;
and the organism can in subcultures be transferred to
solid media such as ascitic fluid agar.

The treponemes can be separated from the bacteria
that usually contaminate the primary culture by letting
them grow through filters which retard the passage of
other organisms, or by growth in stab cultures where
the treponemes grow away from the line of puncture
into the surrounding medium while other bacteria fail
to do so.

In ulcerating syphilitic lesions Spirochaeta refringens
sometimes accompanies T. pallidum. McDonagh (Lancet,

THE PATHOGENIC PROTOZOA 181

1910, i. U20) says the question of distinguishing the T.
pallidum from the S. refringens need not arise, since the
latter is rare in the secretion of a chancre, and does not
occur in other venereal diseases until a stage so late that
a wrong clinical diagnosis is impossible. S. refringens
is thicker, has fewer and wider turns to its corkscrew,
and is more refractile; while on the other hand, the
T. pallidum is finer, has more turns, tapers at both ends,
and appears dead white.

The secretion of a primary sore examined by means of
the ' dark ground illumination ' is said to hardly ever
fail to reveal the T. pallidum. A chancre is cleansed
by washing with normal saline and a drop of clear exudate
squeezed out. This is taken up in a capillary tube, placed
on a microscope slide and covered with a cover-glass.
A drop of cedarwood oil is placed on the under surface
of the slide, and contact made with this and the upper
surface of the parabolic dark ground condenser. The
preparation is examined with a /;-inch objective.

The ' dark ground illumination ' allows no rays of light
to reach the eye of the observer, except those reflected by
certain objects, such as the treponeme, under observation.
The treponeme is seen twisting its corkscrew-like form,
which is brightly illuminated, across the dark, non-trans-
parent background. Neither form nor size is distorted.
T. pallidum appears moderately stiff and rigid. Move-
ments of flexion and oscillation are not so marked as in
some allied species. With the exception of S. dentium,
T. pallidum, almost alone among the spirochsotes, retains
its spiral arrangement not only during movement but
also when at rest. In other words, its spirals represent
a permanent arrangement. Dark background illumina-
tion can be carried out almost as well on dried as on living
preparations.

Material for microscopic preparations may be obtained
by the method recommended by Phillips and Glynn.
The lesion is cleansed with cotton-wool, rubbing it with
wool, and then swabbing with wool soaked in methylated
spirit. At the end of four minutes the spirit is wiped off,
and the clear serum which exudes collected in a capillary
tube.

The organism may be stained by Giemsa's stain :
Azur II. eosin, 3 grammes; Azur III., 0-8 gramme;

i82 AIDS TO BACTERIOLOGY

glycerin, 250 c.c.; methyl alcohol, 250 c.c. Blood films
or smears from chancres are fixed in alcohol for fifteen
minutes and then stained in Giemsa stain, diluted to
the extent of 1 drop in 1 c.c. of distilled water, and made
alkaline with dilute potassium carbonate solution, for
five hours or longer. The slides are washed in distilled
water and dried. T. pallidum is usually stained a light
rose-pink and other spirochsetes and organisms blue.

Burri's Indian-ink method (p. 48). Gunther-Wagner
ink is sterilised by steaming. Sommerville recommends
centrifuging the ink until a film made from the upper
portion of the column appears under the jVinch ob-
jective quite homogeneous. Equal quantities of exudate
and ink are mixed and spread on a slide. The preparation
is allowed to dry and examined by the oil- immersion
lens without a coverslip. The T. pallidum appears as a
white, wavy thread in a dark field. Sometimes treponema
cannot be discovered in chancres after repeated examina-
tions and the cases ultimately prove to be of syphilis.

The Wassermann reaction (p. 21) is used for diagnosis,
and appears to be of most value in the secondary stage
of the disease. It is inadvisable to place reliance on a
negative reaction in the primary stage, but as the method
improves the cases of definite untreated syphilis in which
a negative reaction occurs are becoming fewer and fewer.
The reaction is positive in both the blood and cerebro-
spinal fluid of general paralytics but not at every examina-
tion. D'Arcy Power says it is not usually positive until
five to eight weeks after infection when the disease has
ceased to be local and has become generalised in the body.
McDonagh thinks it doubtful whether our present inter-
pretations of its results are correct, and succeeded in
making a reaction positive or negative at will. H. C.
French states that the test may be negative, and later
positive, or vice versa, without any material change in
the immediate surroundings of the patient.

Positive Wassermann reactions have been found in
cases of non-syphilitic (soft) chancre (French), and also
in alopecia areata, gonorrhoea, yaws, leprosy, malaria,
puerperal eclampsia, lupus erythematosus, trypano-
somiasis, enteric fever, tumours, herpes, aortic disease,
relapsing fever, in some persons under narcosis and in
serum from dead bodies. Positive results of a transient

THE PATHOGENIC PROTOZOA 183

nature have also been reported in pneumonia and scarlet
fever.

Luetin. Cultures of T. pallidum are ground up with
normal saline, heated to 60° C. for an hour and 0'5 per
cent, phenol added. Luetin is injected into the skin
of the arm and a positive reaction consists of the produc-
tion at the site of inoculation in forty-eight hours of a
diffuse erythema with sometimes the development of
a papule or pustule later on. In non-luetic cases a slight
erythema occurs. A positive result means no more
than that the patient has had syphilis, it does not indicate
that the disease is actually active.

As a result of the administration of arsenical prepara-
tions in treatment, arsenic-resistant treponemes may be
produced.

The Malarial Parasite.

Plasmodium malarice or Hcemamceba malar ice is con-
veyed from man to man by mosquitoes of the genus
Anophelina, the commonest species of malaria carriers
being the Anopheles maculipennis, A. funestus, A. cos-
talis, A. sincnsis, and A. argyrotarsus. When at rest the
proboscis, thorax, and abdomen of the Anopheles form
a straight line, with the proboscis almost touching the
wall, and thus form an angle with the wall or other resting
surface. The resting CuUx, on the other hand, rests
with its body parallel to the resting surface. The ova of
the Anopheles are much smaller than those of other
mosquitoes, and are boat-shaped. Down the centre of
the body of a larva run two breathing tubes, the opening
of which is in the anterior part of the last segment, which
necessitates them floating horizontally at the surface of
water. The larvae of other mosquitoes hang down from
the surface. The female alone bites, generally at night,
and, in the event of the subject being malarial and the
parasite being ingested, its development is complete in
eight or ten days, when it is ready for successful inocula-
tion into a healthy subject. After the mosquito bite the
organism enters a red blood- corpuscle, being at first a
small, pale, nebulous, ill-defined body, which grows in
size and alters in shape, sometimes to a ring-like form,
developing black pigment granules, which exhibit slow
movements, and tend to concentrate while the amoeboid

i84 AIDS TO BACTERIOLOGY

movements slacken. Ten to twenty segments develop,
which form round a central clump of pigment, like a
rosette, the blood-corpuscle breaks down, and the seg-
ments, which have now become spherical, are liberated.
Some enter red blood-discs and repeat the cycle (the human
phase, endogenous or asexual cycle), the bodies in the
corpuscles being known as amcebulse or myzopods, and
the liberated spheres as sporocytes or merozoites. The
liberation of the latter is coincident with the appearance
of a paroxysm.

Varieties of the Malarial Parasite. — Varieties of the
parasite supposed to be biologically distinct organisms
are associated with respective types of fever, which are
distinguishable clinically.

(a) Quartan. — This is a benign variety, and depends on
a parasite (Hcemamceba malarias) which takes seventy-
two hours to pass through its cycle of development. The
parasite is feebly amoeboid at first. The pigment is
abundant and coarse-grained, and the symmetrical rosette
that forms next produces six to twelve merozoites. The
red corpuscles invaded by the parasite do not become
decolorised or markedly enlarged.

(b) Tertian. — The cycle of development of this parasite
(H. vivax) takes forty-eight hours for completion. The
organisms within the corpuscles show much greater move-
ment than in the quartan type. The pigment granules
are fine, and show much movement. The sporulating
body consists of fifteen to twenty segments, which form
a mulberry mass. The corpuscles are frequently decolor-
ised and hypertrophied.

(c) Malignant Tertian. — This form (Hwmomenas prce-
cox) requires about forty-eight hours for development.
The organism is relatively minute, and is very actively
amoeboid when young, the parasite assuming a ' signet-
ring ' form after a time. Its more advanced or sporula-
ting stage is completed in the bloodvessels of the deeper
viscera.

(d) Malignant Quotidian. — The cycle of development
of this variety takes twenty-four hours. The parasite is
always small, even in the adult state, and frequently
assumes the ' ring ' form in the corpuscle. The spores
are generally formed by irregular segmentation.

There is some disagreement as to the existence of

THE PATHOGENIC PROTOZOA 185

different parasites in the malignant form, and they are
often classed together as ' the parasites of sestivo- autumnal
fever.'

The main distinction between the benign and malign
species is that in the case of malign parasites flagella are
produced from crescent bodies, while in the case of the benign
parasites they are produced from simple spheres. Crescent
bodies are always found in malign fevers, though not in
the early stages. They continue in the blood for many
days or weeks, and are much less affected by quinine
than the other varieties.

The Sexual Phase. — Within the human host asexual
development alone takes place, but a sexual phase occurs
in the mosquito. Amoabulae become specialised into male
cells (microgametocytes) and female cells (mac regain eto-
cytes). The microgametocytes develop four to eight
filaments (gametes), which break away to fertilise the
large spherical and granular macrogametes, forming a
' zygote,' spoken of at this stage as a ' travelling vermicule.'
This becomes encysted in the stomach wall. This grows,
divides into eight to twelve ' zygometres,' each of which
becomes a spherical ' blastophore ' and develops numerous
slightly sickle-shaped, radially disposed bodies, or zygoto-
blasts. When mature the blastophore disappears, the
capsule ruptures, the zygotoblasts are poured into the
body cavity of the insect, and make their wajr to all parts,
including the salivary glands, whence they are discharged
by the middle stylet of the proboscis when the insect
' bites ' its next victim. The ' blasts ' then become
attached to corpuscles, and develop into the ordinary
form of malaria parasite.

Flagellated bodies do not, apparently, occur in fresh
human blood, but are found when wet specimens have
been under the microscope for some time. The pigment
granules become arranged in the form of a central ring,
and afterwards show a peculiar vibratory movement,
which is apparently caused by the flagella which have
formed within the sphere. At this stage the flagella
shoot through the walls of the envelope of the sphere.
The flagella, which are usually from three to six in number,
are very delicate, actively moving filaments often having
a bulbous swelling at their ends. They frequently break
away and swim free in the blood.

186 AIDS TO BACTERIOLOGY

Examination of Fresh Blood. — A droplet of blood (p. 46)
is touched with the centre of a cover-glass, which is im-
mediately placed on a flip. If cover-glass and slide are
clean the blood runs out in a delicate film. The slide is
examined with TVinch immersion lens, in a not too bright
light, and the interior of every corpuscle is searched for
specks of black pigment, surrounded by a pale, hyaline,
slightly or markedly amoeboid, substance; also for smaller,
pale, unpigmented, hyaline, and more actively amoeboid
bodies in the same situation.

Stained Blood Preparations. — A film is made (p. 46),
allowed to dry spontaneously, fixed by immersion in a
mixture of equal parts of absolute alcohol and ether, and
stained with Lomer's methylene blue.

Leishmaris stain (0-15 per cent, in the purest methyl
alcohol) is used for staining the malarial parasite. Blood-
films, unfixed, are flooded with a few drops of the stain,
which is spread by tilting, no attempt being made to
check evaporation. After half a minute about double
the quantity of distilled water is added, allowed to mix
with the stain on the film, and staining is continued for five,
or in some cases for ten, minutes. The film is then washed
in distilled water, some of the water allowed to remain on
the film for one minute, and it is then dried and mounted.

Jenner's blood-stain may be used. After the film has
dried spontaneously, without fixing it is placed in a pot
of the stain for five minutes. The film is washed in dis-
tilled water (already coloured with a drop or two of stain)
till pink.

Haematoxylin and eosin is also a good stain, and very
permanent.

The Coccidia.

The coccidia are sporozoa possessing thick cuticles.
While some species produce fatal disease in sheep, game
birds, and poultry, human infections are rare. The liver
and intestines are sites for their activity, and the parasite
occurs in large numbers in the droppings.

Coccidium oviforme infects the epithelium of the
intestines and bile-ducts in young rabbits with fatal
results. In the liver it occurs as a large (30 fj, to 40 JLL)
oval, encapsuled organism in yellowish nodules, consisting
of an adenornatous proliferation of the biliary epithelium.

THE PATHOGENIC PROTOZOA 187

The Piroplasmata.

Piroplasma bigeminum, the cause of Texas fever or
bovine malaria, develops in the red blood- corpuscles, first
as a minute double body like a diplococcus, which ulti-
mately grows into two pear-shaped bodies. The disease
is transmitted through the ticks (Rhipiceplialus annulatus]
which live on the beasts. The female ticks feed on infected
animals, and after impregnation fall off and lay their
eggs, from which the young ticks develop. The infection
passes from the mother through the egg to the young tick,
which then infects the animal on which it feeds. It is
probable that the parasite passes through a developmental
cycle in the ticks. Cattle indigenous to the district are
immune.

Piroplasma parvum is found in Rhodesia (red-water)
fever of cattle. Prophylactic vaccination is successful.

Amakebe, a disease affecting calves in Uganda, has as
the chief symptom the swelling of the lymphatic glands.
After recovery from the disease a calf is immune for life.
In addition to P. bigeminum and P. mutans, to which
diseases Uganda cattle are immune, the blood contains a
small piroplasm indistinguishable from Piroplasma parvum,
and the Blue Bodies of Koch are found in the spleen.
Rhipicephali are carriers of the disease (R.A.M.C. Journal,
May, 1910).

Piroplasmosis of dogs, horses, and sheep are caused
respectively by P. canis, P. equi, and P. ovis.

The Microsporidia.

Nosema apis causes the ' Isle of Wight ' bee disease
(microsporidiosis). Certain bees act as parasite ' carriers.'

Nosema bombycis causes pebrine in silkworms. It
occurs as small glistening, roundish corpuscles, in the
caterpillar, butterfly, and egg.

For further information on protozoan diseases see
Castellani and Chalmers' ' Manual of Tropical Medicine '
Bailliere, Tindall and Cox (1913).

1 88 AIDS TO BACTEBIOLOGY

CHAPTER XVIII

FERMENTATION— ENZYMES— SULPHUR AND
IRON BACTERIA

Fermentation.

FERMENTATION by yeasts, see p. 162 et seq. Fermentation
by moulds, see p. 170. Bacterial fermentation may
involve oxidation, hydration, reduction, or simple de-
composition processes^

Acetic Acid Bacteria. — Mycoderma aceti, the oxidising
agent used in the manufacture of vinegar, consists of two
species of bacteria — B. aceti (Hanseri) and B. Pastorianum
(Hansen).

B. aceti (Kutzing} Hansen forms a gelatinous film on
' double ' beer when grown at 34° C. for twenty-four hours.
The film consists of chains of hour-glass-shaped indi-
viduals. The mucilage surrounding the organisms is not
stained by iodine. On wort-gelatin at 25° C. grey, waxy,
well-defined colonies are formed in four days, but no chains
are produced.

B. Pastorianum (Hansen} forms a dry, wrinkled film
on ' double ' beer in twenty-four hours at 34° C. The
mucilage is stained blue by iodine. The growth on wort-
gelatin consists chiefly of chains.

B. Kiltzingianum (Hansen} on ' double ' beer at 34° C.
in twenty-four hours forms a dry film, which,"* unlike
B. Pastorianum, grows high above the level of ^the^liquid,
along the sides of the flask. The mucilage stains blue
with iodine, and long chains are seldom found in the
colonies on wort-gelatin.

The term ' acetic acid bacteria ' is confined to those
that give off acetic acid as a main product. Their number
is small, though many bacteria produce minute quantities
of this acid.

Micrococcus urese produces an enzyme (urease) that
hydrolyses urea to ammonium carbonate. The cocci
vary from 08^ to l'0/u, in diameter, occur as pairs, tetrads,
or chains, and sometimes assume a bacillary form.

B. acidi lactici is aerobic, non-motile, and non-sporing.

FERMENTATION 189

The bacillus is 1 p to !•! p long by about 0-3 p to 0-4 IJL
broad, generally occurring in pairs and in strings of four
elements. (See also p. 108). It converts milk sugar
into lactic acid. Fermentation ceases after a certain
amount of lactic acid has been formed, but will recom-
mence if the liquid be neutralised with calcium carbonate.
Fermentation will also recommence if the milk is still
further left to itself, for moulds neutralise acid.

The MassoJ bacillus (B. Bulgaricus), sometimes in con-
junction with Streptococcus lebenis* and a yeast, is used
for the production of ' Bulgarian Soured Milk,' for use in
diseases arising from auto-intestinal intoxication due to
noxious bacterial growth. When acclimatised in the
bowel this organism hinders multiplication of other
organisms, and, ipso facto, the circulation of their meta-
bolic products. It is of prime importance that the milk
used should be sterile before inoculation with the Massol
culture, and many makers remove more or less of the fat
to produce a better-looking product. Only quick-growing
strains should be used for the purpose, such as will curdle
milk in ten hours. Cultures of the organism are also
administered per os in the form of tablets or bonbons.
Some preparations are useless.

Young organisms are Gram-positive; older ones maybe
Gram-negative, or only partially retain the stain. Growth
is difficult on most media, but good in milk, Cohendy's
milk-serum medium, and on milk peptone agar. Growth
is said to be best at 105° to 108° F. Currie says the
bacilli of B. Bulgaricus type in human faeces and human
saliva are identical. Some strains produce small amounts
of succinic acid, which may account for the presence of
this acid in Cheddar cheese.

B. bifidus (Teisser) and B. acidophilus (Moro) (see
p. 92) are other lactose-fermenters that grow in acid
media.

Saccharobacillus Pastorianus is the cause of the ' turn
ing ' of beer, producing lactic acid.

B. butyricus is an aerobic, short or long thin rod, with
rounded ends, which forms spores, but seldom threads.
It is actively motile, and rapidly liquefies gelatin, with
formation of a pellicle. It coagulates and decomposes

* Sewerin considers S. Ifbenis to be identical with B Bulgaricus ,
but his view is not generally accepted.

igo AIDS TO BACTERIOLOGY

the albumin of milk, forming peptones and ammonium
butyrate.

Clostridium butyricum (B. amylobacter) is a long, thick,
motile, anaerobic rod, often forming chains. A terminal
«pore is formed. Butyric acid is formed in solutions of
sugar?, lactates, and in cellulose-containing plants. The
young organisms of the form described by Prazmowski
contain granulose, which stains blue with iodine. (See
also p. 87.)

Greasiness in Cider .— Kayser finds this disease to be
caused by an anaerobic bacillus which ferments sucrose
with production of carbon dioxide, alcohol, lactic and
acetic acids, also mannitol and laevulose.

Fermentation of Cream. — ' Natural starters ' for ripen-
ing cream naturally contain a mixture of organisms,
those producing lactic acid being in greatest number. It
is now commonly the practice to use an artificial starter
after a preliminary Pasteurisation. In some cases the
organism employed appears to be Streptococcus lacMcus.
Similarly, cultures may be added to the cream to produce
a good aroma in the butter.

Butter is liable to certain bacterial diseases: turnip
flavour (B. fostidus lactis), bitter butter (B. fluorescens
liquefaciens, Oidium lactis, and other organisms), etc.

Bacteria in Cheese Manufacture. — Conn considers that
two moulds are necessary for the production of Camem-
bert cheese — a white Penicillium (candidum ?), and
Oidium lactis (p. 170). Slimy whey, containing Strepto-
coccus Hollandicus (see p. 219), is used as a starter for
Edam cheese. Penicillium glaucum is the chief agent
employed in the ripening of Roquefort, Stilton, and Gor-
gonzola cheeses. Moulds also play an important part in
the production of Brie and other soft cheeses; the growth
of certain organisms is encouraged upon the surface of
these cheeses, so that the special ferment which they pro-
duce can penetrate the body of the cheese and bring about
certain characteristic changes.

Tobacco-Curing. — The leaves are suspended in barns,
when the leaf enzymes convert the starch into sugar.
The browning of the leaves which takes place during this
period is a complicated process, and is the outward expres-
sion of a large number of very little understood processes
which are not bacterial. The dried tobacco-leaves are

FERMENTATION 191

stacked to undergo heating, and fermentation occurs,
possibly by the action of members of the proteus, subtilis,
and mycoides groups, or by one of the Bacilli tobacci,
but the main trend of opinion is to regard the process as
due to oxidising enzymes.

Tanning. — Hides m&y be freed from hair by treatment
with sulphites or milk of lime, or by allowing hides to
putrefy. This is followed by a ' pickle ' of bran and
canine or avian excreta. Lactic acid is developed, which
combines with the lime to form a soluble lactate. Gas-
forming bacteria (B. furfuri is the only one yet isolated)
grow and cause the hides to swell. The changes that take
place in the next stage, in the tan-pit; are due to bacteria;
the process is essentially a souring process, in which
success lies in the proper regulation of the amount of
lactic acid produced.

Retting of flax appears to be due to the growth of
bacteria, one of which has been isolated by Winogradsky,
and is used for the work as a pure culture.

Fermentation processes are involved in the preparation
of tea, coffee, cocoa, and indigo. On the Continent low-
grade wines are improved by the use of pure cultures of
bacteria obtained from high-class vintages, whereby much
of the characteristic aroma and bouquet of the latter are
communicated to the wine.

The Enzymes.

The enzymes are unorganised ferments, often secreted
by bacteria and the cells of animals and plants. In many
cases (groups 1, 2, 3, 5 and 6) they act by hydrolysis,
or, more correctly, by zymolysis. The most important
groups are the following:

1. Proteolytic, changing proteins into proteoses, pep-
tones, and further products of hydrolysis, as in the case
of pyocyanase, trypsin, and pepsin.

2. Amylolytic, converting amyloses (starch, glycogen)
into sugars, such as diastase, ptyalin, and amylopsin.

3. Inversive, which convert disaccharides (lactose,
maltose, cane-sugar) into glucose. Invertase, which
occurs in the cells of yeasts (p. 162) and in the intestinal
juice, is an example of this class.

4. (Joagulative, converting soluble into insoluble proteins,
such as rennet (chymosin), the fibrin and myosin ferments.

192 AIDS TO BACTERIOLOGY

5. Steatctyiic, splitting fats into fatty acids and glycerin.
Lipase (steapsin), which occurs in the pancreatic juice
and in many plants, is an example of this class.

6. Peptolytic, splitting proteoses and peptones into
polypeptides and amino-acids.

Other groups bear oxygen and produce oxidation
(Oxydases), produce reduction (Reductases), or break off
amino-groups from amino-compounds (Deamidases).

While some enzymes can act alone, others require the
stimulus of an activating agent or co-enzyme (p. 163).
Antecedent substances (zymogens) which produce the
enzyme exist in the cells. While an enzyme will only act
under specific conditions on a specific substance, its
power in this limited sphere is inexhaustible, as is that
of an inorganic catalyst. Enzyme action is generally
reversible, and during an analytic or splitting reaction
there goes on at the same time a synthesis reproducing
the original substrate.

Tissue enzymes may be defensive; the blood of a
pregnant woman elaborates a specific defensive enzyme
in response to the entry of placental proteins into her own
blood-stream.

The Sulphur Bacteria.

David Ellis classes under this heading those organisms
which have the power of assimilating sulphuretted
hydrogen and effecting its oxidation: the Beggiatoa (see
p. 160); the Thiothrix, which differ from the Beggiatoa
in being non-motile, and having a common thread mem-
brane; the Thiophysa, which form no threads; and the
Purpur bacteria, which contain a colouring matter in the
cells, bacterio-purpurin, which seems to have a function
in abstracting oxygen from the medium.

The Iron Bacteria.

These organisms occur in ferruginous waters, and
convert the soluble bicarbonate, FeH2(CO3)2, into ferric
hydroxide, Fe2(OH)6, the organisms becoming coated
with the latter. In addition to Crenothrix polyspora
and Cladothrix dichotoma (p. 160), the following organisms
act in the manner indicated:

Leptothrix ochracea (Chlamydothrix ochracea) averages
in length from 100^ to 120 /t. Reproduction takes place

BACTERIAL DISEASES OF PLANTS 193

by fission or by formation of conidia. The organism
takes the form of a straight cylindrical rod.

Qallionella ferruginea (Chlamydothrix ferruginea} assumes
the shape of a hairpin, with the prongs twisted round one
another.

Spiropliyllum ferrugineum (Ellis) is a flat, tape-like
organism, spirally twisted, which multiplies by conidia
formation.

These higher bacteria have not the power to dehydrate
and reduce the ferric hydroxide to bog ore. ' Bacillus
M. 7 ' (Mumford) not only precipitates ferric hydroxide
from iron solutions, but, by an anaerobic action, also
transforms this to bog ore with partial reduction of the
iron to a ferrous state. Mumford thinks that to this
organism are probably due the deposits of bog ore. ' Bacil-
lus M. 7 ' is a motile, sporulating bacillus that occurs
singly or in chains of three or four units.

For further information on the iron and sulphur bacteria,
and also for the bacteriology of fermentation processes
in the arts, see David Ellis's ' Outlines of Bacteriology.'

BACTERIAL DISEASES OF PLANTS.

Black Rot of Cabbage (Pseudomonas or B. campestris)
is a disease of the fibro-vascular bundles, which become
dark brown or black. On cutting across the petiole of a
diseased leaf the affected bundles are seen as dark points,
When so many of the bundles are affected as to cut off the
supply of water to a leaf, the blade dries up and resembles
a piece of brown parchment, the blackened veinlets stand-
ing out sharply against the brown background. As the
leaves in the head become affected they decay, producing
a dark vile-smelling mass.

Commercial seed is a factor in the distribution of the
disease. The organism may gain entrance through
broken roots at the time of transplanting, or through the
water pores at the margin of the leaves. All cultivated
plants of the Crucifer family are liable to be attacked.
Slugs and insects may convey the disease. B. campestris
is a short, motile, Gram-negative bacillus, which liquefies
gelatin, digests casein, and produces a yellow pigment
on potato.

13

194 AIDS TO BACTERIOLOGY

B. solanacearum affects members of the Solanacece
(tomato, egg-plant, and potato), producing discoloration
of shoots, stem, and leaves (brown rot). The fruit and
tubers are attacked also. B. solanacearum rots the skins,
and then, assisted by other bacteria and fungi, rots the
centres too. The Colorado potato beetle is sometimes
the traumatic agent, and can spread the disease. Cucur-
bitaceous plants are immune. The organism grows on
ordinary culture media, forming a yellowish-brown
pigment.

B. tracheiphilus produces a wilting of the leaves (wilt
disease) in cucumbers, pumpkins, and other cucur-
bitaceous plants, but does not affect solanaceous ones
It forms a white viscid growth on agar, does not affect
milk, and produces acid in glucose and saccharose.

B. amylovorus produces brov/ning and death (pear
blight) in the twigs of pear-trees. Peritrichally-arranged
flagella are present, and the organism is motile. A tur-
bidity is produced in broth, with formation of a pellicle.
Transmission is effected by bees.

B. olese produces nodular formations on the olive
(olive-knot disease). B. hyacinthi causes the yellow
disease of hyacinths.

Pseudomonas destructans, a short rod, with a single
terminal flagellum, causes ' white rot ' in turnips. Several
bacterial diseases occur in the cultivated potato — viz.,
Micrococcus nuclei, M. imperatoris, M. pellucidus, al-
ways found associated with the ' scab,' M. albidus and
M. flavidus.

Penicillium glaucum most commonly causes the rotting
of fruits. In apples and pears this is accompanied by
Mucor pyriformis, and in the case of medlars the latter
is much the most common fungus. In lemons, oranges,
and other tropical and subtropical fruits, P. glaucum is
associated with two other closely-allied species, P. italicum
and olivaceum. In plums Mucor racemosus has been
observed. In grapes P. glaucum, and Botrytis cinerea are
the most common fungi. The latter species forms the
grey tufts on walnuts.

B. tumefaciens is the primary cause of many of the
tumour-like growths on the roots of plants (crown gall).

Gum-producing Bacteria. — Greig Smith and Edie have
practically established as a fact_the_bacterial origin of

DTSEASES OF QUESTIONABLE ORIGIN 195

gum. The resistance of trees requires to be lowered
by fire or mechanical injury before the bacteria can work,
and it is thought that ants carry the gum-producing organ*
isms from tree to tree.

For further information on phytopathology, see Jakob
Eriksson's * Fungoid Diseases of Agricultural Plants '
(Bailliere, Tindall and Cox, 1912).

CHAPTER XIX
DISEASES OF QUESTIONABLE ORIGIN

Scarlet Fever. — Streptococcus scarlatince (Klein), or
8. conglomerate, occurs in desquamating particles of skin,
blood, sputa, tissues, urine, and mucous secretion of the
throat of patients (see p. 134). Mallory has found small
rounded bodies resembling protozoa, which others regard
as degenerate leucocytes. They sometimes show rosette
forms and stain with methylene blue. Jordan regards
them as characteristic of the disease. Perhaps, at one
stage at any rate, the virus is filterable.

The disease is more prevalent in the northern part of
Europe than in other parts of the Continent. Epidemics
exhibit a quinquennial recurrence, the mortality being
about 3 per cent., and greatest at the age of five. One
attack is usually protective.

Formerly the chief danger of dissemination was thought
to lie in the desquamating particles of skin, but the faucial,
nasal, and aural secretions are now regarded as the more
important sources of infection. Milk is a frequent medium
for the conveyance of the disease. Much difference of
opinion exists as to whether the disease can arise from a
bovine source.

Malignant Disease. — Hitherto, although a large number
of organisms have been described, none has been shown
to produce either carcinoma or sarcoma. According to
Rous, the aetiological agent of a spindle-celled sarcoma
in chickens proved to be a filterable virus. »*•• *,

Epilepsy. — Reed thinks this disease is an infection with
a gas-producing^bacillus.

Pellagra. — Sambon believes this to be of protozoan

196 AIDS TO BACTERIOLOGY

origin, and to be transmitted by a biting sandfly of the
genus Simulium which haunts running streams.
• Rheumatic Fever is perhaps an acute specific disease
(see also p. 133). Various micro-organisms have been
isolated, particularly staphylococci and streptococci, and
a large bacillus (by Achalme) resembling the B. Welchii.
Hewlett believes the bacillus of Achalme to be identical
with B. Welchii, and says it is probably a terminal infection
or a contamination.

Cholera Nostras, or English Cholera. — Klein found the
colon bacillus and Proteus vulgaris to be abundant in the
dejecta of patients, and the disease has been attributed
to abnormal putrefaction by these and other organisms
in the bowel.

Summer Diarrhoea of Infants. — Booker was unable to
identify any specific organism, but found the colon bacillus,
Proteus vulgaris, and streptococci very abundant. B.
dysenteries, is present in a large number of cases. The
symptoms of tyrotoxicon poisoning resemble those of
cholera infantum. Vaughan, who discovered this body,
also obtained toxic bodies from cultures of Booker's
bacteria, which produced vomiting, purging, and some-
times death in dogs. Sidney Martin has shown that the
products of Proteus vulgaris injected into rabbits cause
depression of temperature and watery evacuations.

Scholberg and Mackenzie Wallis (L.G.B. Med. Off.
Rep., 1911) found peptones and peptone-like substances
of toxic nature in milk in the summer, and in milk that
had been incubated at 37° C. for fifteen to twenty- four
hours. These substances arise from bacterial activity,
and are thought to have some relation to the epidemic
diarrhoaa of infants.

See also Saccharomyces ruber (p. 218) and Morgan's
No. 1 bacillus (p. 97).

Louping 111. — The cause of this disease, which is common
among Scotch sheep, is unknown. The disease is trans
mitted by a tick.

Hay Fever. — Mouneyrat thinks that hay fever is an
infection provoked by an unknown micro-organism trans-
ported at certain times of the year by dust of the pollen
of flowers.

THE FILTERABLE VIRUSES 197

THE FILTERABLE VIRUSES.

Some diseases are produced by organisms so minute
that they will pass through the pores of a kieselguhr or
porcelain filter, and remain invisible under the microscope
(ultra-microscopic organisms). In some cases, the organ-
isms are only ultra-microscopic and filterable at one stage,
the filtered virus producing organisms more or less well
within the limits of visibility.

Some filterable organisms (those of rabies, variola,
vaccinia, and anterior poliomyelitis, for instance) will
live in glycerin for weeks without losing their virulence.
According to Cockayne (Medical Press, February 12,
1913), some are readily destroyed by weak antiseptics
such as 2 per cent, phenol, menthol, or hydrogen per-
oxide. Some resist very low temperature: —2° C. to
- 12° C. Many are readily killed by exposure to 50° to
65° C. for a few minutes. With the exception of the virus
of cattle pleuro-pneumonia and the possible exceptions
of those of infantile paralysis and typhus, none has been
cultured on artificial media (see also Twort, Lancet,
December 4, 1915).

Cockayne (loc. cil.) says: ' In those filterable organisms
which can only pass through a kieselguhr filter, and are
therefore visible under the microscope, there is a resem-
blance in staining reactions so far as these have been
carried out. They stain well with Loffler's methylene
blue, less well with haematoxylin, and there is round them
a clear halo— an appearance altogether different from that
obtained by controls of crushed liver pulp passed through
a similar filter.'

Pleuro-pneumonia in cattle is characterised by the
peculiar marbled appearance of the lungs, accompanied
by great distension of the connective tissue, with a yellow-
ish albuminous fluid. The serious symptoms are due to
toxins, and death frequently ensues. Roux and Nocard
describe a minute refractile body just visible with the
highest and best powers, but small enough to pass
through the pores of a Berkefeld filter. It was found
to stain with gentian violet, methyl violet, and hot carbol
fuchsin, though never more than half the particles took
up the stain.

igS AIDS TO BACTERIOLOGY

Hydrophobia (Rabies). — Infection in man and animals
takes place as a result of bites from dogs, cats, wolves,
jackals, cattle, or other animals. The virus is present in
the saliva and in the central nervous system. The
incubation period in a dog is from about three to eight
weeks, though it may extend to six months. In man the
disease usually develops in from eight days to six weeks,
up to perhaps two years. In the majority of cases it is
in the neighbourhood of ten weeks.

Negri bodies, varying from small structureless round
bodies to larger ones, which may be round or ovoid and
show a circle of chromatoid granules round a central
granule, are constantly present, and afford a means of
diagnosis. The virus is probably ultra-microscopic.
The Pasteur method of treatment commences with in-
oculation of the spinal cords of rabbits which have been
attenuated by suspension in a drying chamber for fourteen
days, and then made into an emulsion. Virus of gradu-
ally increasing strength is injected until a much more
virulent emulsion from a cord that has only been dried
for three days is tolerated, which, without previous treat-
ment, would be dangerous. Such is the success of these
antirabic inoculations, that among the persons treated
(over 1,000) at the Pasteur Institute, during 1912, 1913
and 1914, no death occurred. The immune serum of a
protected animal is said to have a marked curative effect.

Swine Fever (see also p. 96). — Swine fever may be
disseminated by the excreta of infected pigs or by contact
with infected pigs. Stockman mentions that pigs which
to all appearance had recovered from the disease may,
in a small proportion of cases, be capable of spreading
infection for months. The Departmental Committee on
Swine Fever found that rats are not pathological carriers
of the disease, nor is the disease propagated by external
parasites, and that, while infection may be carried locally
by persons, vehicles, or animals in contact with infected
pigs, wide dissemination is due to the movement of pigs.

The Board of Agriculture considers that, if proper
precautions are taken, immunity from swine fever can be
established by serum treatment. The Committee recom-
mended the immunisation of herds by simultaneous
administration of serum and virus. They consider the
lapse of a short period of time (a fortnight is suggested)

THE FILTERABLE VIRUSES 109

may be relied upon for disinfection of premises, and should
be regarded as preferable to chemical disinfection in the
case of large quantities of manure and of premises not
readily capable of being disinfected by artificial means.

Typhus Fever. — Hort (Medical Press, October 28, 1914),
after filtration of typhus urine through Berkefeld filters,
found small cocco-bacillary forms which grew on human
blood- agar. He obtained the same organism in cultures
from blood and cerebro-spinal fluid, and these cultures
produced high continued fever in bonnet monkeys. The
small coccal and bacillary forms give place to large coccal
and bacillary forms when urine, blood, or cerebro-spinal
fluid, is incubated, which forms appear incapable of pro-
ducing fever in bonnet monkeys. Hort regards the
filter-passing cocco-bacillus as infective, and thinks it
probably capable of mutation into the larger cocco-
bacillary forms. Sambon found the body louse was the
transmitting agent of typhus.

Measles. — The virus appears to be filterable. It may
lose its infectivity after fifteen minutes at 55° C., and
resist freezing for twenty-five hours (Goldberger and
Anderson). These workers and others have shown that the
blood and the nasal and oral secretions of patients contain
the infective agent from twenty-four hours preceding to
twenty-four hours following the appearance of the eruption.
Rinderpest (cattle plague) occurs in India and South
Africa. The virus is filterable. Sir George Turner
stamped out rinderpest in Cape Colony by the simultaneous
inoculation with virus and serum.

Foot-and-Mouth Disease, aphthous fever, or eczema
epizootica, is an infectious disease of cattle, characterised
by a vesicular eruption of the mouth, teats, and about the
feet. It affects also sheep and pigs, and may be com-
municated to man by milk or cheese and butter made
from it, and usually produces in the human subject an
aphthous condition of the mouth. The organism is
filterable through a Berkefeld filter. The blood is infective
only in the earliest stage of the disease. The virus is
present in the contents of the vesicles which contaminate
the saliva, litter, milk, etc. The virus is very susceptible
to desiccation and light, is killed by a temperature of
56° to 70° C., but under natural conditions may remain
active for months. Horses, dogs, and cats occasionally

200 AIDS TO BACTERIOLOGY

contract the disease, and with birds, rats, and fowls, may
mechanically carry infection.

^Canine Distemper. — Bacillus bronchisepticus (M'Gowan)
is described as present in the respiratory tract in distemper.
In culture a powerful nerve toxin is produced, and the
disease simulates infantile paralysis of man. Some cases
of distemper, at any rate, appear to have a filterable virus.

Dikkop (blue tongue or horse sickness) is prevalent up
the Blue Nile. The virus is ultra- visible, and appears to
be conveyed by mosquitoes (Balfour).

Sheep-pox. — Cockayne says the organism passes through
a Berkefeld, but never through the Chamberland F.

Yellow Fever. — Sanarelli's B. icteroides (p. 96), previ-
ously supposed to cause this disease, is now regarded as a
casual invader. The virus proves to be filterable, ultra -
microscopic, and probably a protozoon. A species of
mosquito — Stegomyia fasciata (calopus) — is the transmitting
agent, and cannot convey the infection until twelve days
after biting a fever patient. The organism must have a
life cycle in the mosquito as well as one in man. Research
is in progress upon the relationship of the so-called
' Seideliii bodies ' to the virus. Prophylactic measures,
aiming at the destruction of the insects and larvae, have
proved successful in the highest degree.

Dengue. — Ash burn and Craig passed dengue blood
through a porcelain filter, and with the filtrate produced
the disease in healthy men after an incubation period of
three and a half days. The disease is conveyed by a
mosquito, Culex fatigens, Wiedemann, which, after biting
an infected person, is not capable of conveying the disease
until a week has elapsed.

Phlebotomus (Sandfly) Fever. — The virus passes Berke-
feld and Chamberland F. filters. It is conveyed by a
sandfly, Phlebotomus pappatasii, which is not infective
until a week after biting.

Mumps. — Cocci have been found in this disease by many
authors, but Gordon found that in a proportion of cases
a virus that passes through a Berkefeld filter occurs in the
saliva.

Variola and Vaccinia (Smallpox and Cowpox).— Opinions
as to the relationship of these diseases are controversial,
the tendency being to regard vaccinia as a modified
form of variola. No specific organism can be distinctly

THE FILTERABLE VIRUSES 201

connected with either disease, although many organisms
have been isolated, notably Bacillus alb us variolce, of Klein
and Copeman, and what is believed to be a protozoon,
the Cytoryctes variolas. It seems probable that the viruses
of both diseases are filterable.

Acute Anterior Poliomyelitis (Infantile Paralysis). —
Most cases of this disease (which may occur in a sporadic
and also in an epidemic form) affect children, principally
in the second and third years of life. The epidemic type
may attack older children and adults. The virus is
filterable, and for many months in pure glycerin keeps
without loss of virulence.

Flexner and Lewis have shown that the virus can enter,
and is eliminated by, the mucosa of the naso-pharynx.
Transmission may take place by ' carriers ' who them-
selves remain healthy. The virus is carried by the house-
fly both on the surface of its body and within its gastro-
intestinal tract. It can also be transmitted by bed- bugs
and stable flies, but not, as far as is known, by fleas or
mosquitoes. The virus remained potent in sterile milk
and sterile water for at least thirty-one days, and was
found in a patient seven months after the onset of the
disease. Zappert found it in the dust of a patient's bed-
room. It is destroyed at 45° to 50° C. in thirty minutes.
The virus of infantile paralysis has not been definitely
cultivated, but Flexner and Noguchi thought it to multiply
in a medium of broth cum human serum under anaerobic
conditions. Diseases similar to poliomyelitis have been
described in horses, cows, pigs, chickens, and dogs. As
it is impossible, apparently, to infect these animals with the
human virus, the diseases are probably not identical with
the human one. Alexander suggests that the virus of
poliomyelitis may remain in the water and slime of public
baths. In one epidemic over 50 per cent, of one series
of cases had been swimming or wading in water con-
taminated by sewage.

Epidemic Nephritis. — Langdon Brown (R.A. M.C. Journ.,
July, 1915) says bacteria could apparently be excluded
as the infective agent in the acute nephritis that occurred
among the soldiers of the British Expeditionary Force in
France, but a filter- passing ultra-microscopic organism
could not be excluded. The point of entrance of the
infection was possibly the tonsils.

202 AIDS TO BACTERIOLOGY

In addition to diseases already mentioned, Cockayne
gives the following list of diseases proved or suspected
on strong grounds to be due to a filterable virus: African
horse sickness and the nearly allied catarrhal fever of
sheep (both S. African). Myxomatosis of rabbits (also
affecting dog and man, S. America). Agalasie con-
tagieuse de brebis. Infectious or pernicious anaemia of
the horse. Trachoma and a non-gonococcal urethritis
probably due to the same organism. Epithelioma con-
tagiosum of fowls and diphtheria of wood-pigeons, prob-
ably forms of the same disease. ' Farcin crytococcique.'
A disease (jaunisse) of silkworms. Mosaic disease of the
tobacco plant. Variola of carp. Disease of the lips of
barbel. Cyanolophia gallinarum, or chicken typhus, and
an allied disease affecting three species of thrush and the
starling in Italy.

CHAPTER XX

THE BACTERIOLOGY OF SEWAGE, SHELLFISH,
MEAT, SOIL, AIR, AND MILK

The Bacteriology of Sewage.

THE number of bacteria in ordinary town sewage varies
considerably, according to the age of the sewage, the
season, temperature, locality, and sometimes with the
nature of the waste liquors from factories. The number
often reaches several million per cubic centimetre. The
number of B. coli is given by Klein and Houston as from
90,000 to 2,000,000 per cubic centimetre, 100,000 being
an average; of B. Welchii, as from 100 to 1,000 per cubic
centimetre; and of streptococci, as at least 1,000 per c.c.
In addition to the above and those mentioned seriatim
at the end of the monograph, B. pyocyaneus, B. mycoides,
B. subflavus, B. cloacce, staphylococci, Cladothrix diclio-
toma, Beggiatoa alba, B. fluorescens stercolatus, B. fila-
mentosus, M. urece, and many others, are present. Re-
searches on the longevity of the typhoid bacillus in sewage
point to the fact that in crude sewage it cannot be expected
to survive longer than a fortnight, while the cholera vibrio,
on the other hand, was found by Houston to survive for
a month in one instance.

BACTERIOLOGY OF SEWAGE 203

The following scheme (adapted by Moor and Hewlett,
after Houston) is applicable to sewage and effluents. A
fair average sample of the sewage or effluent must be
obtained by mixing portions obtained at intervals. The
mixture should be strained through muslin.

Tests.

Procedure.

1. Total number Gelatin and agar
of bacteria i cultivations

'2. Num bor of 'Gelatin plate cultures

spores of ae- with material pre-

robes viously heated to

30° C. for ten minutes

plate 0-001, 0-0001,
0-00001

1-0, 0-1, -0-01

3. Number of Agar plate cultures with
spores of an- material previously
aerobes heated to 80° C. for ten

minutes and incubated
anaerobically

4. Number of Surface gelatin plates

organisms
liquefying
gelatin

5. Spores of B. Milk cultures (see p.

Wdchii

6. Number of B. Surface Conradi-Drigal-

coli ski plates, or bile salt

broth

7. N u m b e r of Surface plates of Conradi

streptococci Drigalski medium

Amount of Sewage
in c.c.

1-0, 0-1, 0-01

0-001, 0-000 1,
0-00001

87) 0-1, 0-01, 0-001

0-001, 0-0001,
0-00001

0-01, 0-001, 0-0001

In addition, effluents should be submitted to the two
following tests:

1. Incubate some in beakers at 22° C. and 37° C. for
some days. A good effluent should yield little or no
unpleasant odour (an unpleasant odour indicates the

204 AIDS TO BACTERIOLOGY

presence of decomposable organic matter, and such an
effluent might give rise to a nuisance).

2. Place a goldfish or two in a bowl of the effluent.
The fish will live in, and be unaffected by, a satisfactory
effluent. In the eyes of the law a fish is an animal, and
this test may only be performed by a licensee under the
Vivisection Act.

An effluent of sewage treated by land filtration should
show a considerable reduction in the total number of
bacteria, and in the number of B. coli, compared with the
original sewage ; but an effluent from a bacterial system
of treatment may contain as many bacteria as, or actually
more bacteria than, the original sewage.

Apart from the nitrifying organisms, very little of
practical value is known of the bacteria concerned in
sewage treatment, and their ability. Especially is this
true of those acting in septic tanks and contact beds.
With further enlightenment there will be fewer failures.

Houston, in his capacity of bateriologist to the Royal
Commission on Sewage Disposal, while recommending as
' counsel of perfection ' the ' complete sterilisation of
sewage effluents ' in the case of drinking-water streams,
suggested as a ' practicable standard ' ' partial sterilisation
(absence of B. coli from 1 c.c. of the effluent).' Houston
also suggested ' provisional ' standards to apply to sewage
effluents for non-drinking-water streams, as follows :

Total number ^ Gelatin at 20° C., less than 100,000 per c.c.

of bacteria / Agar at 37° C., less than 10,000 per c.c.
JB. coli, less than 1,000 per c.c.
B. ertteritidissporogenc-stcst . . . . . . \Negative results

' Gas ' test (twenty-four hours at 20° C. ) . . J with 0*1 c.c.

Indol test (five days at 37° C. ) ]

Neutral red broth test (two days at 37° C. ) . . (Negative results
Bile-silt broth test (two days at 37° C.) . . j with 0-001 c.c.

Litmus milk (modified) test ( two days at 37° C. ) J

These are primary standards; Houston's secondary
standards are arrived at by rendering the primary stan-
dards ten times more lenient. He does not suggest these
standards in an administrative sense; they are merely
arbitrary, and designed solely for comparative purposes.

Prescott and Winslow think even the above primary
standard is far too lenient, and argue that ' either organic

BACTERIOLOGY OF SEWAGE 205

purity alone is necessary, as at many sewage disposal
plants, or a higher grade of purity than this (the primary
standard) should be attained.'

The Royal Commission deprecate the adoption of stan-
dards of bacteriological purity or of sterilisation processes,
both on the ground of the serious additional cost which
these would entail, and of the ' false sense of security '
which the adoption of such measures would be apt to
engender.

Some Organisms met with in Sewage. — The granular
or sewage variety of Proteus Zenkeri is a non-liquefying,
non-sporing, aerobic, non-motile bacillus, and occurs in
the form of short and long chains and filaments. It grows
well in phenolated gelatin and phenolated broth, does
not form gas in a gelatin-shake culture, does not coagulate
milk, and gives no indole in broth after three days' growth.

P. vulgaris, P. mirabilis, P. Zenkeri, are found in putre-
fying substances, sewage, and water. The first two are
motile, produce involution forms, liquefy gelatin and
blood-serum, and give off a decided putrefactive odour
from cultures. P. Zenkeri is non-motile, does not liquefy
gelatin or blood-serum, and the putrefactive odour is
absent from the cultures. All three species produce local
abscesses and symptoms of toxaemia when injected into
small animals.

Herter and Broeck (Jour. Biol. Chem., 1911, 491) say
that P. vulgaris ferments dextrose and sucrose, but not
lactose; it destroys some native proteins, producing
ammonia, primary amines, hydrogen sulphide, fatty acids
oi high molecular weight, aromatic hydroxy-acids, indole,
and indole-acetic acid. They also obtained thermostable
toxic material from the bacteria. P. vulgaris was formerly
called Bacterium termo. The varieties of proteus men-
tioned above are now generally referred to as Bacillus
proteus.

B. fluorescens liquefaciens, B. fluorescens non-lique-
taciens. — These water organisms are short Gram-negative
bacilli. The former liquefies gelatin, and is motile; the
latter does not liquefy gelatin, and is non -motile. Gelatin
streaks show a fluorescent green, and the growth on
potatoes is brown in both cases (see p. 130).

B. subtilis (hay bacillus] occurs in hay, air, water,
tseces, etc. It is about 2 // to 3 ^ long by 1 /* broad, about the

206 AIDS TO BACTERIOLGOY

same length as, but somewhat narrower than, the anthrax
bacillus. It has rounded ends, grows into long threads,
and is very motile, having long flagella; forms ovoid
spores about I '2 /LI long by 0*6 // broad, which germinate
at right angles to the long diameter, and are very resistant
to heat, surviving dry heat of 120° C. for one hour (see
p. 9). The bacillus is strictly aerobic and Gram-positive.
The colonies on gelatin plates become visible in about
two days, as small white dots in the depth, whereas on
the surface they show small greyish liquefied circles.
In a gelatin stab a liquefied funnel-shaped depression
forms, the lower part throwing out lateral feathery
extensions. The whole of the gelatin is soon liquefied,
and a tough pellicle forms on the surface, and a quantity
of flocculent matter collects at the bottom of the tube.
A white, opaque, moist expansion is formed on agar,
which afterwards becomes dry and furrowed; a uniform
turbidity with pellicle develops in broth, and a moist
cream-like expansion, dull and never shining, forms over
the whole surface of potato.

B. ramosus (wurzel bacillus] strongly reduces nitrates
to nitrites. The bacillus is Gram -positive, about 7 ju
long and 1-7 p broad, with rounded ends. It occurs in
long threads, and has resistant spores. On gelatin plates
cloudy centres, with root-like branches extending in every
direction, are seen; the gelatin is slowly liquefied. In
gelatin stabs a slight depression is seen after the second
day, whilst the needle track in the depth has a greyish
woolly appearance. The whole contents of the tube then
becomes liquid, a tough pellicle forming on the surface.
It grows on carbolated media. It grows rapidly over
the whole surface of agar; in the depth is seen the charac-
teristic woolly appearance. A white dry expansion is
formed on potato.

B. mesentericus fuscus and B. mesentericus vulgatus
(' potato bacillus ') are found on vegetables and in water.
They are short, motile, sporulating, Gram-positive organ-
isms, which liquefy gelatin. Fuscus forms a yellowish-
brown growth on agar, while vulgatus gives a dirty white
growth. Bed (ruber) and black (niger) varieties are also
known.

B. tholoeideum. — An intestinal organism invariably
found in sewage. It produces a septicaemia in mice and

BACTERIOLOGY OF SHELLFISH 207

guinea-pigs, and gives yellowish growths on potato and
gelatin streaks.

Hydrogen- Sulphide- forming Bacteria. — Faeces, both
human and animal, contain bacteria capable of producing
hydrogen sulphide from peptone v/ater. Chamot and
Redfield (abst. Analyst, 1915, 351) found this group do
not actively ferment carbohydrates.

The Bacteriology of Shellfish.

Epidemics of typhoid have arisen through the ingestion
of bivalves arid molluscs obtained from beds polluted
with sewage containing typhoid bacilli (see p. 100).
Oysters are examined for sewage pollution by Houston's
method. Ten oysters are taken from a sample, thoroughly
cleansed by scrubbing in tap-water and rinsing in sterile
water. They are opened aseptically, and the fish is
minced and added, together with any fluid contained in
the shell, to sterile water in a sterile graduated cylinder,
and the bulk is made up to a litre with sterile water.
Each 100 c.c. of the emulsion represents one fish. With
the liquor thus obtained cultures are made in —

(a) Litmus lactose bile-salt peptone-water for detecting
B. coli, amounts being taken varying from 100 c.c. to
O'OOOl c.c., representing quantities of the fish from one
fish to one-millionth of a fish.

(b) Sterile milk for detecting B. Welcliii, amounts
being taken of from 10 c.c. to 0-001 c.c., representing
quantities of fish from one-tenth of a fish to one-hundred-
thousandth of a fish.

Agar and gelatin plates are also made for enumeration.

Houston's standards are— To reject oysters containing
1,000 (lenient standard) or 100 (stringent standard)
B. coli, or 100 (lenient standard) or 10 (stringent standard)
spores of B. Welchii, respectively per oyster.

On the other hand, Hewlett, finding that oysters from
pure layings contain no B. coli, is of opinion that even
ten B. coli per fish should be viewed with suspicion.

Nash is convinced that large numbers of ' indication '
organisms will not always justify the conclusion that the
shellfish have been exposed to what is generally under-
stood by ' sewage ' pollution — that is, human sewage
coming down through a sewer outfall. ' Cattle, sheep,
and gceso are grazed on low-lying marsh-lands, which

208 AIDS TO BACTERIOLOGY

at exceptional tides, and sometimes even with ordinary
tides, are covered at high- water. These animals (and not
least among them the geese) deposit on the marsh-lands
a very large amount of excrement, which is washed off
the marsh-lands at high-water. Cockles and mussels
especially abound in the vicinity of these grazing grounds,
which are often intersected by creeks. The sand in these
creeks will often be found quite black under the surface,
and even the shells of the cockles quite discoloured, and
yet there may be no sewer outfall within several miles.'

Letts attributed the excess of Ulva latissima in Belfast
Lough as being due to its sewage-polluted waters. Nash,
however, demonstrated that Ulva latissima may be found
in creeks or on shores adjacent to extensive tracts of low-
lying marsh-lands which are used for grazing purposes,
but yet miles from any large sewer outfall. He therefore
concluded its presence on mussels to be no indication of
a polluted source.

The Bacteiiology of Meat.

In addition to examination for trichina, vermes, etc.
meat may have to be examined for tuberculosis and other
animal diseases which are dealt with under their re-
spective monographs, or for organisms causing food
poisoning (see B. enteritidis, p. 93, B. botulinus, p. 83,
B. paratyphosus ft, p. 95, and B. suipestifer, p. 96). A
filterable virus, a variant of B. coli,B. proteus, and JStaphy-
lococcus pyogenes aureus, have also been described as organ-
isms producing food poisoning. The isolation of proteus
from suspected material, even if it gives a positive agglu-
tination, requires cautious consideration before it is
connected with the disease, as McWeeney has shown that
it tends to undergo auto-agglutination.

Varieties of Phycomyces, Penicillium, Mucor, Verti-
cillium, and Oospora, are the principal moulds that are
found on refrigerated meat. ' Red spot ' is due to
B. prodigiosus, and Cladosporium herbarum, causing ' black
spot/ is frequent. Klein investigated some brown spots
on frozen meat, and found a variety of yeast. Experi-
ments with animals showed that the yeast was not harm-
ful. Black spots are sometimes present in Argentine
beef on the lower or thin parts of fascia, in the fatty
portion of the thick parts, and in the fat belonging to

BACTERIOLOGY OF MEAT 209

the inner surface of the flanks. Klein found Oldium
carnis to be the cause, and states that it is harmless to
animals.

B, fcedans (Klein) causes taint in miscured hams, which
have undergone ' dry curing ' only. It is a short cylindri-
cal rod producing chains and filaments, and is an obliga-
tory anaerobe, non-motile, Gram-positive, and apparently
non-sporing. Growth does not take place on ordinary
media, but glucose gelatin, glucose broth, and particularly
glucose pork broth, were found suitable. Milk acquired
a nauseating odour, but guinea-pigs were unaffected by
subcutaneous injection.

Beveridge, Fowler, and Fawcus, describe an organism
isolated from blown tins of corned beef, which is appa-
rently identical with B. cadaveris sporogenes (Klein) and
B. putrificus coli (Bienstock). This organism is an
obligatory anaerobe, motile, with a slow wavy movement,
and forms large terminal spores. In four or five days
at blood-heat litmus-milk is decolorised, and sometimes
clotted. Later slow digestion of the casein takes place,
leaving a clear yellow whey. Digestion is complete in
seven to fourteen days, merely leaving a granular deposit.
Plentiful gas is formed in neutral red glucose agar, and
in all cultures it gives off an exceedingly putrid odour.
The organism is apparently non-pathogenic, but decom-
poses tinned meats and renders them unfit for consump-
tion. The spores are only killed by a temperature of
112° C. in twenty-five minutes, 115° C. in ten minutes,
or 117° C. in less than five minutes. Tinned meats often
contain sporing organisms of the B. subtilis (p. 205) and
mesentericus (p. 206) groups. Wanhill and Beveridge
incubate tins at 37° C. for fourteen days, when bulging
indicates the presence of gas-forming bacilli, probably
cadaveris.

In some cases of tinned meats which have caused
food poisoning nothing unusual is noted in the appear-
ance, but in others the gelatin is liquid at ordinary tem-
peratures, or discoloration, sickly odour, or a soapy taste,
is noted. For further information see Savage's Report
to L.G.B. on Bacterial Food Poisoning and Food Infec-
tions, 1913.

As food is still sent to analysts to be examined for
' ptomaines,' the following quotation from a Memorandum

210 AIDS TO BACTERIOLOGY

by the Local Government Board (September, 1911) is
given as representing the considered opinion of the best
authorities on the subject: ' Ordinarily little is to be gained
by so doing. It is by no means certain that " ptomaines "
in the sense of alkaloidal substances produced by bacterial
action are present in meat foods which have caused
poisoning, and the significance of the reactions which are
held to demonstrate the presence of these substances is
a matter of considerable doubt.'

The Bacteriology of Soil.

Surface soil is very rich in bacteria, the number below
a depth of 1 metre being small. Frankel found that the
superficial layers of the soil of a fruit orchard contained
from 50,000 to 350,000 organisms per gramme. The
greatest number was at | to £ metre below the surface.
At a depth of from f to 1| metres there was a very abrupt
diminution in the number of bacteria. From 200,000
organisms at a depth of £ metre, the number fell to 2,000
at a depth of 1 metre, from 250,000 at f metre to 200
at 1 metre. In virgin soil there is a dividing-line at a
depth of from f metre to 1| metres, below which very
few bacteria are found, thus showing the ground water
region is quite free, or nearly free, from micro-organisms,
notwithstanding the vast number upon the surface of the
soil.

Houston found surface virgin sandy soil to contain less
than 100,000 bacteria per gramme, other virgin soils
about 1,000,000, garden soil from 1,000,000 to 2,000,000,
and grossly-polluted surface soils up to 115,000,000
bacteria per gramme. Houston regards B. mycoides and
the Bismarck brown cladothrix as especially characteristic
of soil.

James Buchanan Young found the soil of graveyards
to be very rich in micro-organisms, particularly those of
a liquefying type, Proteus vulgaris being present in
great numbers. Their action is so effective that he found
no notable quantities of organic carbon and nitrogen in
the upper layers, and not so very much more in the lower
layers than are found in virgin soil. He found, on the
whole, that the bodies do not greatly influence the number
of micro-organisms found.

Two peat organisms, * O ' and ' Q' (Houston), produce

BACTERIOLOGY OF SOIL 211

acid when grown in peat-infusion, which has a great
solvent action upon lead. It is to these organisms that
the plumbo-solvency of waters from peaty districts is
attributed. Experiments to determine the longevity of
the typhoid bacillus in various soils have given variable
results. Thus, Savage found it to live in highly-polluted,
unsterilised mud from a tidal river for two weeks in one
experiment, and for five weeks in another. Firth and
Horrocks found that in a sewage -polluted soil recovered
from beneath a broken drain the organism survived for
sixty-five days.

When completely frozen, there is an unexpectedly
rapid multiplication of organisms. H. J. Conn thinks
there may be two groups of soil bacteria, one flourishing
in summer, the other in winter.

Nitrogen Fixation. — A number of organisms absorb or
* fix ' nitrogen from the atmosphere, notably Clostridium
Pastorianum, a sporulating anaerobe, some thermophilic
bacteria, and the aerobic ovoid organisms Azotobacter.
Botrytis cinerea, Aspergillus niger, Penicillium glaucum
— in fact most fungi — and a pseudo-yeast, Tulare No. 466
(Lipman), also fix nitrogen. The fixation of free atmo-
spheric nitrogen by Lolium temulentum, or darnel, is due
to the fungus which infests it. This grass has no sym-
biotic root organisms. Leguminous plants possess on their
roots little tubercles containing ' bacteroids.' Bacteroids
may occur as rods or branching structures. When
inoculated into suitable media, they give rise to the nodule
bacteria (B., Pseudomonas or Rhizobium radicicola) which
measure about 5 /n by l/u. The latter do not form spores,
are actively motile, and are strict aerobes. The organisms
enter the plant through the root hairs, where on multi-
plication they produce a filamentous zooglcea which
grows into the root and causes production of nodules.
These take up atmospheric nitrogen and convert it into
a form assimilable by the plant. Atmospheric nitrogen
is absorbed in greater quantity in the legume than in
artificial media. The plant, so far from welcoming the
organisms, offers resistance to their attack; this resistance
is most evident where free potassium nitrate is present,
when nodules do not form, because the ' nitrogen hunger '
is lessened. The association of the plant and bacteria
cannot, therefore, be regarded as true symbiosis.

212 AIDS TO BACTERIOLOGY

Success has not always followed the use of pure cultures
of the nodule bacteria. Bottomley's preparation of Azo-
tobacter is in the form of a powder. It remains virulent
for months, and has proved highly successful when
intelligently used. Bottomley has shown that by asso-
ciating together Azotobacter smdPseudomonas a very much
greater quantity of nitrogen is collected from the air,
and fixed in a form available as a plant food, than can be
fixed by them acting independently. He treated moss
litter with the nitrogen-fixing bacteria and other soil
organisms, and incubated for three weeks. Ammonia
was formed, which combined with the humic acid present,
making the whole alkaline and producing a large quantity
of soluble humates available for plant growth. The peat
was then sterilised by heat, and a pure culture of Bacillus
radicicola and the Azotobacter chroococcum added. After
a further period of incubation, the peat contained weight
for weight about fifteen to twenty times as much humates
as were present in two-year-old rotted farmyard manure.
With soil rich in nitrogen, bacterised peat is of little use,
but on poor soils experimental croppings after its use
have proved highly satisfactory. Professor Bottomley
describes substances which he calls auximones, which are
comparable to vitamines, in the treated peat.

The small amounts of iron and silicates in humus have
been shown to assist Azotobacter chroococcum, which
explains the favourable action basic slag has on the
organism. Bottomley says a good medium for both
Azotobacter and Pseudomonas, or for a mixed culture of the
two, may be obtained by adding to distilled water 1 per
cent, of dextrin, 0-2 per cent, of dipotassium phosphate,
0-02 per cent, of magnesium sulphate, and 0-4 per cent,
of basic slag.

A mild activation of the air by pitchblende has been
found to increase the amount of nitrogen fixed by Azoto-
bacter. Brilliantly successful results have sometimes
followed the addition of soil from a place where the
nitrogen bacteria were abundant to an area where develop-
ment of plants was slow.

Nitrification. — The conversion of nitrogenous substances,
as found in dejecta, cadavers, etc., into nitrates (the form
in which plants absorb nitrogen), takes place in three
stages, each being produced by bacteria, (a) Ammonisation:

BACTERIOLOGY OF SOIL 213

The first products of the putrefaction of proteins by
Proteus organisms are further broken up by B. mycoides,
B. subtilis, B. mesentericus, B. putrificus, B. fluorescens
liquefaciens, B. tumescens, and other organisms, to produce
ammonia. (6) Nitrosation: The ammonium salts are
converted into nitrites by Nitrosomonas, a variety of
Pseudomonas (short, fat, motile organisms). These or-
ganisms differ from ordinary bacteria, but have definite
life-cycles, which, however, differ with the locality. They
appear to derive their supply of carbon from carbon
dioxide alone, (c) Nitratation: An apparently different
class of organism (Nitrobacter) from those concerned in
nitrosation converts the nitrites into nitrates.

Denitrification. — A few organisms reduce nitrates with
production of free nitrogen — e.g., B. denitrificans and
Denitrobacterium thermophilum, the latter growing at
65° C. Others can only carry the action so far as the
formation of nitrites and ammonia — e.g., B. butyricus,
B. mycoides, B. subtilis, and B. tumescens.

Partial Sterilisation of Soil. — By heating soil to 55° to
60° C., protozoa and nitrifying organisms are destroyed,
while denitrifying organisms and those that produce
ammonia remain alive, the result being enhanced ammonia
production. Effects vary very much, according to the
nature of the soil and the temperature employed. Speak-
ing generally, the tendency is for both organic and in-
organic matter to become more soluble. At high tem-
peratures, such as 250° C., calcium and magnesium become
less soluble. It appears that a toxin is produced which,
though inimical to plant growth at first, eventually
benefits it. Steam has proved most effective, and Russell
and Buddin consider that in vegetation experiments a
steamed soil should be included as a standard.

Partial sterilisation may also be effected by the applica-
tion of antiseptics. Lime, formaldehyde, chloroform,
toluene, pyridine, calcium sulphide, carbon disulphide,
and cresol have been used for the purpose of destroying
protozoa and increasing ammonification.

Examination of Soil. — When the deeper layers are to
be examined, care must be taken to prevent contamination
with the other portions, particularly the upper layers.
Frfinkol devised an instrument for taking samples from
various depths. A borer contains at its lower end a small

214 AIDS TO BACTERIOLOGY

cavity, which can be closed up by turning a handle one
way, or opened by turning in the opposite direction. The
borer is pushed down to the necessary depth; the handle
is then turned; the earth enters the cavity; the handle is
again turned, enclosing the sample of earth completely;
the borer is then withdrawn. The soil is thoroughly
mixed with melted nutrient gelatin, which can be poured
into a Petri dish, or, better, made into a roll culture by
Esmarch's method. Another method is to wash the soil
with sterile water, which is examined, as usual, by the
plate method.

The existence of faecal pollution, and whether it is recent
or distant, is regarded by Houston as being shown by the
following factors: Total number of aerobic organisms;
number of spores present; estimation of B. coli, B. Welchii
and streptococci. Houston found -that streptococci dis-
appear rapidly, and regards them as indicating very
recent pollution; the spores of B. V/elchii, on the other
hand, persist for longer periods.

Tetanus and malignant oedema bacilli are detected by
inoculating glucose formate broth, heating to 80° C. in
a water-bath for twenty minutes, and then cultivating
anaerobically.

The Bacteriology of Air.

No particular organisms can be regarded as character-
istic of air. Those that do occur are derived from dry
surfaces, and are carried with the dust by air-currents.
A diminution in numbers occurs with an increase in alti-
tude, with an increase of distance from towns into country,
and in mid- ocean the air is nearly sterile. The species
generally found are spores of moulds, yeasts, bacterial
spores, and chromogenic bacteria. Owing to the influence
of desiccation, B. coli is seldom met with. The presence
of tubercle bacilli in dust has been abundantly proved.
In sputa found on the ground tubercle bacilli were present
in 60 per cent. Examinations of sewer air have given
discordant results. Some find half the number of organisms
present in external air, and that organisms normal 1o
sewage are comparatively rare. Andre wes found strepto-
cocci, generally corresponding with the S. salivarius
type, while the S. equinu-s type is most abundant in fresh
London air. He also found numerous B. coli. Horrocks

BACTERIOLOGY OF AIR 215

has shown that in the drainage system of an ordinary
private house splashing allows the disengagement of
bacteria passing down soil-pipes, which bacteria can be
carried along a 4-inch drain, against the flow of sewage,
for at least 50 feet by ventilation currents.

Gordon uses the Staphylococcus epidermidis albus as
an index of pollution of the air with material detached
from the skin. He also detects pollution by material
brought in from the street on boots by the presence of

B. coli, spores of B. Welchii, certain streptococci, and
sometimes B. mycoides.

Gordon uses a streptococcus as an indicator of con-
tamination of air with saliva. He found the chief or-
ganism present in saliva to be a streptococcus, present to
the extent of never less than 10,000,000, and sometimes at
least 100,000,000, per cubic centimetre. This strepto-
coccus was of the brevis type, growing well anaerobically,
and best at 37° C., generally clotting milk with the pro-
duction of acidity, and producing acid in glucose, lactose,
fructose, maltose, and galactose media. It reduced
neutral red much in the same manner as the colon bacillus
— viz., the cherry-red became changed to a yellowish -green,
and was generally non- virulent to mice. Its growth in
neutral red broth served as a means for isolating it. The
method adopted to examine for contamination with saliva
was to use ' broth * plates — i.e., a tube of neutral red broth
was poured into a Petri dish, exposed to the air, then poured
back into the tube, which was incubated anaerobically at
37° C. The change in the broth, together with a micro-
scopical examination, sufficed to show the presence of the
streptococcus.

Streptococci appear to bulk largely in domestic dust.

C. A. E. Winslow obtained 22,700 acid-forming streptococci
per gramme from an average of nineteen samples of dust
taken from New York schoolrooms.

Filtration of Air. — Dry cotton-wool, a dry Pasteur -
Chamberland filter, a sufficient number of bends in a
narrow tube, or, best of all, glass-wool and sugar, prevent
the passage of organisms.

Examination of Air. — For qualitative or comparative
examinations, Petri dishes containing a solid medium
may be exposed.

Hesse's apparatus consists essentially of a glass cylinder

216 AIDS TO BACTERIOLOGY

70 cm. by 3-5 cm., covered at one end by two rubber
caps, the inner one having a hole in its centre 10 mm.
in diameter, and at the other end a rubber cork fits in the
cylinder. Through this cork a glass tube 100 mm. in
length passes, which is plugged with cotton-wool. The
cylinder is sterilised for one hour in the steam steriliser.
The rubber stopper is removed, and 50 c.c. of nutrient
gelatin in a fluid condition is introduced into the tube and
rolled out on the sides, as in the preparation of an Esmarch's
tube, leaving a somewhat thicker coating along the under
side of the cylinder. The cylinder and its fittings are
mounted on a tripod stand, and the glass tube which passes
through the rubber stopper is connected by means of a
rubber tube with an aspirator, the cotton having first
been removed from its outer end. The aspirator most
suitable for the purpose is the double wash-bottle arrange-
ment, which is conveniently attached to the stand by means
of hooks. The outer rubber cap is then removed and the
aspirator started. Air is drawn through the tube by
suction, the micro-organisms contained therein falling
on the gelatin. The amount of air entering is estimated
by the capacity of the flasks forming the aspirator. The
rate at which it enters is controlled by the flow of the water,
which can be regulated by a pinchcock. Hesse advises
that the amount and the rate of flow for rooms and closed
spaces should be about 1 to 5 litres, passed at the rate of
\ litre a minute. For open spaces 10 to 20 litres is passed
at about four minutes per litre. The tube is then capped
and the colonies allowed to develop, after which they can
be further examined by subcultures.

Petri's Method. — The air is led through glass tubes
packed with fine sand, kept in place by plugs of glass-
wool, the organisms being retained by the sand filter, and
afterwards the sand is pushed out of the tube with a
sterile wire and plated out in gelatin. In this method the
sand, being insoluble, is troublesome, interfering with a
clear view of the colonies.

FranklaruTs Method. — Except for the substitution of
powdered cane-sugar for the sand, the process is the
same as that of Petri.

Sedgwick and Tucker's Method. — A special tube, re-
sembling a narrow cylindrical separating funnel, without
a stopcock, is used. Powdered cane-sugar is packed into

BACTERIOLOGY OF MILK 217

the narrow part, and kept in place with glass-wool plugs
The volume of air having been aspirated through, the
sugar is pushed into the wide portion, a tube of melted
gelatin poured in, and after the sugar has dissolved a
roll-culture is made in the tube.

Chattaway and Wharton's apparatus measures the air
driven through a glass tube by a rubber puff-ball bellows.
The air is met by a jet of nutrient medium automatically
sucked up by the current of air, as in a scent spray.

The Bacteriology of Milk.

Although the 'fore-milk' (the first portion drawn)
generally contains bacteria, which have obtained access to
the milk ducts since the previous milking, the milk in the
udder of a healthy cow is sterile. After leaving the cow ,
excremental and other particles from the cowshed, the
hide, and the milker's hands, start a contamination which
does not cease to be augmented from extraneous sources
till the milk is consumed. Especially in poor neighbour-
hoods, flies are prominent factors in the pollution of milk.
Cox, Lewis and Glyn have shown that a housefly, while
drowning, may shed from 2,000 to 350,000 bacteria.
Flies are garbage feeders, and the bacteria carried in or
on their bodies are never desirable, and often pathogenic.
It is now proved beyond all doubt that flies can and do
infect milk with the organisms producing epidemic
diarrhoea of infants, typhoid, cholera, and dysentery.

Milk is an admirable medium for the growth of bacteria ,
and rapid multiplication takes place. Eyre states that
the numbers in London are about 3,000,000 to 5,000,000
in December, January, and February; and 20,000,000 to
30,000,000 in June to September. Milk may be the
medium by which the following diseases are conveyed
to man: Tuberculosis (pp. 62, 64), typhoid fever (p. 101),
sore throat (p. 134), diphtheria (p. 115), scarlatina (p. 195),
bacillary dysentery (p. 107), cholera (p. 154), Malta fever
(goat's milk, p. 145), foot-and-mouth disease (p. 199), and
infantile diarrhoea (p. 196). Milk is, in addition, subject
to various diseases peculiar to itself.

Blue Milk. — Blue patches are formed on the surface
by B. cyanogenus, a small motile multi-flagellate bacillus.
The organism does not liquefy gelatin, which, however, is
stained bluish-green, finally becoming of a dirty greyish

218 AIDS TO BACTERIOLOGY

tinge. The growth on potato is a thick, dirty yellow layer,
which afterwards becomes blue; the medium is dis-
coloured. It gives an alkaline reaction in ordinary milk,
and does not produce coagulation. It usually yields two
pigments— one of the ordinary fluorescent type, and the
other of a bluish to greyish colour, which becomes more
strongly blue up to azure in milk with an acid reaction.
Blue milk may also be caused by other organisms.

Red Milk. — Haemorrhage from the udder, B. prodigi-
osus, Sarcina rosea, or Saccharomyces ruber, may give
rise to red milk, and the last may cause infantile diarrhoea.
Generally B. lactis erythrogenes (Hueppe) is to be found —
a short bacillus, liquefying gelatin. The colonies are of
a yellow colour when first seen, but after liquefaction
they become rose -red. A yellowish deposit occurs on
agar, which soon changes to yellowish-red. The cultures
give rise to an unpleasant, sweet smell. In milk the red
coloration is developed best when the medium is slightly
alkaline and kept in the dark, and is checked by acidity
and light. On standing, the cream rises as a yellowish
layer and the casein is precipitated, though the reaction
remains alkaline and the clear serum is pink.

Yellow Milk. — The best-known organism causing this
disease is B. synxanthus, a motile rod, curdling milk by
means of a rennet-like ferment, which afterwards redis-
solves the curd and produces a yellow pigment.

Bitter Milk may be due to the ingestion of certain
plants by the cow. Among the several organisms capable
of producing bitterness is the bacillus of Bleisch, a flagel-
lated, facultative anaerobe, rapidly liquefying gelatin, and
producing a thin, flat, greyish growth on agar and potato.
In milk it will, after a week, produce transparent yellow
streaks below the cream, the milk itself coagulating, and
the coagulum being subsequently, earlier or later, almost
completely dissolved. The bitter taste arises after the
second week; there is no smell; the reaction is acid. At
higher temperatures the milk becomes bitter, and gives
the biuret reaction after twenty-four hours, while spores
are produced which resist boiling for six hours. Conn's
micrococcus of bitter milk coagulates milk, and then pro-
duces a slimy solution, with a slightly sour and very bitter
taste. Trillat and Sauton found bitter milk to contain
aldehydes and ammonia.

BACTERIOLOGY OF MILK 219

Stringy or Ropy Milk. — When poured from a jug, ropy
milk takes a rope-like form. If a few drops are placed
between finger and thumb, and these separated, the ropy
milk draws out in a thread. Ropiness may be due to
milk coming from an inflamed udder ('garget' milk), in
which case the milk is unfit for food. In other cases,
where the bacteria concerned have an extracorporeal
origin, the milk, ' though abnormal, is quite wholesome,
and does not endanger public health ' (Board of Agri-
culture Leaflet No. 266).

Water used for washing churns sometimes transmits
the organisms to the milk, or the cows may stand in
ponds containing them. They may be derived from dust,
straw, mouldy hay, and butterwort.

The following organisms produce stringiness:

B, lactis pituitosi (Loffler). — A stout, slightly curved
rodlet, which does not liquefy gelatin (see below).

B. lactis viscosus (' the viscid milk bacillus ' — Adametz)
is a very short rod, aerobic, and does not liquefy gelatin.
Its effect is apparent after four or five days, and is con-
tinued for four weeks, by which time the milk corpuscles
have practically disappeared, and the milk is transparent.
The casein is not precipitated; no acceleration of the
process occurs on a rise of temperature, and there is no
special smell.

Streptococcus Hollandicus is used in the manufacture of
Edam cheese. It does not liquefy gelatin; it renders
milk stringy within twelve to fifteen hours at a tempera-
ture of 77° F., the milk becoming sour at the same time.

Norwegian taettemaelk is made with a ' stringy ' milk
bacillus.

Soapy Milk. — B. lactis saponacei (Weigmann and Zirn)
has been found in straw used as litter. It does not
coagulate milk, but makes it slimy and slightly ropy, with
a faint soapy taste. It grows best at 10° C.

Slimy Milk is attributable to various organisms. Micro-
coccus viscosus (Schmidt-Miihlheim) is of 1 ^u diameter,
often occurs in wreathed chains of fifteen or more cells,
and produces a slime from the milk-sugar. The process
seems to differ from that of slime production in wine,
in that it forms no mannite and no carbonic acid. B. lactis
pitintosi of Loffler gives a specific smell, and renders the
milk slimy and slightly acid, especially at the lower part.

220 AIDS TO BACTERIOLOGY

Salty Milk may be watery (1027 to 1029 specific gravity).
It is stated to occur only in connection with inflammation
of the udder. It is detected by taste, its high percentage
of ash, and by its low percentage of milk-sugar. Accord-
ing to Klenze, 2-4 per cent, of small deposits of calcium
carbonate in the milk glands may give rise to sandy milk.

White Mould. — See O'idium lactis, p. 17.

For practical purposes, it may be said that any milk
which goes sour rapidly is a bad milk, although the con-
verse is not necessarily true. According to Schatzmann,
if a sample of milk be kept for twelve hours at 40° C., and
within that time coagulates, it is to some extent defective,
and in nine hours no change whatever should appear to
have occurred. The presence of colostrum is a ground
for the condemnation of milk; it can usually be detected
by the presence of long, elastic, yellowish threads.

Sterilisation. — Fractional sterilisation is too lengthy a
process for use commercially, so recourse is had to Pas-
teurisation (see 'Aids to the Analysis of Food and Drugs') —
i.e., the milk is heated to 62° or 68° C. for thirty minutes.*
This kills all, or nearly all, the pathogenic bacteria that
are likely to be present in milk, but also destroys the
lactic acid bacteria. After pasteurising, the spore-
forming bacteria which will escape develop, unchecked
in the absence of lactic acid bacteria, and may turn the
milk rotten, often with no outward sign of its condition.
For this reason milk once pasteurised should be consumed
within eighteen or twenty-four hours — i.e., before sporu-
lating bacteria have had time to multiply to an objection-
able extent. Pasteurisation affords milkmen a con-
venient method of preserving surplus milk till the follow-
ing day, but the period during which milk remains in
a house is so short that, as a general rule, probably twenty -
four hours do not elapse between Pasteurisation and
consumption.

Reporting on the electrical treatment of milk as applied
by E. W. Hope, Beattie says that there is a reduction in
content of bacteria of 99-25 per cent., and colon and
tubercle bacilli are destroyed.

* See also p. 60. Sir John McFadyean (Veterinary Congress, 1914)
also advocates raising milk to 85° C., and considers there is evidence,
already ample in amount, that milk so treated is from a nutritive
point of view in no way inferior to uncooked milk.

BACTERIOLOGY OF MILK 221

The Budcie process involves the addition of hydrogen
peroxide, and the heating of the milk for three hours to
52° to 53° C. Meat extract removes the peculiar taste
left by the treatment. In Behring's method 1 ounce of
perhydrol is added to 6 gallons of milk, and the milk
is then heated to 122° F. Behring believes that milk
loses some of its best qualities when exposed to daylight.
He advocates green or red milk-bottles.

In the summer months it is a common practice to run
the milk as soon as possible through a cooler, which serves
to delay bacterial development. One of Houston's
' counsels of perfection ' is the cooling to, and maintaining
at, a temperature of 10° C. of the milk. The standard of
the Academy of Medicine of Toronto requires the milk to
have been cooled to 45° F. within half an hour after milk-
ing, and kept at a temperature not exceeding this till
delivered. They also impose an age limit of twenty-four
hours. '

Bacteriological Examination of Milk.

Sediment. — This examination is carried out, not only
as a qualitative test, but also to obtain an idea of the
number of leucocytes present. It may safely be said that
no one can differentiate between a pus cell and a leuco-
cyte in a milk sediment, and it is only when the cells
occur in excessive numbers, or accompanied by pyogenic
organisms, that pus can be certified as present. At the
same time, it must be remembered that Revis and others
have shown that a very large leucocyte count can be
obtained from the milk of healthy cows. Uncertainty
exists as to the significance to be attached to the relative
numbers of leucocytes and streptococci. Savage (Journal
of Hygiene, April, 1906) has shown the absence of any
relationship between these in the milk of healthy cows;
but he also calls attention to the fact that the number
of pus cells present in milk from inflamed udders would
be much higher than in the milks he examined.

A number of different processes for estimating leuco-
cytes, allowing varying degrees of inaccuracy, are in use,
with the consequence that the data have little significance
to anyone except the operator. Savage (ibid.) dilutes
the milk with Toisson's fluid, and, after centrifuging,
counts the leucocytes in the Thonia-Zeiss counting

222 AIDS TO BACTERIOLOGY

apparatus used in haematological work. This method seems
to allow a closer approach to accuracy than most others.
Houston, in his report to the L.C.C., dilutes the ' filth '
obtained by his apparatus, and makes definite-sized
cover-glass preparations with 0*01 c.c. of the sediment.
The number of cells found in the sediment bears no con-
stant proportion to the total number present in the milk
(Breed).

Examination of Sediment. — The strippings, or milk last
drawn, is the best for examination purposes (Mettam).
The milk is centrifuged for twenty minutes at 1,500 revo-
lutions per minute. At about half-time, the centrifuge
is stopped and the cream stirred up in the upper part of
the milk. This disentangles a good many organisms,
cells, and other matter, that have been lilted up by the
fat globules. The centrifuging is completed, and the fat
and liquid tipped out. Three slide preparations are made,
each with four drops of the sediment, which are spread
evenly over three-fourths of the slide. The slides are
air-dried, and then treated with a mixture of absolute
alcohol and ether for ten minutes. One slide is stained
with Loftier' s blue, another by Gram's method, and a
third by the Ziehl-Neelsen method. The L6 flier's blue,
specimen gives a general idea of the number and kinds
of bacteria and cells present. The number of leucocytes
in twenty fields should be counted, and if they average
more than twenty per field (with a j1., inch), it is pretty
certain that pus is present. This is confirmed if in this
and the Gram specimens large numbers of streptococci
of the longus type are found. Confirmatory evidence is
obtained if in a fresh specimen (undried) of the sediment
mounted in a drop of Gram's iodine solution red blood-
cells are seen in addition. The sediment should also be
examined fresh with the low powers (§ and ^ inch) for
gross and filth contaminations — e.g., hair, straw, sand,
vegetable matter, etc.

The slide stained by the Ziehl-Neelsen method should,
after treatment with acid, be treated with alcohol
(p. 74) to decolorise any smegma bacilli, which are some-
times found in milk. Unless tubercle bacilli are found
before, this slide should be searched for at least half an
hour. It must be noted that milk may contain such
acid-fast saprophytes as the Timothy-grass bacillus, and,

BACTERIOLOGY OF MILK 223

on the contrary, tubercle bacilli may be missed. Should
organisms morphologically resembling the tubercle bacillus
be found in a sample from a herd, inspection of the beasts
will often reveal an animal showing signs of the disease.
The milk from such must be proscribed, pending the
result of an animal experiment: Two guinea-pigs are
inoculated, one subcutaneously, one intraperitoneally,
each with 1 to 2 c.c. of the sediment.

Although probably mistakes do not often occur, the
guinea-pig test is not infallible. The experimenter may
be misled by the lesions produced by Timothy-grass
bacilli, Rabino witch's bacillus, the ' Mistbacillus ' or
Johne's bacillus. Conversely, error may occur in the
opposite direction, for negative results may be obtained
with guinea-pigs inoculated with milk from tuberculous
udders. The test usually occupies four or six weeks.
In spite of these disadvantages, it is the most reliable
method at present known for detecting tubercle bacilli.

Examination for the Diphtheria Bacillus. — A number
of serum-tubes are inoculated. If an organism resembling
the Klebs-Loffler bacillus be isolated, it must be sub-
mitted to the test of inoculation, since bacilli are not
infrequently present in milk and milk products which,
though resembling the Klebs-Loffler bacillus morpho-
logically and culturally, are non- virulent.

Examination for the Typhoid Bacillus. — The milk can
be examined by one of the methods described under
' The Examination of Water.'

Enumeration of Organisms. — This is not generally per-
formed as a routine test, and there are various objections
to attempts to use it as an indication of purity. Should,
however, a count be desired, plates must be made on the
decimal system to show a range of organisms from 20,000
to 20,000,000 organisms per cubic centimetre. Dilutions
of from 1 in 1,000 to 1 in 100,000 should be made. Eastes
recommends distilled water agar.

Counts made directly on microscopical preparations o£
milk are higher than those obtained by plating (Brew).

Estimation of B. Coli. — Quantities of 10 and 1 c.c. of
milk are inoculated into MacConkey bile-salt lactose
peptone medium tubes, and decimal dilutions containing
from 0-1 to O'OOOOOl c.c. of milk are inoculated into tubes
of the same medium, using the same sterile 1 c.c. pipette,

224 AIDS TO BACTERIOLOGY

and commencing with the lowest dilution (No. 6) and
working up to the dilution containing 10 per cent, of milk
(No. 1). Tubes showing gas and acid are worked up for
typical B. coli, as described under ' Water.'

Enumeration of B. Welchii.-^Quantities of 100 and
10 c.c. of milk are placed in sterile tubes, and into milk-
tubes the following quantities are inoculated: 1 c.c.,
0*1 c.c., 0*01 c.c., and O'OOl c.c. The tubes are then
treated as described on p. 87.

Enumeration of Streptococci. — The dilutions used in
the examination for B. coli can be plated out on such
solid media as Conradi-Drigalski or bile-salt neutral red
lactose agar plates (see remarks on bile-salt media for
streptococci, p. 233). Streptococci appear on both as
small colonies, which should be subcultured and further
examined. Or the dilutions of milk may be inoculated
into glucose neutral red broth (first recommended by
Savage), and the tubes examined for chains, after incuba-
ting at 37° C. for twenty-four to forty-eight hours.

Houston separates the tests into two classes : multiply-
ing factors (B. coli and streptococcus tests, and enumera-
tion of organisms), and non-multiplying factors (B. Welchii
[Enteritidis sporogenes] and readings of volume of sedi-
ment).

The Toronto Academy of Medicine has fixed the fol-
lowing standard : Milk shall not contain during the months
of June, July, August, and September, more than 10,000
bacteria per cubic centimetre, as shown by a forty-eight-
hour culture on nutrient agar medium at 37° C.; nor in
the remaining months of the year more than 5,000 bacteria
per cubic centimetre, as demonstrated by the same test.

CHAPTER XXI
THE BACTERIOLOGY OF WATER

CERTAIN organisms are so frequently found in water that
they may be regarded as normal inhabitants thereof,
others are recognised as soil bacteria that have been
washed in, while the third group (bacterial indicators of
pollution) comprises organisms of intestinal origin. The
bacterial alvine flora of animals and birds resembles that

BACTERIOLOGY OF WATER 225

of man, with the consequence that it is often impossible
to identify the source of pollution by bacteriological
results.

Typhoid, cholera, paratyphoid, dysentery, and perhaps
anthrax, are the principal water-borne diseases. Polio-
myelitis, gore throat, conjunctivitis, suppurative otitis
media, and frontal sinus suppuration, are all supposed
sometimes to follow visits to swimming-baths used by
infected persons.

Several factors contribute to the increase or decrease of
the number of bacteria in water. Sunlight (p. 9) plays
an unimportant part in reducing the number, unless the
depth of water is very shallow and exposure is prolonged.
Cold inhibits growth of bacteria, and, ceteris paribus, a
higher bacterial content should be found in warm weather.
Other influences may, however, cause the winter content
of bacteria to be greater than that of summer. When
food material is ample in quantity more organisms are
met with. Most bacteria tend to settle to the bottom of
a bulk of water, and this sedimentation constitutes an
important factor in the self-purification of waters. Storage
is an important process in the purification of water on
a large scale. By storage time is given for any typhoid
or cholera bacilli to die out, owing to lack of nourish-
ment, to a temperature unfavourable to development (in
temperate climates), and to antagonism of normal water
bacteria.

In the M.W.B. reservoirs, Thames and Lea waters can
be stored for fifty-two days. The reduction in the number
of organisms is sometimes over 99 per cent. It has been
suggested that, owing to bacteria clumping during storage,
and each clump producing a single colony, and therefore
being counted as one organism, the diminution is more
apparent than real. While this may be the case, clumps
of bacteria settle quicker than do individuals, and the
clumping may accelerate sedimentation (see p. 99).

Houston found that when typhoid bacilli or cholera
vibrios were added to raw Thames, Lea, or New River
waters, a reduction of 99-9 per cent, of these organisms
in each case was attained in a week. Cholera vibrios
could not be found in three weeks, but eight weeks'
storage was found to be necessary for the disappearance
of B. typJiosus (cf. p. 99). This bacterial purification is

15

226 AIDS TO BACTERIOLOGY

known as the ' safety change.' Perhaps the tendency for
these two organisms to rise to the top of a bulk of water,
where the influence of light is possible, allows sunlight
to materially contribute to their ultimate disappearance.

Houston considers that the ' safety ' of an adequately
stored water may possibly come to be ' accepted so fully
as even to afford justification for filtration through mechan-
ical filters at a specially rapid rate, with the result of
thus compensating to a large extent for the initial cost
of the storage reservoir.' Houston's final conclusion is
that raw river water should be stored antecedent to
filtration, preferably for thirty days. Prescott and
Winslow (1913) conclude ' that any water which has been
stored for four weeks is practically safe.'

A pure water when freshly drawn, even if kept in a
sterile flask free from aerial contamination, will (after
a slight diminution in bacterial content during the first
three to six hours) exhibit a remarkable increase in the
content of bacteria. Less pure waters, such as those of
ordinary rivers, which contain initially a large number
of bacteria, exhibit, when similarly treated, a much less
conspicuous increase in their bacterial population. Frank-
land points out, however, that whilst in sewage the number
of organisms only gradually diminishes, in pure waters
' after the rapid increase in numbers follows a correspond-
ingly rapid decline, so that the numbers again fall below
those found in impure surface waters.' Multiplication
after sampling appears to be less in a large sample than
in a small one, and less when no air is admitted than with
free aeration. The composition of the bottle and the
temperature at which it is kept also influence the rate of
multiplication.

Collection of Water Samples. — Samples should be
collected in sterile glass-stoppered bottles. In sampling
a lake or stream, the bottle should be immersed with the
neck a foot below the surface and away from the edge
before the stopper is removed. A tap should be allowed
to run for five or ten minutes before a sample is taken,
as bacteria may multiply while water stands in the pipes.
When sampling a public supply, a tap directly off the
main or on a hydrant should be used, as passage through
a cistern introduces a factor of contamination unfair to
the water. Samples should only be taken from pumps

BACTERIOLOGY OF WATER 227

after pumping has been carried on for some time. A
sample from definite depths can be collected by small
vacuum bulbs, which are let down to the necessary depths
by means of a weighted wire or string; the drawn-out
point of the bulb is then broken by a suitable mechanical
arrangement (Sclavo's ' smash bottles '). If more than
three hours must elapse between sampling and com-
mencement of examination, the bottle must be kept or
packed in ice.

Bacteriological Examination of Water.

Although examination for a specific pathogenic organ-
ism is sometimes required, bacteriological examination
of water is generally directed at the demonstration of
absence or presence of sewage contamination. The
important water-borne diseases being usually of intestinal
origin, absence of sewage contamination shows fitness
for drinking purposes, unless of course chemical examina-
tion has shown metallic or other poison or excessive
mineral salts to be present. Of course, no examination
is a criterion of permanent fitness for use where inter-
mittent or sporadic pollution is possible.

A bacteriological examination may fail to indicate a
dangerous water where urine, but not faecal, contamination
occurs. In such a water there may be a high count,
but the absence of intestinal organisms will probably
produce a favourable bacteriological opinion. This form
of contamination is not so rare as is usually supposed,
and epidemics have followed when typhoid bacilluria has
contributed to it (see p. 99). Colon bacilli and streptococci
will also be absent from water receiving drainage from a
disused cesspool.

Bacteriological examination will also fail when water
has been heated, treated with antiseptics, or filtered, for
the purpose of misleading the bacteriologist.

A scheme drawn up by the committee appointed by
the Royal Institute of Public Health is very generally
followed in this country, according to which the irreducible
minimum of procedures should be —

(a) Enumeration of bacteria capable of growth at room-
temperature (18° to 22° C.). (6) Identification and enumera-
tion of B. coli, if present.

The majority of the committee recommend, in addition:

223 AIDS TO BACTERIOLOGY

(c) Enumeration of bacteria capable of growth at blood-
heat, (d) Enumeration of streptococci, if present.

B, Welchii is not often included among the bacteria
estimated, but cases occur where an estimation is de-
sirable.

Enumeration of ' Cool ' Organisms. — All media used
in counts should have a reaction of 4- 10 (Eyre's scale).
Nutrient gelatin is most often used for the ' cool ' organ-
isms, as it gives a relatively larger number of organisms
with a polluted water than does distilled water gelatin.
Conversely with an unpolluted water, a larger count is
usually obtained with distilled water gelatin than with
nutrient gelatin. So, although nutrient gelatin should
be used when only one gelatin medium is employed, the
use of both gives comparative figures of value.

Three tubes of nutrient gelatin are melted in a water-
bath at blood-heat or else in a blood-heat incubator.
Three sterile Petri dishes are inoculated with 0-5, 0-3,
and 0-2 c.c. of the water respectively and labelled. After
wiping the adherent water from the outside and flaming
the mouth of the tube, the contents of a gelatin tube is
poured into each inoculated Petri dish, and the water
and gelatin mixed by tipping the dish back and forth a
few times. The plates are allowed to solidify on a flat
surface, and then placed in the cool incubator. Counting
should be done at the end of seventy-two hours, but
plates should also be inspected daily, as the count may
have to be made earlier should too many liquefying
organisms be present. Counting is done with the naked
eye, preferably in daylight, any doubtful colony being
determined with the aid of a lens or low-power objective.

In a pure water it will generally be found that less than
10 per cent, of the organisms liquefy gelatin. With a
polluted water the ratio of the number of organisms
developing at room -temperature to those developing at
blood-heat approaches 10 to 1, and frequently becomes
10 to 2, 10 to 3, or even less. But, as Horrocks has
pointed out, the number of B. fluorescens liquefaciens and
non-liquefaciens may be abundant, and as they grow well
at blood-heat, not only may the ratio of liquefying to
non-liquefying organisms be misleading, but also the ratio
of ' blood-heat ' to ' cool ' organisms be rendered of no
value for an opinion.

BACTERIOLOGY OF WATER 229

In the purest upland streams and lakes the number of
bacteria in 1 c.e. is frequently under 100, while in town
sewage there are many millions in the same volume. In
ordinary rivers the number is generally between 1,000
and 100,000 per c.c. In water from deep-seated springs
the presence of more than 100 organisms per c.c. is con-
clusive evidence of some contamination with surface water.

In waters of poorer quality the numbers may approach
500 per c.c. Anything over this casts suspicion on the
water, and 1,000 per c.c. or more should probably condemn
the sample, always supposing, of course, that multiplica-
tion in vitro can be excluded by the proper storage of the
sample bottle in ice. In Victoria aerated waters may not
contain more than 40 bacteria per c.c.

Before taking a portion of the sample for any purpose
the water should be thoroughly shaken, the American
committee interpolating the procedure, ' Shake at least
twenty-five times the bottle which contains the sample.'
They also stipulate that the ' cool ' incubator should be
well ventilated, and that its atmosphere should be practi-
cally saturated with moisture. To avoid fictitious
accuracy they stipulate that numerical results should be
returned to the nearest 5, 10, 25, etc., number of organisms
depending on the number found. Counting a fractional
part of the organisms is sometimes done when plates are
crowded, by means of a Pakes's disc, but it is far prefer-
able to count all the organisms. As a thousand organisms
can be counted on a 10-centimetre dish, there is generally
little need to dilute, and if the organisms exceed this on
a plate made from 0*2 c.c., they can be truly returned as
" uncountable.'

Crowded plates are to be avoided, as, quite apart from
the labour of counting, the results tend to be low: some
colonies may be hidden under others, two colonies may
coalesce and only count as one, and the metabolic
products excreted by quick-growing species may diffuse
into the medium and inhibit growth of neighbouring
colonies. Therefore it is desirable that 2CO colonies
should be the maximum allowed on a dish, and when
dealing with a water known to contain a large number
of organisms, plates should also be made from dilutions
of the sample. It is very desirable that duplicate sets
of plates should be made for all counts.

2 3o AIDS TO BACTERIOLOGY

Enumeration of ' Blood-Heat ' Organisms. — Two sterile
Petri dishes are inoculated with 1 and O'l c.c. of the
sample respectively. Two tubes of nutrient agar are
melted in a bath of boiling water and allowed to cool to
45° C., when the tubes are wiped on the outside, the
mouths flamed, and the contents poured into the Petri
dishes. The agar and water are mixed by tilting, and
placed on a flat surface to cool. This needs to be done
with expedition (p. 42). The American committee
recommend the use of Petri dishes with ' porous earthen-
ware covers, in order to avoid the spreading of colonies
by the water of condensation.' Otherwise the agar plates
should be inverted in the incubator. Counting should
be done at the end of forty to forty-eight hours.

The actual number of organisms capable of develop-
ment at blood-heat is subordinate in importance to the
ratio of organisms developing on the agar plates to those
developing on the gelatin plates. In a pure water this is
generally considerably less than 1 to 10 — i.e., I to 20, 30,
or 40 — while in an impure water the ratio becomes 1 to 8,
5, 3, or more.

Estimation of B. Coli. — Quantities of the sample amount-
ing in all to 50 c.c. for a shallow well or surface water,
or to 100 c.c. for a deep-well water, are added to tubes
of a medium containing some substance that is fermented
by the colon bacillus, and some other substance that,
while inhibiting the growth of most water organisms,
allows the growth of the colon bacillus. Savage prefers
neutral red glucose broth, but MacConkey's bile-salt broth
is generally used, and the carbohydrate incorporated is
usually lactose. The colon bacillus ferments this sugar
with production of acid and gas, a fermentation reaction
not given by a number of organisms that produce acid
and gas from glucose. The composition of the broth as
described on p. 237 is of ' single strength.' A quantity of
medium containing double the amounts of the constituents
is also prepared—' double strength.'

If the water from a shallow well is to be examined,
quantities of O'l and 1 c.c. of the sample are inoculated
respectively into two tubes containing 10 c.c. of single
strength medium. Tubes containing respectively 5 c.c.,
10 c.c., 15 c.c., and 20 c.c. of double strength broth are
inoculated with 5 c.c., 10 c.c., 15 c.c., and 20 c.c.

BACTERIOLOGY OF WATER 231

respectively of the sample. When a water to which a
more stringent standard for B. coli can be applied is to be
examined, quantities of 10 c.c., 20 c.c., and 20 c.c. are, in
addition, added to double-strength tubes containing
similar quantities of the broth. This will give a total
quantity of water examined of over 100 c.c. The tubes
are then incubated at 37° C.— or, better, at 42° C.—
for twenty-four to forty-eight hours. The production of
acid and gas is only presumptive evidence of the presence
of B. coli. Other organisms are frequently responsible
for the change, hence the necessity for the isolation and
identification of the organism. Contrariwise, the colon
bacilli may not give the reaction for seventy- two hours,
or they may be inhibited or killed by the bile-salt, but
in these cases the bacilli may be regarded as attenuated
forms and their non- detection as of no serious consequence.
Negative results are very occasionally due to death or
inhibition of colon bacilli through overgrowth of strepto-
cocci. This only seems to occur with grossly polluted
waters, especially when large quantities of water (such
as 100 c.c.) are tested (Prescott and Winslow).

The tubes which received least water, and which
show acid and gas, are used for making the secondary
cultures for which neutral red bile- salt agar or Conradi-
Drigalski agar are convenient. The use of Petri dishes
for these secondary cultures is not necessary. A loopful
of liquid from a primary tube showing acid and gas is
added to a test-tube containing 10 c.c. of sterile water.
This is agitated, and a loopful transferred to another
test-tube of sterile water. The latter is agitated, and a
loopful added to a third tube of sterile water. A loopful
of the first dilution is smeared over the surface of a sloped
tube of medium, and similar quantities of the other two
dilutions are smeared over the surfaces of two other sloped
tubes. One or other of these secondary cultures is pretty
certain to give discrete colonies. Characteristic colonies
are subcultured into tubes of nutrient broth, and when
growth is sufficient the broth cultures are examined for
the presence of motile organisms, and preparations are
examined by Gram's method. The attributes of a typical
colon bacillus are given on p. 90. From the broth culture
inoculations into the various media necessary to identify
the organism are made. The estimation of B. coli is

232 AIDS TO BACTERIOLOGY

unanimously regarded as the most important datum
obtained in the ordinary bacteriological examination of
water. If the colon bacillus is found, it is usual to report
it as absent in such a quantity, but present in so many
cubic centimetres. Thus, if typical B. coll be found in
the tube to which 10 c.c. of the water had been added,
but that receiving 5 c.c. remained unchanged, the organ-
ism is present in 10 c.c., but absent in 5 c.c.

Savage came to the following conclusions (1902), the
justification of which is very generally conceded: Waters
which show no B. coli in 50 c.c. are of a high degree of
purity, and therefore the proved absence of this organism
in this amount, and still better in larger quantities, is
of great value. B. coli should be absent from at least
50 c.c. of spring water, possibly from greater amounts.
In upland surface waters the presence of B. coli in 40,
10, or even 2 or 1 c.c., means contamination, but not
necessarily a contamination which it is essential to pre-
vent. It may be from contamination with the excreta
of animals grazing on the gathering areas, and is by no
means necessarily from sewage or other material contain-
ing specific organisms of infection. If B. coli are present
in numbers greater than, say, 500 per litre (or even in
that amount), such a water is suspicious, as it is rare to
get so many B. coli in a water from the kind of animal
contamination indicated, and further investigation is
desirable. In filtered samples the number of B. coli is
as a rule considerably reduced. In surface wells B. coli
in large numbers indicate surface or other contamination,
generally very undesirable, if not actually dangerous.

Deep-well or spring water should not usually contain
B. coli in 100 c.c.

As regards atypical B. coli, we cannot do better than
again quote Savage: ' The nearer these . . . approach
typical B. coli in their characters, the more nearly are our
numerical standards for that organism applicable to them,
while if they lack essential characters, a proportionately
greater number must be present to justify an adverse
opinion.'

Enumeration of Streptococci. — Quantities similar to
those used in the estimation of B. coli are inoculated into
glucose broth or glucose neutral red broth, and incubated
at 37° C. for forty-eight hours, when hanging- drop or

BACTERIOLOGY OF WATER 233

Gram-stained preparations are made from the bottom
liquid. Or the actual tubes used for the primary examina-
tion for B. coli may be examined.

Not all streptococci will grow in the presence of bile
salt, but as the group that is important in the sanitary
analysis of water— i.e., those from human excrement —
does, omission to find others is of little consequence.

For the isolation of streptococci, Conradi-Drigalski agar
is convenient. Houston showed that fsecal streptococci
from human sources produce acid from lactose and
saccharose media, and generally from salicin too. They
produce acid and clot in milk, produce a uniform turbidity
in broth, and reduce neutral red — that is, they correspond
to the S. fa'calis of Andrewes and Horder (p. 134). Hous-
ton found no reliable distinction between the streptococci
of man and animals. With the horse, sheep, rabbit,
and cow, excrement is deficient in streptococci, and
especially in lactose-positive streptococci as compared
with human excrement.

It is important to note that the human excrement
group of streptococci is of a brevis type. If, in the examina-
tion of a Gram-stained slide, Gram-positive diplococci
are noticed, the search should be kept up, since perhaps
these diplococci are streptococci that have not grown
out. In most of such cases definite chains will be found
sooner or later.

In contradistinction to B. coli, streptococci do not
multiply in water after sampling (non-multiplying factor).
Previously regarded as indicators of recent pollution,
owing to an idea that they soon died out in water, they
are now regarded by Horrocks and most other workers
as living as long as, if not longer than, B. coli. Their
number should not exceed that permissible for B. coli.

Enumeration of B. Welchii. — Quantities amounting to
250 c.c. for a surface water, and up to a litre for a deep-
well water, are examined as described on p. 87. This
organism is a ' non-multiplying factor.' Savage considers
that it should be absent from 100 c.c. of a surface water
and from a litre of a deep-well water. The estimation
is not usually included in routine examinations, owing
to the uncertainty as to its interpretation. Thresh has,
however, suggested a number of standards (see * Applied
Bacteriology ').

234 AIDS TO BACTERIOLOGY

Detection of the Typhoid Bacillus. — The incubation
period of typhoid fever is generally ten to fourteen days,
a period often long enough for the disappearance of the
organism from the water before an examination is sug-
gested, leaving out of the question the period elapsing
before the water is suspected. A water containing typhoid
will almost certainly contain very much larger numbers
of the colon bacillus and other organisms which will
flourish on the media used for isolation of the typhoid
bacillus, crowd the plates, and perhaps outgrow the
organism sought. The last reason is less likely where
typhoid urine alone is responsible (see pp. 99 and 227),
and this class of cases happens to be that where typhoid
bacilli have been isolated and successfully identified.
Apart from the urine contamination cases, there has been
constant failure to find the typhoid bacillus in incrimi-
nated water, and considering all things this is no matter for
surprise. A negative result is worthless, and great danger
to the community may result from failure to realise this.

The amount of actual typhoid pollution is almost
invariably very small, and direct plating of a suspected
water is waste of time. One of the following methods of
concentration is therefore adopted :

(a) Filtration through a Porcelain Filter. — A large
number of the organisms get entangled in the filter candle,
and the process is seldom used.

(6) Willsori's Precipitation Method. — A 10 per cent,
solution of alum in sterile distilled water is added to the
water till it contains 0-5 gramme of the salt to the litre.
The precipitate is allowed to settle, or, after the precipitate
of aluminium hydrate has formed, the vessel is well shaken
to distribute its contents evenly, and centrifuged for
fifteen minutes. The clear supernatant fluid is then
siphoned or poured carefully off from the precipitate,
and the mass of precipitate in the conical extremity of
the tube stirred up with the little fluid remaining. The
suspension is then plated out. If the water is very soft
it is advisable to add a little lime-water, or the precipitate
will not form well. The alum is said to have no bacteri-
cidal effect, but if too much alum be allowed on the surface
of a plate growth will be retarded. This method is the
most satisfactory.

(c) Serum Agglutination (Schepilewsky). — Ten to twenty

BACTERIOLOGY OF WATER 235

c.c. of the water are added to 50 c.c. of nutrient broth,
to which, after three or four days' incubation at 37° C.,
an addition of the typhoid serum is made, and after
standing for some hours and centrifuging, the deposit is
plated out.

(d) Hoffmann and Ficker's Enrichment Method. — The
water itself is converted into a nutrient medium by the
addition of 1 per cent, of nutrose, O5 per cent, caffeine,
and O'OOl per cent, of krystal violet. The mixture is
incubated at 37° C. for twelve to thirteen hours, by which
time the typjioid bacilli should have multiplied to such
an extent as to permit of direct isolation by plating, the
B. coli being inhibited. It is very doubtful whether this
medium is really satisfactory. The action of caffeine on
B. coli is uncertain, while krystal violet merely inhibits
water organisms.

(e) Process of Cambier. — A special alkaline peptone
medium is placed in a glass jar. In this is immersed a
Pasteur-Chamberland filter candle half filled with the
same solution, to which is added a little of the fluid to be
examined, and the whole is incubated at 37° C. Sooner
or later growth appears in the fluid outside the candle,
and Cambier states that if typhoid bacilli be present
they will make their appearance before B. coli. Most
of those who have tried this process find that organisms
will grow through, but they are usually not B. typliosus.

(/) Wilson adds 10 c.c. of nutrient broth to every litre
of the water, and evaporates at reduced pressure at 42° C.

Isolation of the Typhoid Bacillus. — The concentrated
deposit obtained is plated out on a medium chosen for the
more or less characteristic growth of typhoid bacilli
thereon, and for its ability to inhibit the growth of most
other organisms. Conradi-Drigalski agar, litmus lactose
bile-salt agar, Fawcus's medium, China green agar, or
one or other of a host of media all devised for this purpose,
may be employed. Of one thing a bacteriologist may
be certain: he will always get colonies portraying the
characters of a typhoid colony on that medium. These
colonies must all be subcultured into broth and their
attributes worked up (see pp. 97 and 108). If plenty of
typhoid serum is available; it is advisable to try agglutin-
ating reactions on each subculture as soon as possible.
Pfeiffer's test should be done to clinch a diagnosis. It

236 AIDS TO BACTERIOLOGY

must be borne in mind^that^typhoitTfever, as diagnosed
clinically, is not always due"* to a typical Eberth-Gaffky
bacillus, and should an organism be isolated differing in
one or two respects from the recognised characters of
this bacillus, the agglutination test should be performed
with the serum of a case supposedly infected through
the water. With an atypical typhoid bacillus a negative
Pfeiffer reaction with an animal receiving this bacillus
and the serum from an animal immunised against a typical
typhoid bacillus is of doubtful significance, as the reaction
is such a markedly specific one.

The following organisms resemble the typhoid bacillus
morphologically and in cultural characters:

Bacillus aquatilis sulcatus is distinguished from the
typhoid bacillus (1) by developing at 5° C.; (2) by growing
feebly at 37° C.; (3) by not agglutinating with typhoid
serum; and (4) by the colonies after a time acquiring a
yellowish colour. Several varieties have been described.

Bacillus fcecalis alkaligenes does not agglutinate with
typhoid serum, and produces alkali in milk. Fuerth
describes a disease simulating typhoid produced by an
organism of this type. Savage mentions a single sporadic
case of gastro-enteritis that was shown by Ridder to be
probably caused by this organism.

B. coli anaerogenes is dealt with elsewhere (pp. 93 and
108).

Detection of the Cholera Vibrio.— See p. 151.
Media used in the Examination of Water.— The media
used for enumeration purposes should preferably be not
more than three weeks old, as changes occur in the reac-
tion with age. Such media are to have a reaction of
+ 10.

Nutrient Agar and Nutrient Gelatin. — See p. 36.
Distilled- Water Gelatin. — Ten per cent, gelatin in dis-
tilled water.

Distilled- Water Agar. — Powdered agar 1| per cent., dis-
solved in distilled water.

Peptone-Water and Carbohydrate Peptone- Waters. — See
p. 37.

Litmus Milk. — See p. 38.

Neutral lied Broth. — A J per cent, solution of neutral
red in water is added in the proportion of 1 c.c. to
every 100 c.c. of a broth containing 0'5 per cent, of glucose.

BACTERIOLOGY OF WATER 237

MacConTcey and Hill's Bile-Salt Broth. — Sodium tauro-
cholate 0'5 gramme, glucose or lactose 0*5 gramme,
peptone 2'G grammes,* are dissolved in 100 c.c. of water
by heating; the mixture is filtered, and after filtration
sufficient neutral litmus solution is added to give a dis-
tinct colour. The medium is then distributed into
Durham's fermentation tubes, and sterilised for twenty
minutes on three successive days. This is of ' single
strength.'

Neutral Red Bile-Salt Lactose Agar (MacConkey). —
Sodium taurocholate 0*5 per cent., peptone 2 per cent.,*
lactose 1 per cent., agar 2 per cent. To be made with
tap-water, and 0-5 c.c. of a 1 per cent, solution of neutral
red added.

Organisms, such as B. coli, which ferment lactose gener-
ally, but not invariably, give bright red colonies. B. ty-
phosus, which does not ferment this sugar, produces white
colonies, unless the peptone contains glucose.

Conradi- Drigalslci Agar. — Add peptone 10 grammes,
nutrose 10 grammes, sodium chloride 5 grammes, to 1 litre
of acid beef broth. Steam for one hour, and add 25
grammes of powdered agar. Steam for three hours,
bring to a reaction of + 10, and filter through papier
Chardin (A).

Boil for a few minutes 100 c.c. of Kubel-Tiemann
litmus solution, add 15 grammes of pure powdered lactose,
and boil again for a few minutes (B).

Add B to A, and to this mixture add 2 c.c. of a hot
10 per cent, solution of anhydrous sodium carbonate and
10 c.c. of a 0*1 per cent, solution of krystal violet. The
medium is then tubed, 10 c.c. being placed in each
test-tube, and sterilised. On this medium in forty-eight
hours B. coli forms large red colonies, typhoid small blue
' dewdrop ' colonies, and streptococci small delicate red
colonies.

This medium and the one preceding are employed as
surface plates. Tubes are melted in a water-bath, and
their contents poured out into sterile Petri dishes. When
set, the plates are placed in the blood-heat incubator for
two hours, with the lids slightly tilted at one edge, so that
the surface of the medium may dry somewhat. Other-
wise the moisture present would probably cause the
* The peptone used must be free from glucose.

238 AIDS TO BACTERIOLOGY

colonies to run together. The matter to be plated is
sufficiently diluted, and one or more drops are run on to
the surface, and spread by means of a sterile glass rod
bent at a right angle.

China Green Agar (Werbitzki). — An ordinary 3 per
cent, agar neutralised to + 13 on Eyre's scale, with 1-4
to 1-5 c.c. of a 0'2 per cent, solution of China
green added to each 100 c.c. contained in a flask.
McWeeney says that China green agar suppresses about
75 per cent, of the coli colonies, whilst the survivors
are much inhibited in their growth, remaining small,
opaque, and point-like. The typhoid colonies, on the
other hand, develop most luxuriantly, and are decidedly
more numerous than on any of the other media. Their
appearance is of a delicate transparent green, deeper
in the centre, with a thin, filmy, peripheral layer that
spreads out like a veil over the substratum, and presents
under a low power a typically sulcate appearance. The
motility of the typhoid bacilli is much diminished, and
they present a filamentous aspect. Subcultures in broth,
however, are typical by the next day, and can be sub-
jected to the usual agglutination and other tests.

Filtration of Water.

Domestic Filters. — The primary object of a filter is to
remove suspended matter, including micro-organisms.
The filtering media include carbon (animal and vegetable
charcoal, silicated carbon, and manganous carbon), felt,
sponge, car feral (charcoal, clay, and iron), spongy iron
(Bishofs filter), magnetic iron, cellulose, earthenware,
and natural stone. Animal charcoal oxidises and absorbs
part of the organic matter, removes colour due to organic
impurities, and is credited with the power of removing
lead. Vegetable charcoal is less efficient. Iron may
reduce nitrates to ammonia. With the exception of
earthenware types, these filters only remove part of the
bacteria, and, unless the filtering medium be often re-
newed, may actually be sources of danger. If once
polluted with typhoid or cholera, they may subsequently
convey the bacilli to unpolluted water for long periods
after contamination. The initial efficiency of these filters
is due to the bacteria being temporarily arrested in the
filter media, where they multiply and are gradually

BACTERIOLOGY OF WATER 239

washed through, the bacterial content of the water in-
creasing until sometimes it is greater than that of the
unfiltered water. Sponge filters form an excellent nidus
for bacterial growth, and are particularly reprehensible.
Bacterial filters of the earthenware type are by far the
most satisfactory. These are made in tubes or ' candles,'
to offer as large a surface as possible to the water.

The Pasteur-Chamberland candle is a composition of
unglazed porcelain, the Berkefeld of a diatomaceous earth
(kieselguhr); the Slack and Brownlow, and the Doulton
filter, are also composed of porcelain. None continues to
give sterile water indefinitely, and should be submitted
to a weekly scrubbing with a nailbrush, and subsequent
boiling in water containing sodium carbonate. Kieselguhr
filters vary considerably in the length of time for which
they will give sterile water, some allowing direct con-
tamination, while others are satisfactory. The Berkefeld
filter has a small portion of its surface removed every
time it is scrubbed, and thus may in time become faulty.

The inevitable appearance of organisms in the filtrate,
in the case of all filters after shorter or longer periods of
effectiveness, is, according to Craw, partly dependent on a
mechanical acceleration, caused by the water current
sweeping the micro-organisms through the filter mass.
This indirect contamination appears to depend partly on
the grain of the filter. The most efficient makes of filters
examined by Craw — the Pasteur - Chamberland and
Doulton — contain very small-sized pores; the Berkefeld
pores are larger, and the Slack and Brownlow larger still.
The characters of the organisms (motility, size, etc.) and
the chemical composition of the water may also be factors
in determining the period of sterility. When water is
filtered under pressure an extra strain is put on the filter.
The soundness of the Pasteur-Chamberland filter can be
ascertained by compressing air in the candle at a pressure
of one-half to one atmosphere, when, if held' beneath
water, no air will escape from a sound tube. A stream of
bubbles will issue from any spot capable of passing
bacteria.

Recognising their limitations, bacterial filters, especially
the Pasteur - Chamberland, offer a perfect protection
against infection from drinking-water. The slow rate of
filtration is not surprising considering the size of bacteria,

24o AIDS TO BACTERIOLOGY

and by using a batter}7 of filters the supply can be in-
creased at will.

Sand Filtration. — Filter-beds at waterworks are con-
structed of a layer of large stones, with unjointed pipes
placed at intervals at the bottom; smaller stones are
placed above these, then gravel and f ough sand, and lastly
a layer of sand from 2 to 4 feet deep. The fineness and
contour of the grains of sand, thejiepth of the filter, and
the rate of filtration, all affect the" working of the filter in
the removal of organisms. The fineness of the sand is
per se no criterion of filtering power, a comparatively
coarse sharp-angled sand acting better than a finer article
with rounded edges. Sea sand is for this reason unsuit-
able for filtration. Koch's coefficients for safe working
are filtration through a sand layer not less than 30 centi-
metres thick, at a rate not exceeding 100 millimetres per
hour, and giving a filtrate containing not more than
100 bacteria per c.c. growing on gelatin. These coeffi-
cients, however, take no account of the class of sand used
or character of water filtered, and they are no longer
regarded as trustworthy. When freshly constructed,
organisms are washed through a filter-bed with great
rapidity, but after a certain quantity of water has passed
through or the water has been allowed to stand upon it
for a certain time, a slimy coating of detritus, bacteria,
and other lowly organisms, is formed on the surface.
If water is slowly passed through the filter when sufficient
of this coating has formed, the majority of the bacteria
will be retained by this surface, either by sticking to it
or by being strained off. The increasing thickness of
this coating will reduce the velocity with which the water
passes, and at the same time some of the bacteria will
tend to grow downwards into the lower strata of the
filter, and, if the process were continued long enough,
would be washed through into the filtrate, and ultimately
become more numerous there than in the unfiltered water.

This layer also serves as a culture ground for oxidation
bacteria, which to a large extent tend to prevent the multi-
plication of the other bacteria, and consequently their
growth through the filter, In summer, when the tem-
perature is more favourable to the growth of the organisms,
the purification is more complete than in winter.

The indication for scraping usually adopted is that the

BACTERIOLOGY OF WATER 24!

filter-bed no longer passes the required quantity of water
under the maximum permissible head. The sufficiency of
this practice has not been clearly shown. No general rule
can be given for the depth to which the top layer must be
removed, as it varies with the nature of the water and
sarid, temperature, etc. The epochs of scraping and other
interference, as by storm water, with the filter-beds, have
been frequently observed to synchronise with groups of
typhoid cases in the districts served with the water in
question. A properly working filter should remove 98 or
99 per cent, of the organisms, and a filtered water should
not contain B. coli in 100 c.c.

The sand filters of the Metropolitan Water Board vary
in depth from 2 to 4| feet, and the rate of filtration
varies from 0-89 to T94 gallons per square foot per hour.
Each acre of filtering surface requires to be cleaned on
the average seven times during the year. Filtered
Thames and Lea waters contain two to three colon bacilli
per litre (Houston).

The Sterilisation of Water.

The use of bacterial filters is dealt with on p. 239.
Water may be efficiently sterilised by heat. In apparatus
for this purpose, the hot sterile water passes through
tubes or compartments surrounded by the incoming
water, with the result that the sterilised water is cooled
and at the same time the unsterilised water is heated
to such a temperature that comparatively less fuel is
required to bring it to the necessary temperature. Water
sterilised by heat loses part of its natural salts and part
of its dissolved gases, and may be mawkish in flavour.
For such an apparatus to be safe it must be fitted with
an automatic valve which does not allow water to leave
the apparatus until it has been raised to the required
temperature. Such an apparatus would be safe, but it
cannot be cheap in first cost, and its weight and the cost
of fuel are against its popularity except on a small scale.

The distillation of water is sometimes practised where
water contains too great a quantity of salts to be drink-
able. The distillate is, of course, sterile if properly collected,
but, like all boiled water, it must be aerated to make it
agreeable to the palate.

Bisulphate of sodium (15 grains per pint) is said to

242 AIDS TO BACTERIOLOGY

sterilise water in thirty minutes, but the use of an acid
compound is to be deprecated. In the British Army
tabloids of sodium bisulphate, flavoured with lemon and
saccharin, are supplied for the use of cavalry. In
practice it is found that men soon get tired of the taste
of water treated in this way, and, though the tabloids
may be issued, they are not regularly used.

Potassium permanganate is effective, but it is best
followed by treatment with alum. Copper sulphate in
a very weak solution kills algae (p. 254), and its use has
been suggested for the routine disinfection of water.

Iodine has been employed and is quite effective, but
the water must be subsequently treated with sufficient
hyposulphite of soda to neutralise the iodine.

Of late years sterilisation of water by chlorine has come
into use, and its employment is rapidly increasing. Its
use was suggested by Major Nessfield in about the year
1897, who showed that colon and typhoid bacilli could
be killed by the addition of chlorine to water in high
dilutions. Later it was found that the addition of small
quantities of a dilute solution of bleaching powder was
equally effective. The less a water contains of organic
matter and chemical compounds that combine with
bleaching powder, the less is needed for sterilisation.

The essential point is to add as much bleaching powder
as is required to kill the disease-producing organism
(which is assumed to have occurred if no living coli-
group organisms can be recovered after treatment), but
to avoid excess, because even a very small excess of
bleaching powder gives a most unpleasant taste to water.
This taste is brought out more strongly when water is
made into tea. Some individuals are very sensitive to
small quantities of bleaching powder. Moor and Hewlett,
in a report to the L.G.B. (Report of the Medical Officer
to the Local Government Board, 1909-10), examined
the processes available for the purification of chalk waters,
and reported in favour of sterilisation by bleaching
powder. In those investigations it was shown that
0'25 part of chlorine (equivalent to approximately 0-75
part of good bleaching powder) per million killed colon
bacilli in chalk water in thirty minutes. The amount of
chlorine necessary varies. Sims Woodhead found the
amount of chlorine necessary to kill the whole of the

BACTERIOLOGY OF WATER 243

non-sporulating bacilli in Cambridge water is usually
1 part per 7 ,003,00 J parts of water. Less pure waters
may require 0'7 to 2 parts of available chlorine per
million.

The army directs that 2 grammes of bleaching powder
shall be added to the contents of water-carts which hold
100 to 110 gallons, at the discretion of the medical officer.

Many water-supplies in America and some in the
United Kingdom are now sterilised by bleaching powder.
In order to remove any traces of the reagent after treat-
ment, the water may be run through iron turnings or
charcoal, or an addition of an appropriate quantity of
sodium hyposulphite may be made.

One of us (C. G. Moor) has applied the process of sterili-
sation of drinking-water on a large scale for camps and
hospitals at a base in France, with satisfactory results.

At present it appears that sterilisation by minute
quantities of bleaching powder, or by chlorine produced
electrically, is the coming method, and its cheapness
and simplicity make it applicable everywhere. Before
it is applied, waters should be strained or filtered to take
out all visible particles, otherwise the sterilising solution
may fail to kill bacteria enclosed in such particles. For
army use a valuable piece of apparatus has been devised
by Colonel Horrocks, in which water is clarified and
sterilised by bleaching powder at one and the same time.

Water may be sterilised by allowing it to trickle down
a tower through which ozonised air is caused to pass.
This is a method which has much to recommend it, inas-
much as the water is rendered sterile, but loses none
of its salts or gases in the process. The apparatus is,
however, expensive, requiring elaborate electrical ap-
paratus and skilled attention. It is in use in some towns
abroad.

Sterilisation of water may be effected by the ultra-
violet rays created in Cooper-Hewitt lamps made from
transparent quartz. A continuous supply of sterile water
is available in five minutes. The water is unaffected as
far as taste is concerned, as it retains all natural gases
and salts in solution. So far the process is not widely
adopted.

In Clark's process for softening hard water, Moor and
Hewlett showed that bacteria were reduced, but results

244 AIDS TO BACTERIOLOGY

were inconstant. In the Porter-Clark process, where the
precipitated calcium carbonate is removed by filtration
through canvas, considerable reduction in bacterial con-
tent results (Hewlett and Nankivell).

Houston's ' excess lime ' method involves the addition
of lime till the water is decidedly alkaline. After a period
of five to twenty-four hours, the time required to kill
colon bacilli, sufficient stored water is added to deal with
the excess of lime. In his Tenth Research Report
Houston mentions that, with small quantities of lime
above the neutralisation point, colon bacilli lived over
two days, whilst with 6 -3 parts of lime in excess they
lived for less than one day.

CHAPTER XXII

DISINFECTION AND DISINFECTANTS
Fire.

DESTRUCTION by fire is the most efficient and simplest
form of disinfection, and should be employed where
articles are of little value, or to remove infection from
surfaces which will not be appreciably damaged thereby.
Forbush and Fernald's apparatus consists of a portable
tank and pump, from which paraffin gas oil is driven
through a hose (such as is used for the delivery of oil), to
which is attached a pole, consisting of an iron pipe 12 feet
long, which is protected by a covering of wood, and
to the end of which is attached a cyclone nozzle. The
fine spray from the nozzle is ignited, and the resulting
torch-fire passed over the surfaces to be disinfected.

Heat.

Most articles sent for disinfection are of an organic
nature, to which injury is inevitable at temperatures not
much above those necessary for disinfection. Death of
the bacteria is determined by the coagulation of the
protein. With unlimited moisture protein free from salts
can be coagulated as low as 50° C.; in the absence of
moisture it can stand exposure to 170° C.

DISINFECTION AND DISINFECTANTS 245

Dry Heat depends for its penetration on conduction
and convection, which are slow processes, and does not
kill all organisms at a temperature which can be borne by
any ordinary fabric, except horsehair, even when the
organisms are exposed on the surface. It is, therefore,
inadmissible for disinfection of fabrics. The temperature
of a dry-heat disinfector must not rise above 120° C., or
woollen material will be damaged. Wide variations of
temperature occur within the disinfecting chamber,
owing to unequal diffusion of the gases and radiation from
the heated surfaces.

Moist Heat. — Steam at any temperature and pressure
which can condense without cooling is called ' saturated
steam,' and will wet the surface on which it condenses.
When moisture is present in a disinfector, steam con-
denses in the pores of fabrics, more steam is sucked in to
fill the place of that condensed, and this process continues
till the interior of the fabric becomes so hot that con-
densation ceases.

When by contact with a hotter surface, or by being
derived from a saline solution, its temperature is raised
above that at which it can condense under its existing
pressure, the steam is called ' superheated.' The dis-
infectant value of strictly superheated steam is about the
same as that of hot air. In practice, the extent of super-
heat present in a disinfector is usually not sufficient to
prevent, the steam from being rapidly reduced to satura-
tion, and acting as saturated steam. It is only in the
later stages of a disinfection that there is risk of the
objects being too hot to cool the steam to saturation, and
of organisms on the surface thus escaping disinfection.
A more certain objection to the use of superheated
steam is that its temperature, not being determined
solely by its pressure, cannot be read off on a pressure
gauge.

If the coagulation theory of heat disinfection is correct,
the disinfectant effect of hot air or of superheated steam
will be considerably less than that of saturated steam or
hot water. The presence of air in steam has the effect of
delaying or preventing the condensation of the steam, and
should have a marked effect in reducing the efficiency of
the steam. The temperature of saturated steam increases
directly as the pressure, and on any theory, therefore,

246 AIDS TO BACTERIOLOGY

steam under pressure must have a higher disinfectant
value than steam not under pressure.

Steam is used either confined under pressure or as a
current with or without a pressure exceeding that of the
atmosphere. The advantage of some amount of pressure
of saturated steam, however small, is that it gives a real
control over the temperature of steam, which in a well-
designed disinfector is practically uniform throughout.
It has also been repeatedly shown that in the absence of
pressure the temperature and disinfectant value of the
steam depend largely on its velocity, and the rate of
stoking will largely affect it — an objection which is
serious because there is no convenient or trustworthy
means of controlling either the velocity of the steam or
the rate of stoking. What the temperature should be is
still a matter of discussion. Taking all the facts together,
the least exposure which can assure general disinfection
is fifteen minutes to air-free, saturated steam at 115° C.
This exposure may leave absolutely no margin of safety,
and the conditions of saturation, freedom from air,
temperature, and time of exposure must be rigidly assured
independently of the operation.

Spray Disinfection. — In spraying, the operator should
begin at the bottom of a wall and work upwards. If the
opposite is done, the deposited liquid runs in streams down
the dry wall, and staining results. Given an efficient
disinfectant, a sufficiently fine spray, and a conscientious
worker, spray disinfection of rooms is most satisfactory.

For descriptions of sprays and steam disinfectors, see
'Applied Bacteriology.' For action of light, see p. 9;
for action of desiccation, see p. 8; for sterilisation by
filtration, see pp. 215, 239.

Chemical Disinfectants.

A disinfectant, or germicide, is a substance capable of
killing bacteria; an antiseptic inhibits bacterial growth;
and a deodorant prevents or absorbs foul smells.

Theories of Chemical Disinfection. *— Paul and Kronig
suggest that the degree of ionisation of a solution has an
important bearing on its disinfectant efficiency. It is

* For further information on the theories of disinfection and on
the influence of the constitution of disinfectants on germicidal power,
see Professor Sommerville's ' Aids to Public Health.'

DISINFECTION AND DISINFECTANTS 247

certain that in most cases the extent and rate of dis-
infection to be had from a solution depend on the extent
and rate of its penetration into the bacterial cell, and that
accordingly an increase of concentration above that of
dilute solutions may not be accompanied by a pro-
portionate increase of disinfectant power. Adsorption
of the disinfectant is the first phase in disinfection,
chemical action of the disinfectant on the micro-organism
being the second phase. The sum, however, of all the
known facts fails to establish any general chemical
criterion by which the germicidal efficiency of a substance
can be foretold.

Where salts of an analogous composition are concerned,
disinfection is found to be more efficient as the amount of
dissociation increases. Paul and Kronig found that solu-
tions containing a toxic ion in the same proportion have
an identical toxic action . Mercuric chloride and mercuric
bromide are almost equally dissociated in solution, and are
of equal power as disinfectants in dilutions of 1 gramme
molecule in 64 litres. Mercuric cyanide undergoes less
dissociation, and is a weaker disinfectant in dilutions of
four times the strength. The efficiency of a solution of
mercuric chloride is considerably diminished by the
addition of sodium chloride, which illustrates the general
principle that, when to a solution containing free ions
similar ions are added, there is a tendency to re-form the
original molecules.

Water ionises most electrolytes; glycerin and acetic
acid are indifferent dissociants; while substances dissolved
in chloroform, ether, or benzene ionise but little or not at all.

Chick found the destruction of bacteria by water
between 45° and 55° to be a consistent time process,
and to run parallel to the heat coagulation of proteins.

Madsen, Nyman, and Chick have shown that relatively
larger number of organisms are killed when contact with
disinfectant commences than after, when the rate of killing
gradually falls. They found that if the results be plotted,
ordinates showing the numbers of surviving organisms and
abscissae the corresponding times, the points lie on a
hyperbolic curve. The curve is expressed by the formula

— log -1 = K, when n-, and 7?2 are the numbers of

*2 — *1 W2

bacteria alive after the times ^ and t2 respectively, and K
is a constant.

248 AIDS TO BACTERIOLOGY

Delepine defines an ' antelethal inhibitory period '
existing before actual death. He also shows that some
disinfectants, when dilution is pressed beyond the
' ultimate lethal dilution,' actually excite growth of
bacteria.

Gossl found some disinfectants to owe their efficiency
towards yeast cells to the fact that they are soluble in
the cell-lipoids.

Behaviour of Chemical Disinfectants. — In oil or alcohol
disinfectants lose all or most of their activity. The value
of alcohol itself as a disinfectant is also lessened when it
holds other things in solution, but tincture of iodine is
an exception. Among fats, lanolin alone seems com-
patible with the disinfectant efficiency of substances
exhibited in it, probably because it holds them in a fine
emulsion of their watery solutions (Gottstein). Some
disinfectants form an emulsion on the addition of water.
Rideal and Walker found that a resin soap emulsion of
tricresol had three times the germicidal value of an oleate
solution of the same disinfectant.

The temperature at which the organism is exposed to
the disinfectant has a considerable influence on the extent
or rate of disinfection. Up to the optimum temperature
at whicl> the organism to be disinfected grows on the
medium in which it is exposed, the activity of a disinfectant
may fall off as the temperature rises, owing to the increased
vigour which the organism derives from the improvement
in its conditions in respect of temperature. On the con-
trary, as cooling below the optimum temperature proceeds,
the organism gradually passes into what Christian calls
' a state of coma,' where the bacterial cell has a corre-
spondingly low tendency to undergo chemical change.
In practice, the latter alternative seems more frequent
— that is, diminution in temperature lessens disinfectant
action. A relatively small difference of temperature —
two or three degrees — may make an appreciable differ-
ence in the activity of disinfectants, and in their examina-
tion failure to remember this has led to serious error.
Above the optimum a rise of temperature increases the
activity of the disinfectant, sometimes to an enormous
extent.

Fasson, Ponder, and Sims Woodhead, comparing
emulsified disinfectants (cresols, etc.) with carbolic acid,

DISINFECTION AND DISINFECTANTS 249

found that at lower temperatures the activity of the
emulsion is raised more rapidly than that of the solution,
but at the higher temperatures used the activity of the
emulsion is no longer increased in proportion to the
increase in the activity of the carbolic acid.

Two disinfectants used together may be more efficient
than either separately. Conversely, mixture of others
ma}* diminish or neutralise the efficiency of the com-
ponents. One species of bacteria may be many times
more sensitive to one disinfectant than to another, when
both substances exert an equal effect on a second species.
In a less degree, varying degrees of susceptibility to the
same disinfectant are exhibited by different strains of
the same organism. Repeated subculture on favourable
media will often increase resistance to chemical dis-
infection. By culture in media containing non-toxic
proportions of a disinfectant, an enhanced degree of
resistance to this disinfectant may be acquired temporarily.

Chick and Martin find that finely particulate matter
affects the value of emulsified disinfectants containing
the higher phenols more than it does phenol, and the loss
is the greater the finer the emulsion. The removal of
an emulsion of higher phenols by organisms is a process
of adsorption at first, in which the organisms are sur-
rounded by concentrated disinfectant, and so dis-
infectants of this class possess a higher efficiency. King-
zett and Woodcock, however, found that the fall in
germicidal value generally shown when disinfectants
are examined by the Lister Institute method was not due
to the mechanical action of the solid matter (fseces). They
showed that when kieselguhr, powdered pumice, and
precipitated chalk were introduced into the water used
to dilute the disinfectant (Sanitas Bactox), there was no
depreciation in its coefficiency.

Little is known of the destruction of the filterable
viruses by chemical agents. Saponine does not affect
bacteria, but Cockayne (Medical Press, February 12, 1913)
says it will kill filterable viruses, with the exception
of those of trachoma and Cyanolophia gallinarum. These
two also resist the action of taurocholate of soda, which
destroys the others.

Points of Efficient Disinfectants. — 1. Capability of
killing bacteria. A disinfectant capable of efficient action

250 AIDS TO BACTERIOLOGY

when diluted many times is to be preferred, not only
because of the saving in storage and transit, but so that
in cases of necessity, as when sporing organisms require
disinfection, a stronger disinfectant dilution is permitted.

2. Retention of efficiency in the presence of such
organic matter as is likely to be met with in practice.
Organisms are seldom offered for disinfection separate
from appreciable amounts of oxidisable organic matter,
and a disinfectant dependent for its efficiency on oxidation
properties wastes itself on dead matter more readily than
it attacks living protoplasm.

3. Production of emulsion or solution in all proportions.

4. Permanent homogeneity. Disinfectants of a certain
type separate out on standing or in cold weather into
layers very different in efficiency, and although a shilling
bottle may be rendered homogeneous by shaking, the
matter is less easy when a 40-gallon cask is concerned.

5. Solvent power for grease.

6. Stability at all reasonable temperatures, so as to
allow its use when heat is also available.

7. Freedom from toxicity in all dilutions necessary for
complete disinfection.

8. Non-caustic, non-corrosive, harmless to fabric, and
without action on dyes.

Acids. — All acids are disinfectants, the efficiency being
directly proportional to the degree of acidity. Sulphur
dioxide is obtained by burning sulphur ( ' Sulphur candles '
are convenient), or from the liquefied gas. Plenty of
moisture is necessary in the atmosphere of the room or
ambulance to be disinfected, and a high temperature assists
the action. Its power of penetration can be taken as
practically nil, and its chief application at the present
time is the destruction of vermin, notably rats, which in
plague-infected ships and houses may often be desirable.
For this purpose the Clayton apparatus has been used,
but its high cost and somewhat complicated construction
have prevented it from obtaining an extended application.
Christian mentions that air containing 5 per cent, of
Clayton gas kills rats, fleas, and other vermin in a few
seconds, and when 1 per cent, only of the gas is present
the rats cannot reach their runs. (As an alternative,
Nocht and Giemsa proposed carbon monoxide for rat
destruction. This gas is not a disinfectant, though

DISINFECTION AND DISINFECTANTS 251

quickly poisonous to mammals and birds. As it may be
inhaled without the slightest indication of its presence
till the man falls senseless, its use is attended with serious
risk.)

Some use has been made of crude sulphuric and hydro-
chloric acids in dilute solution for the disinfection of
fseces, and strong sulphuric acid has been used for the
disinfectant destruction of infected carcasses.

Sulphur is used with some success in destroying Oidium
on vines, and other fungi parasitic for plants. Marcille
attributes the effect to the small amount of sulphuric
acid present in the preparations employed.

Alkalies and Soaps.— Efficiency here depends on the
character of the metal, but is also affected by the degree
of alkalinity. Faeces may be disinfected by caustic lime,
used generally as a 20 per cent, milk, but lumps must be
broken up. Kaiser's method consists in adding enough
hot water (50° to 65° C.) to cover the motion, and then
adding about one-fourth of the entire bulk of calcium
oxide, covering the receptacle, and allowing it to stand
for two hours. The hydration of the lime generates
sufficient heat to destroy typhoid. Lime is inefficient
against the more resistant organisms, and lime-whiting
is no sufficient precaution against them. Addition of
a suitable disinfectant renders the process efficient.
Ordinary size distemper has been shown by us to have no
action on the colon bacillus. Few disinfectants are
compatible with soap, binoxide of mercury and the best
coal-tar products being notable exceptions. Disinfectant
soap in the removal of grease, dirt, pus, etc., certainly
assists in eradicating bacteria, but the period of contact
of the soap in ordinary ablutions is insufficient to pro-
duce sterility. Nevertheless, as Rideal has pointed out,
organisms perhaps of an infective type left on the soap
by one person may remain long enough for the disinfectant
incorporated to act before another uses the soap. This
point is of importance in the prophylaxis of barber's
rash, for which purpose disinfectant shaving-soaps are
prepared. Commercial carbolic soaps are generally
worthless.

Halogens. — When dry, chlorine, bromine, and iodine
are poor disinfectants. Their use is practically confined
to solutions, and in practice they have to be used in an

252 AIDS TO BACTERIOLOGY

excess proportionate to the amount of organic matter
present. When, as in the case of water, organic matter is
practically absent, disinfection is assured by a very small
percentage. ' Chloride of lime ' (a mixture of calcium
hypochlorite, hydrate, and chloride), hypochlorite of soda
(chloros), and hermitine are the best known sources of
available chlorine. The last is prepared for distribution at
Poplar by the electrolysis of a solution of sodium chloride
and magnesium chloride. Sodium hydroxide is added to
fix free hypochlorous acid, and acts as a preservative.
Much dissension has arisen over the value of this disin-
fectant, some authorities regarding it as stable and
powerful even in the presence of sewage or other organic
matter, and others as unstable and, in the presence of
organic matter, untrustworthy.

The use of chlorine as a gaseous disinfectant has pro-
duced bronchitis and laryngitis, with a fatal ending.
Iodine employed as a 2 per cent, solution in potassium
iodide solution, or, even better, as an alcoholic tincture,
is a disinfectant of the first order for the primary treat-
ment of wounds, and for the disinfection of skin previous
to operation. The tincture is recommended in the
War Office memorandum. Its use in the field has ex-
tended its popularity. It is true that it stains the skin,
and that serious inflammations have resulted from the
injudicious use of tincture of iodine as a local application,
but these are trivial details compared with its efficiency
(see also p. 248). Delorme, Medical Inspector-General
of the French Army, points out that dressings impregnated
with perchloride of mercury and carbolic acid while iodine
is being used are apt to cause a troublesome dermatitis,
which does not occur when a simple aseptic dressing is
resorted to. The iodine of iodoform is gradually liberated
in contact with the enzymes of pus, blood, etc. Its
success is largely due to this continual production of
fresh iodine. Iodoform by itself is inert. Antiformin
(p. 68) actually dissolves proteins, mucus, and most
bacteria, and for many purposes is an excellent disinfectant.
Christian mentions a 5 per cent, solution as suitable.

Metals. — Some metals, especially copper and its alloys,
gradually kill adherent bacteria. In a liquid containing
bacteria, a sterile zone develops round the metal. Twenty-
four hour contact with bright copper plates has been

DISINFECTION AND DISINFECTANTS 253

suggested in America for the destruction - of typhoid
bacilli and cholera vibrios in drinking-water.

When the metal is very finely subdivided, as it is in
the colloidal state, its disinfectant powers increase.
Colloidal silver and colloidal mercury are credited with
amazing efficiency.

Inorganic Salts. — Solutions of mercury salts are strong
disinfectants, efficiency depending on the proportion of
mercury in dissociation. As pointed out by Sommerville
and Ainslie Walker, mercuric chloride has been assigned a
much greater disinfectant value than is justified, as a
result of workers failing to recognise that when the
medicated organisms or spores are transferred to the sub-
culture tube a small quantity of mercuric chloride is
carried over, which exerts an inhibitory action on the
organisms, and the subculture tube shows no growth. But
if a drop of sulphuretted hydrogen water be added, the
mercury is converted into an inert sulphide, and permits
the growth of organisms that otherwise would not take
place within the time limits of the experiments. Mercuric
chloride is poisonous, and with proteins or soaps forms
compounds which have no germicidal action. The
addition of hydrochloric acid (as in the L.G.B. formula)
largely counteracts this depreciation. Cheatle (Medical
Annual, 1915) appears to regard this combination with
protein as a point in favour of the salt: ' Mercuric chloride
is adsorbed by skin, and will remain in a soluble condition
for a long time. In wounds it is adsorbed by its own
precipitated albuminate, and is given up into solution
slowly. Therefore it possesses a marked and most
desirable depot action.' The binoxide is strongly dis-
infectant in a potassium iodide solution, and is not affected
by proteins to the same degree (see also p. 251). Soluble
silver salts, chiefly used in ophthalmic work and for
cauterising dog-bites, are powerful disinfectants, weaker
than perchloride, but far less sensitive to proteins. They
are incompatible with chlorides, except in certain organic
combinations, from which silver chloride is only partially
precipitated.

Iron and zinc salts have been credited with useful dis-
infectant action, but their value is of no practical account.
Zinc chloride, copper sulphate, sodium fluoride, and zinc
fluoride are used for preserving timber.

254 AIDS TO BACTERIOLOGY

dopper sulphate in a proportion of 1 part per million
has been used for the destruction of algse in reservoirs.
Bordeaux mixture, a popular insecticide for plants, is
a mixture of calcium hydrate and copper sulphate.
Springer finds that copper salts are highly selective,
being most efficient in inhibiting the action of putre-
factive organisms.

Oxidising Disinfectants. — The halogens (pp. 242, 251)
are examples of oxidising agents. The permanganates
have considerable germicidal power when in strongly
acid or alkaline solution, but the readiness with which
they are affected by organic substances makes them
unreliable for practical use. Peroxides and ozone are
open to the same objection, and have less disinfectant
power.

While ozone is a deodoriser, it masks rather than destroys
smells. Hill and Flack found that one part in a million
irritates the respiratory tract, and even less reduces
respiratory metabolism, and rapidly causes a fall of body
temperature. They describe its beneficial effect, as
popularly believed in, as a myth, and think the irritation
of the olfactory nerves may relieve the monotony of close
air. Ozone may conceal faults in ventilation when used
for removing frowsiness in the atmosphere of a closed
place.

Hydrogen peroxide has been used as a milk and cream
preservative (p. 221). Hinks (Analyst, December, 1915)
has shown that it persists in such material for months,
and does not, as might be thought, quickly break up in
the presence of organic matter. It is used for bleaching
purposes, as a mouth-wash, and as lotion for infected
or suppurating wounds. It is non-toxic, practically
non-irritant, and does not precipitate albumen, but a
free exit must exist for the escape of the oxygen, which
is so rapidly liberated when the peroxide comes into
contact with blood or pus. A small quantity of mineral
acid or acetanilide is added to render it more stable.

Disinfectants of the Aliphatic Series. — Formaldehyde is
supplied either in the form of a 40 per cent, solution
(formalin) or as solid para-formaldehyde. As a gaseous
disinfectant little more than superficial disinfection is
possible. Disinfection is best insured by saturation of
the air with moisture, maintenance of a good room-

DISINFECTION AND DISINFECTANTS 255

temperature, sealing of the room, the use of at least GO
grammes of formaldehyde per 1,000 cubic feet (preferably
more, up to 120 grammes), and, in the case of large
rooms, mixture of the gas with the air of the room, either
mechanically or by the provision of a multiplicity of inlets
for the gas into the atmosphere. In the Alformant lamp
twenty-five tablets of paraform are volatilised for every
100 square feet of floor space. The Kuhn lamp generates
formaldehyde by the partial oxidation of methyl alcohol.
In the Trenner-Lee retort and Lingner apparatus form-
aldehyde gas and water vapour are passed into the
room. The ' Maine process ' requires only a 10-quart
tin pail, in which 300 grammes of potassium permanganate
are placed and 600 c.c. of formalin are added. The re-
action is violent, and is complete in about five minutes,
a maximum dosage of the room being quickly obtained.
Base modifies the method by using 300 c.c. of water with
375 grammes of potassium permanganate and 600 c.c.
of formalin, but finds the yield of gas less satisfactory.
The Military Commission in Vienna, on the contrary,
got better results by dilution of the strong formaldehyde
solution. Where portability of materials is important,
paraform and sodium bicarbonate solution may be sub-
stituted for formalin.

On evaporation, molecules of formaldehyde condense
to form para-formaldehyde (polymerisation), (HCOH)w,
which has little disinfecting power, but which, on heating,
produces formaldehyde. Polymerisation seems to be
prevented to a large extent by the presence of moisture
(see also above). As a spray, formalin can be used in
any ordinary apparatus. It is stated by McLaughlin
that if the formaldehyde be mixed with phenol vapour,
polymerisation does not take place, and that therefore
the formaldehyde penetrates thoroughly. The mixture
he employs is 3 parts of 40 per cent, solution of formalde-
hyde with 1 part of carbolic acid. It may be volatilised
in a retort, or it may be simply poured on a sheet, which
is then hung up in the room to be ^disinfected. Base
quotes the conclusions of Werner as to the practical
requirements for an effective formaldehyde disinfection:
(1) 5 grammes of formaldehyde (absolute) should be
present in each cubic metre of space (0'1416 gramme per
cubic foot), and should be allowed to act for seven hours;

256 AIDS TO BACTERIOLOGY

(2) the temperature of the room should not fall below
50° F., the best temperature being from 68° to 77° F.;
and (3) the strength of the formalin should be ascertained.

Ethyl alcohol in from 20 to 50 per cent, solution is
germicidal for many vegetative forms of bacteria. - Stronger
mixtures generally have less effect in vitro, but, all the
same, rectified spirit is said to be admirable for the dis-
infection of the surgeon's hands. If bacteria thereon
are not quickly killed, it is argued that the alcohol fixes
them on the skin so effectively that they can only be
removed with difficulty (see also p/ 248).

Picric acid is used for external disinfection in 0*5 per
cent, aqueous solution, especially after burns or scalds.
For wounds, Philip Turner recommends a 1 per cent,
solution in methylated spirit. Chloroform is used for
media (p. 39). Quite recently urea in strong solutions
has been recommended for antisepsis of wounds.

Disinfectants of the Aromatic Series. — Phenol (carbolic
acid) in saturated solution contains 6 to 7 per cent.
Proteins and organic matter generally only slightly
lessen its value. It is very poisonous and caustic, pro-
longed application to a surface producing ' carbolic acid
gangrene.' It is useless for sporing organisms, and is a
comparatively weak disinfectant. Its value in the arts is
such that very little of it is left in commercial carbolic
acid, which consists mainly of the cresols and higher
phenols. Cresols are only slightly soluble in water. A
mixture of ortho-, para-, and meta-cresols (tricresol) is
stronger and more soluble than individual cresols.
Cresols may also be dissolved in alkaline solution.

Cresols are much reduced in efficiency by proteins.
In saturated salt solution the disinfectant value of crude
carbolic acid is greatly increased. With crude sulphuric
acid it forms, if the mixture is conducted under conditions
preventing any great rise of temperature, a substance
niiscible with water, and possessing strong disinfectant
activity. For disinfection and destruction of faeces this
sulphonated cresol may be used in 5 per cent, solution,
not, of course, in metal utensils.

: Ordinarily neutral tar- oils with no appreciable dis-
infectant value are left in or mixed with tar distillate, and
the saponified product produces an emulsion with water.
Innumerable products of this type are made. Their

DISINFECTION AND DISINFECTANTS 257

efficiency varies not only with their active ingredients, but
also with the character of the emulsion which they form,
from about the same as that of phenol to about three
times as much. Commercially they are known as soluble
carbolic acid, soluble creosote, etc. Creolin is a type of
numerous preparations of the same character. They are
all poisonous and sensitive to proteins. If naphthalene
is present in excess, it is deposited on standing in cold
weather. Lysol contains 50 per cent, of the cresols in
fat or linseed oil saponified with addition of alcohol.
It gives a clear solution with water, having slightly less
efficiency on naked bacteria than cresol, much superior
solvency for grease, and equal sensitiveness to proteins.
Many preparations of the same type are now sold, and are
convenient for such laboratory purposes as disinfecting
slides and glass apparatus after use.

Liquor cresolis saponatus (British Pharmacopoeia,
1914) consists of cresylic acid 50 per cent, dissolved
in castor oil and saponified with potassium hydrate.

A new era in chemical disinfection began when Ainslie
Walker first prepared cyllin, which contains oxidised
hydrocarbons possessing a diphenyl nucleus, emulsified
with a neutral hydrocarbon oil. A large number of
emulsified disinfectants can now be obtained — kerol,
M.O.H. fluid, bactox, and cofectant being the most reliable.

It should bo noticed that the use of water containing
very high total solids (e.g., sea- water) reduces the efficiency
of these preparations, and special articles, in which glue
is used as a base, are made for such waters; but all such
preparations require to be agitated before dilution, owing
to the fact that they separate out on standing into layers
of very different efficiency.

Salicylic acid was formerly used as a preservative for
milk and milk products. It is still used for preserving
jams and lime-juice. It has recently been used as a
dressing for wounds. Thymol is a strong disinfectant
that enters into the composition of many mouth-washes
and tooth-pastes.

Disinfectant Powders.

If a suitable powder be sprinkled on manure-heaps,
dustbins, or in pail-closets or privies, not only are foul
odours absorbed, but flies are kept away, and prevented

25B AIDS TO BACTERIOLOGY

from laying eggs or feeding on the refuse. Lime, which
has a high capacity for absorbing sulphuretted hydrogen,
is very well adapted as a base for a disinfectant powder.
This is inadmissible with phenol, but is successfully
employed in the preparation of cyllin and kerol powders.
Examination of Disinfectant Powders. — Robertson and
Severn's process (Kept. M.O.H. Cape Colony for 1906):
One hundred grammes of the powder are thrown into a
litre stoppered measure, and made up to the mark with
sterile distilled water (temperature to be between 15° C.
and 18° C., little or no difference being noticeable be-
tween these limits). The cylinder is shaken violently
and horizontally every fifteen minutes for four hours, and
left then for subsidence. At the expiration of twenty-
four hours, some of the clear supernatant extract is drawn
off with a sterile 50 c.c. pipette into a sterile bottle or
flask. This is used to make the dilutions. (In some cases
a layer of the finer particles of the base will remain ob-
stinately on the surface of the extract, and some will
stick to the pipette. Quickly wipe this away with a
sterile cotton-wool plug.) Each 10 c.c. of the extract is
regarded as equal to 1 gramme of the powder. As a
standard, a 15 per cent, carbolic powder is taken as 1.
Such a powder is difficult to prepare and keep and extract,
owing to its deliquescence and the necessity for using
hot water for its complete extraction. A 10 per cent,
extract of a completely extracted 15 per cent, carbolic
acid powder can be simulated by taking 100 c.c. of a 4*7
per cent, solution of phenol and diluting to 313 c.c. (or
100 c.c. 5 per cent, phenol made up to 332 c.c.). This
solution is called "standard powder 1 in 10." Then a
17 dilution, for instance, is made by taking 100 c.c. and
diluting to 170 c.c. The R.-W. process is then applied
to extract and standard.

Bacteriological Examination of Disinfectants.

Few disinfectants are capable of being examined by
chemical means, so that the disinfectant value is revealed,
and, after all, the determination of the germicidal value
by actual experiment on living organisms is the only
reliable method of gauging the value. Many factors,
however, enter into the question, which must be re-
cognised: the time allowed for action, the temperature of

DISINFECTION AND DISINFECTANTS 259

medication, the age of the culture, the species, and even
the strain, of the organism, the number of organisms pre-
sented for disinfection, the reaction of the culture medium,
and the influence of organic matter, must all be con-
sidered. The desirability of stating the values of
disinfectants in terms of a common standard is obvious,
as it allows comparison, and the standard invariably
used is absolute phenol (first suggested by C. G. Moor).
The examination of a disinfectant involves, first, the
determination of its value in comparison with that of
phenol when the organisms are presented ' naked ' — -
i.e., without any appreciable amount of extraneous organic
matter present; and, secondly, when such organic matter
as is likely to be met with in practice is introduced into
the test to ascertain what, if any, is the depreciation in
value caused thereby. One method only of testing a dis-
infectant against a naked organism (the Rideal- Walker
method) need be considered in any detail, as this particular
method has been universally adopted for the purpose.
It requires a certain amount of dexterity and much care
to perform, but it is remarkable for the manner in which
factors likely to cause discrepancies are recognised and
guarded against.

Rideal -Walker Method.— A special test-tube rack is
used: a lower tier contains five holes for four tubes, hold-
ing the strengths of the disinfectant to be tested, and one
tube containing the standard phenol. The upper tier has
spaces for thirty test-tubes, in six sets of five holes. In
the upper tiers are placed tubes of standard broth, and
the tubes are numbered 1 to 30.

The method may now be briefly described as follows:
To 3 c.c. of a particular dilution of the disinfectant in
sterile water add 3 drops of a twenty-four hours' blood-
heat culture of the organism in broth; shake, and take
subcultures every two and a half minutes up to fifteen
minutes. Incubate these subcultures for at least forty-
eight hours at 37° C. Allowing thirty seconds for each
act of medication and the same time for making each
subculture, four different dilutions of the disinfectant under
examination, together with one standard control, may be
tested against the same culture, under conditions which
make the results strictly comparable. No table is to be
considered complete which does not show a positive result

26O

AIDS TO BACTERIOLOGY

in the first column and a negative result in the last. The
strength or efficiency of the disinfectant is expressed in
multiples of carbolic acid performing the same work —
i.e., when a dilution of the disinfectant has been obtained
which does the same work as the standard carbolic acid
dilution, the former is divided by the latter, and so a ratio
is obtained which the authors call the ' carbolic acid
coefficient.'

The use of the term ' carbolic acid coefficient ' in con-
nection with later tests, giving different results, has led to
much confusion. To guard against this, it is advisable
to express the results as ' Rideal- Walker ' coefficients
when employing the original test.

The following table shows the degree of refinement to
which this test may be carried with a little care:

B. TYPHOSUS (KRAL), TWENTY-FOUR HOURS' BROTH
CULTURE AT 37° C.

Room-Temperature, 15° to 18° C.

Time Culture exposed
to Action of Disin-

Subcultures.

fectant (Minutes).

2*

6

7i

10

12J

15

Period of
Incuba-
tion.

Tempera-
ture.

Disinfectant W

1 : 70

X

X

48 hours

37° C.

J5

1 : 80

X

X

X

s

> >

1 : 90

X

X

X

X

X

)

1 : 100

X

X

X

X

X

X

> 5

»

Carbolic acid...

1 : 80

X

X

...

From this it is seen that a 1 in 70 solution of disinfectant
W has the same action as a 1 in 80 solution of carbolic
acid. Disinfectant W is therefore not quite so active
as carbolic acid, and this is represented by the carbolic
acid coefficient — viz., £g- = 0'87.

Notes on the Test. — For rough work it may be taken
that 110 parts by weight of acidum carbolicum lique-
factum (B.P.) contains 100 parts by weight of absolute
phenol. Carbolic acid sometimes contains alcohol, which

DISINFECTION AND DISINFECTANTS 261

has been said to increase its germicidal power, and there-
fore to give a low coefficient for the sample. But, in
fact, the amounts found are too small to have appreciable
influence. More important is the quantity of cresols
usually found in carbolic acid crystals. As cresol has
approximately three times the germicidal power of
phenol, the error from this may be considerable. The
solidifying-point of the crystals should be not less than
40° C. Ainslie Walker and Weiss (Journ. Franklin Inst.,
1912, 101) say the bromine titration method does not
yield trustworthy results as regards the purity of the
phenol, but it may be employed for checking the strength
of the 5 per cent, stock solution prepared from pure
phenol. The 5 per cent, stock solution should be kept
in a closely-stoppered bottle in a dark cupboard. The
culture must be a Lemco broth one, twenty-four hours
old. The broth should be made as follows:

Lemco . . . . 20 grammes.

Peptone 20 „

Sodium chloride . . . . 10 „

Water 1 litre.

Boil the mixture for thirty minutes, and standardise to
a reaction of +1-5 per cent. (4-15, Eyre's scale), using
phenolphthalein.

When the cholera vibrio or diphtheria bacillus is the
test organism, a neutral or alkaline broth must be used
(' Bacteriological Examination of Disinfectants,' 1907,
p. 25), and for the latter a forty-eight hour old culture is
necessary (ibid., 17). Hewlett and Norman Hall (Journ.
Hygiene, 1912, p. 473) have shown that when working with
anthrax spores agar is preferable to nutrient broth for
the subcultures, as the latter does not show a life very
often when agar does.

Before addition to the medication tubes the culture
should be freed from clumps. This may be attained by
shaking the culture and allowing to settle for twenty
minutes before the test, when the culture required is
pipetted from the top half, or it may be filtered through
filter-paper.

The general laboratory procedure is to keep a stock
culture on agar, and subculture on to a fresh agar slope
at the end of a month From the twenty-four hour

262 AIDS TO BACTERIOLOGY

broth culture a fresh subculture is made in broth before
the former is used for the test. This subculturing into
fresh broth may and should be done every twenty-four
hours, whether a test is to be made or not. After a month
a fresh agar slope is made, and from the month-old agar
streak a fresh series of broth cultures started.

All measures, pipettes, and tubes should be sterile,
and sterile distilled water must be used for making the
dilutions.

The pipette used for delivering the three drops of
culture to the disinfectant dilutions should deliver O'l c.c.
per drop. The needle used should be composed of thin
aluminium rod, with a short piece of thin platinum wire
passed through and twisted round an eye in the end of
the rod. The free end of the wire is then made into
a loop about 4 millimetres in diameter. If the sides of
the loop be depressed so that in side-view it looks like
a cholera vibrio, the loop easily carries a big drop. Use
of a thin wire allows quick cooling.

When subculturing, a good-sized drop (easily obtained
by bringing the loop away from the liquid with a jerk)
should always be taken up.

The desired result in the phenol column is life in two
and a half minutes and in five minutes, and no life
thereafter. Rideal and Ainslie Walker (Medical Press,
September 15, 1915) suggest that a typhoid culture be
rejected if it calls for a phenol dilution higher than
1 in 110, or lower than 1 in 90.

While the test is in progress, the temperature of the
medication tubes should not vary more than 2° C. When
tests are being performed on successive days, it is of
considerable help if the temperature of the room in which
the first test is done be known. Should the temperature
be higher the next time, a weaker phenol solution should
be used, or, if lower, a stronger one.

Ainslie Walker (N.Y. Med. Journ., February 1, 1913)
points out that the use of bullock's heart broth instead
of Liebig broth has the effect of depressing a coefficient
by about 50 per cent. For further details of this test,
see ' The Bacteriological Examination of Disinfectants,'
by W. Partridge.

A coefficient obtained on a freshly prepared emulsion is
termed a ' temporary coefficient,' but as frequently a

DISINFECTION AND DISINFECTANTS 263

disinfectant solution is made up and allowed to stand for
several days before it is completely used, so if the dilution
necessary is calculated upon a freshly made emulsion,
which afterwards separates, and which in this way has a
lower efficiency than that calculated, it is quite conceiv-
able that considerable damage might be done. Rideal
and Walker, therefore, suggest that before applying the
test to a disinfectant a 1 per cent, solution should be

Srepared at normal temperature in a stoppered litre
ask, the portion required for further dilutions in the
test to be pipetted from the top of the flask after twenty-
four hours' subsidence, avoiding the withdrawal of any
insoluble matter which may be floating on the surface.
The coefficient is then referred to as a ' permanent
coefficient.'

The Lancet Commission (see Lancet, November 13, 20,
and 27, and December 18, 1909; Pharm. Journ., July 30,
1910) used a considerably modified Rideal-Walker test.
B. coli was used as the test organism, MacConkey's bile-
salt broth for the secondary subcultures, and platinum
cups were substituted for the inoculating needle. The
test covers thirty minutes as against the fifteen minutes
for the Rideal-Walker test, and more dilutions both of
the disinfectant and phenol are used. In determining
the coefficient, the weakest dilutions of disinfectant and
phenol killing in two and a half minutes are compared,
and a coefficient for the thirty-minutes period is obtained
in the same way. The mean of these two figures is
taken. This method is no advance on the Rideal-Walker.
When bacteria have been exposed to non lethal dilutions
of disinfectant, they are ' sick.' To put these into
MacConkey media, which cuts out weak organisms,
introduces an indecisive factor into the comparison.
Anderson and McClintic, of the United States Public
Health Service, advocate keeping the typhoid bacillus
as the test organism, as there is greater variability
between strains of colon bacilli than between typhoid
strains.

Anderson and McClintic (Journ. Infect. Dis., viii. 1), in
their ' Hygienic Laboratory Method,' compare an average
of two and a half and fifteen minute periods, and use an
increased number of dilutions of both disinfectant and
phenol. So much work has to be done in the time that

264 AIDS TO BACTERIOLOGY

seeding tubes have to be left unplugged. The process
is no improvement on the Rideal- Walker test.

Organic Matter in Disinfectant Testing.— Many forms
of organic matter have been suggested for incorporation
into the bacteriological test. Milk of various strengths,
washings from schoolroom floors, and faeces, have been
suggested and used; but these either (in the case of milk)
never want disinfection in practice, or allow no comparison
between two disinfectants, owing to the impossibility
of different workers using identical material.

Sommerville and Walker are undoubtedly right in
insisting that the forms of organic matter included in the
test should be of a simple form. The false coefficients
obtained with oxidising disinfectants can be revealed in a
most satisfactory manner by the use of urine, mucin,
peptone, casein, gelatin, or blood. Sommerville and
Walker first dilute the disinfectant in the proportion
recommended by the manufacturers or sanctioned by
use, all further dilutions being made with the organic
solution. The disinfectant is allowed to remain in contact
with the organic solution for one hour before adding the
test organism. They prefer as a diluent a solution con-
taining 0*5 per cent, gelatin and 0'5 per cent, rice starch,
the latter being added to meet the needs of adsorption.
A coefficient obtained with this diluent and allowing the
preliminary hour's contact of organic matter with the
disinfectant is known as a ' Sommerville- Walker co-
efficient.'

In the ' Lister Institute method,' the faeces are dried,
first in a water- bath and subsequently at 105° C., ground
to a fine powder in an agate mortar, and passed through
a fine sieve with a mesh of 130 to the inch. Quantities
of 0-15 gramme are added to test-tubes, to which 2 '5 c.c.
of distilled water are added, and the tubes are sterilised.
Different amounts of a suitable dilution of the disinfectant
are added to each tube, together with enough distilled
water to make 5 c.c. This gives different concentrations
of the disinfectant in presence of 3 per cent, of faeces.
The tubes are inoculated in the same way as when the
test is made with distilled water. Fifteen minutes is
allowed for the disinfectant to act; the test is done at
20° C., and an exactly similar experiment is done with
phenol.

DISINFECTION AND DISINFECTANTS 265

A disinfectant must be applied in such a strength as
to leave an excess above the proportion theoretically
needed. In other words, what. Defries has termed ' a
wide margin of safety ' must be insisted on. Sommerville
and Walker suggest ' it might be insisted that the multiple
5 be applied as a minimum to the strength of the various
disinfectants which are found to perform the same work
as 1 in 100 phenol.'

APPENDIX

Insolubility of Enzymes. — Enzymes exist in a colloidal
state, and are insoluble in the true sense of the word.
Bayliss (Jour. PhysioL, December, 1915) shows urease,
lipase, emulsin, invertase, lactase, papain, peroxidase,
and catalase are active in media from which they can
be filtered off by ordinary filter-paper, leaving an inactive
substrate.

Streptococci in Faeces. — Distaso (Lancet, January 8,
1916) points out that though Wright's ' saddle-bag '
streptococcus (see p. 135) is a common organism in fseces,
it is not S. fcecalis.

Tubercle Bacilli. — Eastwood and Griffith (Jour. Hygiene,
January, 1916) found 21*1 per cent, of human bone and
joint tuberculosis cases to be due to bovine bacilli. The
bovine type was also identified in three out of seven-
teen cases of tuberculosis of the human genito-urinary
tract. They emphasise the need for caution when, as
commonly happens, a ' human ' strain not in full vigour
gives a poor growth on glycerinated test media. Should
such a strain cause fairly severe lung disease in a
rabbit, there is a risk of it being wrongly returned as
' bovine.'

Meningococci. — Nankivell (Jour. Roy. San. Inst.,
January, 1916) is convinced that cerebro-spinal fever
is not propagated by personal contact, and believes it to
be conveyed by the bite of some animal parasite; all his
cases had shown flea, bug, or louse marks. Symonds
and others do not think vermin necessary for the spread

266 AIDS TO, BACTERIOLOGY

of the disease. Nankivell also thinks the period of in-
cubation to be short — not more than four days, probably
twenty-four to forty-eight hours. Meningococci having
been found in the blood in pure culture when meninges
and cord were unaffected, Nankivell strongly recommends
blood-culture.

Researches on the meningococcus till the end of 1915
are summarised in a ' Report of the Special Advisory
Committee upon Bacteriological Studies of Cerebro-Spinal
Fever during the Epidemic of 191.5 ' produced by the
Medical Research Committee (National Health Insur-
ance) and to be obtained from Wy man's. Fetter Lane,
E.G. (6d.). In this report, Professors Andrewes, Bulloch,
and Hewlett express scepticism regarding the ' pleomor-
phism ' of the meningococcus. The absence of growth
at 23° C., formerly thought to be of diagnostic value, has
proved unreliable; some meningococci show a degree of
growth at this temperature, though it is never profuse.
Further, certain forms of Micrococr/us flavus refuse to
grow at 23° C., and Gaskell finds that, at this temperature,
Micrococcus catarrhalis gives only a feeble growth that
soon fails. Nasgar and ivar-nasgar have proved unsatis-
factory for throat swabs. Agar or nasgar, smeared with
a few drops of fresh human blood are used by some workers
in preference to other media, but pea-flour-trypsin-agar
(in its final form of Trypagar} is also strongly recom-
mended. The Report considers Shearer's work on the
stimulus of nasal mucus on the meningococcus and Gor-
don's work on its inhibition by salivary streptococci.

Details of Douglas's trypsin-broth, used in the prepara-
tion of trypagar, will be found in the Lancet, October 10th,
1914. For subsequent work the reader is referred to
numbers of The Journal of the Royal Army Medical Corps.

INDEX

ABTOGENESIS, 4
Abortion bacillus, 149
Acetic acid bacteria, 188
Acetobacter melanogenum, 11
Achalme's bacillus, 196
Achorion Schorileinii, 173
Aoid-fast organisms, 28, GO, 157

staining of, 49, 58
Acids, 78, 79, 241, 250, 256,

257

Acne bacillus, 131
ActinobacilluH, 158
Actinomyees, 156
Actinomycosis, 156
Addiment, 20, 22
Aerated water, 100, 229
Aerobes, 6
Agar-agar, 36, 236, 237, 238

plates, 42, 230
Agglutination test, 104, 208,

236

Agglutiriins, 19, 20
Aggressins, 19
Agin bacilli, 90
Air, bacteria in, 100, 140, 214
Alcohol, 248, 252, 256
Alcohol-fast organisms, 61
Alexin, 20, 21, 22, 26
Alkalies, 251
Amakebe, 187
Amboccptor, 20, 22
Amoebre, 174
Amoebuljc, 184, 185
Amphitricha, 2
Amylomyccs Rouxii, 170
Amylopsin, 191
Anaerobes, 6, 43
Anaerobic cultures, 43
Anaphylaxis, 25
Anilin gentian violet, 48
Animal inoculations, 44
Anopheles, 183

Anthrax bacillus, 15, 1C, 28, 75
Orders, 77, 79

Anthrax Regulations, 78
Antibodies, 19
Antidote hypothesis, 17
Antiformin, 68, 252
Antigen test, 21
Antigens, .20, 26
Antimicrobic sera, 17, 22, 77,

132

Antitetanic serum, 81
Antitoxin, diphtheria, 117
Antitoxins, 17, 18, 19
Antityphoid vaccine, 103
Antivenin, 27
Aporrhegma, 10
Apotoxin, 26
Archebiosis, 4
Arsenic-resistance, 177, 183
Arthrospores, 4
Ascosporcs, 161, 165
Aseptic pus, 127
Aspergillinte, 168
Aspergillosis, 169
Aspergillus albus, 168

flavescens, 168 "

fumigatus, 168

niger, 168, 211
Atrcpsy theory, 17
Attenuation, 14
Atypical organisms, 88, 232
Autoclave, 8, 31
Autogenoiis vaccines, 23
Auximones, 212
Azotobacter, 211, 212

Bacillus, 5
aceti, 188

acidi lactici, 93, 108, 188
acidophilus, 92
acnes, 131

aerogenes capsulatus, 85
Aertryck, 96
albus variola;, 201
amylobactcr, 190
amylovorus, 194

267

268

AIDS TO BACTERIOLOGY

Bacillus anthracis, 15, 10, 28

75

aquatilis sulcatus, 236
auris, 112
bifidus, 92
botulinus, 83
bronchisepticus, 200
Bulgarians, 189
butyricus, 189
cadavcris sporogenes, 209
campestris, 193
ceruminis, 112
Chauvoci, 83, 86
choleroe gallinarum, 125
cloacfe, 92, 108, 202
coli. Sec Colon bacillus
anacrogcncs, 92, 108
communior, 90
coryzoe sogmentosus, 112
cyanogenus, 217
denitrificans, 213
diphtheria, 15, 110

columbarium, 121
dysenteric, 107, 108, 196
enteritidis, 93, 96, 97, 108

sporogencs, 85
ftocalis alkaligenes, 108,

236

fllamentosus, 202
fluorescens liquefaciens,
130, 190, 205, 213,
228
non -liquefaciens, 11,

205

stercolatus, 202
foedans, 209
foetidus lacticus, 190
furfuri, 191
fusiformis, 120
Griinthal, 88
hyacinthi, 194
icteroides, 96, 200
influenzas, 145
Koch-Weeks, 146
Kiitzingianum, 188
lactis aerogenes, 93, 108
erythrogenes, 218
pituosi, 219
saponacei, 219
viscosus, 219

lepro). See Leprosy bacil-
lus

levans, 88
M. 7, 193
mallei, 147

Bacillus meson tericus fuscus,

206

niger, 206
ruber, 206
vulgatus, 206
Morgan's No. 1, 97, 108
mycoides, 28, 202, 210, 213
neapolitanus, 92
03dematis maligni, 82, 214
of Blcisch, 218
of Ducrey, 146
of Hofmann, 111, 112, 113
of MacConkcy, ' No. 73,'

88

of Massol, 189
of Morgan, 97, 108
of pseudo-tuberculosis, 125
of Rabinowitch and Petri,

74

olere, 194

oxytocus perniciosus, 88
ozoonte, 93
paralyticans, 113
paratyphosus, 95, 108
Pastorianum, 188
pertussis, 147
phlegmoncs emphysema-

tosa?, 85
phlei, 159

pestis. See Plague bacil-
lus

pneumonia;, 143
prodigiosus, 10, 16, 132,

208, 218
proteus, 11, 16, 196, 205,

208, 210

pseudo-tuberculosis, 125
psittacosis, 96
putrificus coli, 209
pyocyancus, 130, 202
radicicola, 211
ramosus, 206
smegmatis, 28, 73, 61
solanaceannn, 194
subflaviis, 202
subtilis, 9, 13, 205, 213
suicholersc. See B. sui-

pestifer

suipestifer, 96, 108
synxanthus, 218
tetani. Sec Tetanus bacil-
lus

tholocideum, 206
tobacci, 190
trachciphilus, 194

INDEX

269

Bacillus tuberculosis. See Tu-
bercle bacillus

tuincfaciens, 194

tumescens, 213

typhi muriurn, 96

typhosus. See Typhoid
bacillus

vermiforme, 165

Wclchii, 85, 196, 202, 214,
215, 233

xerosis, 111, 112, 138
Bacteria, 5
Bacteriacese, 5
Bacteriolysis, 18, 22
Bacterio-purpurin, 192
Bacterium, 5
Bacteroids, 211
Bang's abortion bacillus, 149

method, 66

necrosis bacillus, 121
Barred staining, 110
Basidia,-168, 169
Bazillenemulsion, 68
Beading, 60, 147
Beef broth, 35
Beer wort, 39

gelatin, 39

Beggiatoa, 6, 192, 202
Beggiatoacese, 6
Behring sterilisation, 221

unit, 118

Bile salt media, 91, 230, 237
Biogenesis, 4
Bismarck brown, 50, 111
cladothrix, 210
Bitter milk, 218
Black-leg, 83
Black rot, 193

spot, 208

Blastomycetes, 161
Blastomycosis, 167
Blast ophore, 185
Blood cultures, 46

films, 46

serum, 38
Blood-heat organisms, 7, 32,

230

Blood-smeared agar, 39
Blue bodies of Koch, 187

milk, 217

tongue, 200
Bordet-Gcngou bacillus, 147

phenomenon, 21
Botrytis ciuerea, 194, 211
Bottcher chamber, 166

Bottom yeasts, 162

Botulism, 84

Bovine malaria, 187 V ,-"..

tuberculosis, 63 , "
Brilliant green, 107
Brown mould, 169

rot, 194

spot, 208

Brownian movement, 2
Buchanan's serum water, 111
Buchner's tube, 44
Buddhe process, 221
Bulgarian bacillus, 189
Bulloch's apparatus, 44
Burri's stain, 48, 182
Butter bacillus, 74

Cadaveric alkaloids, 12
Calf diarrhoea, 92, 97

diphtheria, 121

dysentery, 97.,
Calmette's reaction, 70
Cambier's process, 235
Capsule, 1

staining, 53
Carbol fuchsin, 49

gentian violet, 49

mcthylene blue, 49

thionine blue, 49
Carbolic acid, 31, 78, 249, 256,

260

coefficient, 260
Carcinoma, 195
Carrier cases, 95, 99, 102, 116,

136, 140, 154, 201
Cell membrane, 1

plasma, 1
Centrifuge, 32
Cerebro -spinal meningitis, 138,

265

Chain cocci, 4

Chattaway - Whartou appara-
tus, 217

Chemiotaxis, 18
Chicken cholera, 125
Chick-Martin process, 264
China -green agar, 238
Chlamydobacteriaceee, 5
Chlamydothrix ferruginea, 193

ochracea, 192
Chloride of lime, 242, 252
Chlorine, 242, 251
Chloroform, 39
Cholera, chicken, 125

nostras, 196

270

AIDS TO BACTERIOLOGY

Cholera spirillum, 8, 12, 150,

202, 225

Christmas's medium, 136
Chromidrosis, 10
Ghromogenesis, 10, 128, 130
Chymosin, 191
Cider disease, 190
Cladosporium hcrbarum, 208
Cladothrix, 6, 156

Bismarck brown, 210

dichotoma, 160, 169, 202
Classification, 4, 174
Clostridium, 3

butyricum, 190

Chauvoei, 83, 86

Pastorianum, 211
Coagulative ferments, 191
Coccaceee, 4
Coccidia, 186
Coccidium oviforme, 186
Coefficient, carbolic acid, 260

permanent, 263

Rideal-Walker, 260

Sommerville -Walker, 264

temporary, 262
Co-enzymes, 163
Cold, effect of, 7, 211, 225
Coley's fluid, 132
Coliform organisms, 88, 92
Colloidal metals, 253
Colon bacillus, 16, 88, 99, 108,

133, 196
in air, 8, 214
in sewage, 202
in sewer air, 214
in shellfish, 207
in water, 91, 230
Colonies, 33, 42
Colorado beetle, 194
Colostrum, 220
Columella, 168
Comma bacillus. See Cholera

spirillum

Complement, 20, 21, 22
Conn's micrococcus, 218
Conradi-Drigalski agar, 237
Contact preparations, 51
Contagion, 13
Cool organisms, 32, 228
Cooper-Hewitt lamp, 243
Copper sulphate, 242, 253, 254
Corrosive sublimate, 31, 78,

247, 253
Cover-glass, 46

preparations, 50

Cow-dung bacillus, loi)
Cowpox, 200
Crenothrix, 6

Kuhniana, 161

polyspora, 192
Crescent bodies, 185
Cresols, 248, 256
Culex, 183, 200
Culture media, 33

of bacteria, 33, 40
Ctitaneous reactions, 69, 106
Cytases, 18
Cytolysis, 19
Cytoryctes variola?, 201
Cytotoxins, 19

Dark -ground illumination, 181

Deamidases, 192

Deneke's cheese bacillus, 156

Dengue, 200

Denitrification, 213

Desiccation, 8

Deviation of complement, 22

Diarrhoea, calf, 92, 97

summer, 97, 196
Diastase, 191
Dikkop, 200

Diphtheria bacillus, 15, 110
Diphtheritic roup, 121
Diphtheroids, 71, 112, 115, 138,

142
Diplococcus, 4, 133, 135

crassus, 141

flavus, 141

gonorrhoea}, 136

intracellularis meningiti-
dis, 138, 265

mucosus, 141

of faeces, 135, 265

pharyngis siccus, 141

pneumonia}, 142

rheumaticus, 133, 134
Discontinuous sterilisation, 9
Disinfectant powders, 257
Disinfectants, 31, 246

valuation of, 258
Disinfection, 78, 244
Dissection of animals, 45
Distemper, 200
Distilled water gelatin, 236
Dourine, 178

Drumstick appearance, 3, 79
Dry heat, 8, 9, 30, 244
Dunham solution, 151
Durham tubes, 37

INDEX

271

Dust, 62, 82, 86, 100, 143, 201,

215, 219
Dysentery, amoeboid, 174

bacilli, 107, 108
Dysgonic bacilli, 64

Eberth-Gaffky bacillus. See

Typhoid bacillus
Education of bacteria, 98
Egg cultures, 39
Ehrlich's side-chain theory, 18

unit, 119
Electrolysis, 9
Emulsified disinfectants, 248,

257, 262

Endemic, 14, 77, 153
Endogenous spores, 3
Endotoxins, 12, 19
Endotryptase, 163
Entamoebse, 174
Enteritidis group, 93
Enterococcus, 135, 265
Enzymes, 27, 162, 190, 191, 265
Eosin, 49
Epidemics, 14
Epilepsy, 195
Epizootic abortion, 149
Erysipelas, 15
Esmarch cultures, 41
Eugonic bacilli, 64
Excess lime method, 244
Exhaustion theory, 17
Extracellular toxins, 13
Eyre's scale, 34

Facultative bacteria, 6, 7
Fteces, bacteria in, 80, 85, 88,
92, 93, 94, 95, 97, 98,
106, 109, 150, 155, 26o

disinfection, 251, 256
Farcy, 148
Favus, 173

Fermentation, 12, 28, 37, 188
Ferments, 191
Film formation, 166

preparations, 46
Filterable viruses, 197, 249 "
Filters, 101, 238
Filtration of air, 31, 215

of water, 31, 99, 238
Finkler-Prior spirillum, 155
Fir-tree growth, 76
Fission fungi, 3
Fixation abscess, 127

of complement, 20

Fixation of films, 50, 52

of nitrogen, 211

of tissue, 55

test, 21
Flagella, 2, 175, 185

staining, 52
Flagellated bodies, 185
Flaginac bacilli, 90
Fleas as carriers, 124, 266
Fleming's medium, 131
Flexnef bacillus, 107, 108

serum, 142
Flies as carriers, 62, 97, 100,

154, 201, 217
Fly disease, 177
Foamy organs, 86
Foot-and-mouth disease, 199
Forbush-Fernald torch, 244
Formaldehyde, 78, 254
Formic acid, 78
Fractional sterilisation, 9
Frankel' s method, 44

soil borer, 213

Frankland's method for air, 216
Friedlander's bacillus, 143

Gallionella fcrruginea, 193
Galziekte, 177
Gametes, 174, 185
Garget, 135, 219
Gartner bacillus, 93, 108
Gaseous gangrene, 86
Gelatin, distilled water, 236

glucose, 36

nutrient, 36, 91

plates, 40, 42, 228
Gemmation, 161
Giant cells, 63
Giemsa stain, 181
Ginger-beer plant, 165
Glanders bacillus, 147
Glossina, 175
Glucose agar, 37

broth, 36

gelatin, 36

neutral red broth, 230

peptone water, 37
Glycerin agar, 37

broth, 36
Goldfish test, 204
Gonococcus, 136
Gram-Gunthcr stain, 60
Gram-negative, 28, 59
Gram-positive, 59
Gram iodine, 59

272

AIDS TO BACTERIOLOGY

Gram stain, 59
Granulornata, 63
Granulose reaction, 160
Gum-producing bacteria, 194

Haemamoebse malarise, 183, 184

vivax, 184
Haemolysis, 20, 27

test, 152

Hsemornenas proecox, 184
Hsernorrhagic septicaemia, 121
Ha3morrhagin, 27
Haffkine's prophylactic, 124

stalactite growth, 122

vaccine, 154
Halogens, 242, 251
Hanging-drop preparations, 47
Hankin's salt agar, 122
Hansen's yeast cultures, 166
Haptophore group, 18
Hay bacillus, 205

fever, 196

self -heating of, 10
Heat, 8, 30

production of, 10
Hermitine, 252
Hesse's apparatus, 215
High yeasts, 162
Hiss's serum water, 114
Hoffmann-Ficker method, 235
Hofrnann bacillus, 111
Homogeneity of disinfectants,

250

Horse sickness, 200
Housell's acid alcohol, 61, 6!)
Houston's standards for

oysters, 207
for sewage, 204

peat organisms, 210
Hueppe egg cultures, 39
Hyacinth disease, 194
Hydrochloric acid, 78
Hydrogen peroxide, 221, 254
Hydrophobia, 198
Hyphse, 167
Hyphomycetcs, 167
Hypochlorites, 242, 252

Ice, 8

Immune body, 20, 21, 22

Immunity, 15, 16

Impression preparations, 51

Inactivation, 20

Incubator test for effluents, 203

Jncubators, 32

Indian ink, 48, 182
Indicators of pollution, baC
terial, 87, 91, 99, 214, 215,
223, 224
Indolc, 11

Infantile paralysis, 201
Infection, 13
Influenza bacillus, 15, 145

cold, 130
Infusoria, 174
Inoculating needle, 32
Inoculation of animals, 44

of media, 40-44
Intracellular toxins, 12
Intravitam staining, 48
Inversive ferments, 191
Invertase, 162
Involution forms, 3
Iodine, 242, 251
lodoform, 252
lonisation, 246
Irish moss jelly, 40
Iron bacteria, 192
Isle of Wight bee disease, 187
Isolation of bacteria, 33, 40

Jenncr stain, 186
Johne's bacillus, 70

Kala azar, 178

Kedrowsky 's bacillus, 7 1

Keratomycetcs, 171

Klebs-Loffler bacillus, 110

Koch's blue bodies, 187
comma bacillus, 150
liltration coefficients, 240
postulates, 13
tuberculins, 67, 68

Koch-Weeks bacillus, 146

Kuhne's blue, 49

method for sections, 58

Lancet method, 263
Leishmania Donovani, 178
Irishman's stain, 186
Lemco broth, 35, 36, 261
Leprosy bacillus, 59, 63, 71
Leptothrix, 6, 156, 160

buccalis, 160

epidermidis, 160

innominata, 160

ochracea, 192
Leucocytes in milk, 221
Light, effect of, 9

production of, 10

INDEX

273

Lime, 244, 251

Lipase, 192

Liquor creosolis sapoiiatus, 257

Lister Institute method, 249,

264
Loffler's blue, 49

medium, 39

method for sections, 58
Lophptricha, 2
Louping-ill, 196
Low yeasts, 162
Luetin, 183
Lupus, 9, 62, 64, 182
Lysol, 31, 257

MacConkey agar, 237

bile -salt broth, 91, 230, 237
Macroganietes, 174
Madura disease, 158
Malarial parasites, 183
Mai do Caderas, 177

ducoit, 178
Malignant disease, 195

oedema, 82, 214

pustule, 76
Mallein, 148
Malta fever, 144
Maltase, 162
Maltose agar, 172
Mas sol bacillus, 189
Mastigophora, 174
McCrorie's stain, 53
Measles, 15, 199
Media, nutrient, 33
Meningococcus, 138, 265
Mercuric chloride, 31, 78, 247,

253

Merozoites, 184
Metachromatic granules, 2
Metachromatism, 110
Micrococcus, 3, 5

albidus, 194

calco-aceticus, 11

catarrhalis, 130, 139, 141

cereus, 129

flavidus, 194

flavus, 141

imperatoris, 194

melitensis, 144

nuclei, 194

pellucidus, 194

pyogenes. See Staphy-
lococcus pyogenes

salivarius, 129

tetragenus, 130

Micrococcus urea?, 188, 202

viscosus, 219
Microgametes, 174
Micro -millimetre, 2
Micron, 2
Microscope, 29
Microspira, 5
Microsporidia, 187
Microsporon Audouini, 172

furfur, 173
Microtome, 31
Milk, 16, 217

bitter, 218

blue, 217

cholera vibrios in, 154

diphtheria bacilli in, 115,
223

fermentation, 189

foot-and-mouth disease
and, 199

Malta fever and, 145

para -typhoid bacilli in, 95

red, 218

ropy, 219

salty, 220

scarlet fever and, 195

slimy, 219

soapy, 219

sour, 189

sterilisation, 9, 38

stringy, 219

tuberculous, 62, 63, 222

tubes, 38

typhoid bacilli in, 101, 223

yellow, 218
Miller's spirillum, 155
Mistbacillus, 159
Mixed infections, 15
Moeller's method, 52
Monotricha, 2
Morgan's bacillus, 97, 108
Moro's ointment, 70
Mosquitoes, 176, 183, 200
Motility, 2, 47
Moulds, 167, 190

pathogenic, 168
Mouse septicaemia, 127
Mucor corymbifer, 168

erectus, 170

mucedo, 168

pyriformis, 194

racemosus, 194

ramosus, 168

rhizopodiformis, 168

Rouxii, 170

18

274

AIDS TO BACTERIOLOGY

Mucoriucaj, 1G8
Muir capsule stain, 54
Muir-Pitncld stain, 53
Muiler's fluid, 55
Mumford's bacillus, 193
Mumps, 15, 200
Muscarin, 12
Mussels, 208
Mutability, bacterial, 28
Mycelia, 167
Mycetoma, 158
Mycoderina aceti, 188

cerivisise, 164
Mytilotoxin, 12
Myzopods, 184

Nagana, 177
Nail-head growth, 144
Naked bacteria, 2-59
Nasgar, 138
Necrosis bacillus, 121
Negative staining, 48
Negri bodies, 198
Neisser spore stain, 52

stain, 110

Nephritis, epidemic, 201
Neutral red broth, 236

bile salt agar, 237
Nicolle's blue, 49
Night blue stain, 53
Nitratation, 213
Nitrification, 39, 212
Nitrosatiou, 213
Nitrosornonas, 213
Nocardia, 156
Nodule bacteria, 211
Nosema apis, 187

bombycis, 187
Nutrient agar, 36

broth, 35

gelatin, 36, 91

media, 33
Nutrose ascitic agar, 138

Obligate bacteria, 6, 7

Oidiaceo), 170

Oidium albicaus, 170
carnis, 209
lactis, 170, 190

Olive-knot disease, 194

Opsonic index, 70

Opsouius, 18

Optimum temperature, 7,

Ostertag's method, 66

Oxidation organisms, 11

248

Oxydases, 192
Oxyphobia, 7
Oysters, 100, 207
Ozeena, 93
Ozone, 243, 254

Pabulum hypothesis, 17
Packet cocci, 5
Pandemic disease, 14
Pappenheim solution, 7'4
Paracolon bacilli, 97
Paraffin bath, 56
Parasites, 7

Paratyphoid bacilli, 95, 108
Pasteurisation, 6
Pasteur vaccine, 77
Pathogenesis, 13
Pathologist' s warts, 62
Pear blight, 194
Peat bacteria, 210
Pebrine, 187
Pellagra, 195
Penicilliacece, 168
Pcuicillium brevicaule, 169

candidum, 190

glaucurn, 169, 190, 194,
211

italicum, 194

olivacemn, 194
Pepsin, 191

Peptolytic enzymes, 192
Peptone water, 37

Dunham's, 151
Peritricha, 2

Permanent coefficient, 263
Personal vaccines, 23
Pertussis, 147
Petri dishes, 41

method for air, 216
Pfeiffer's influenza bacillus, 145

pseudo-tuberculosis bacil-
lus, 125

reaction, 22, 236
Phagocytosis, 18, 19
Phagolysis, 18
Phenol, 31, 78, 249, 256, 260
Phlebotomus fever, 200
Photogenesis, 10
Pick-me-up media, 91
Picric acid, 256
Pigeon diphtheria, 121, 202
Pink eye, 146
Piroplasma bigeminum, 187

parvum, 187
Piroplasmosis, 187

INDEX

275

Plague bacillus, 7, 121
Planococcus, 5
Planosarcina, 5
Plant diseases, 193
Plasmodium malaria?, 183
Plate cultures, 40, 42, 228, 230,

237

Pleuro-pneumonia, 197
Pneumo -bacillus, 143
Pneumococcus, 15, 142
Polar staining, 110, 121
Poliomyelitis, 201
Polyvalent sera, 132

vaccines, 23, 92
Potassium permanganate, 242,

254
Potato bacillus, 206

diseases, 194

tubes, 38

Poynton and Paine's organ-
ism, 133

Precipitation of typhoid ba-
cilli, 234
Precipitins, 20
Proteolytic ferments, 191
Proteus. See B. proteus
Protozoa, 174
Pseudomonas, 5, 212

aromatica, 11

campestris, 193

destructans, 194
Pseudopodia, 174
Psittacosis, 96
Ptomines, 12
Puerperal fever, 133
Pugh's stain, 111
Pure culture, 33, 44
Purpur bacteria, 192
Pus in milk, 221
Putrefaction, 11
Pysemia, 14
Pyocyanase, 130
Pyogenesis, 127, 171

Quarter-evil, 83
Quinine, 185

Rabies, 198
Radium emanations, 9
Rat virus, 96
Ray fungus, 156
Reaction of media, 34
Receptor group, IS
Red braxy, 83
milk, 218

Red spot, 208
Red-water, 187
Reductases, 192
Reproduction of bacteria, 3

of protozoa, 174
Retention hypothesis, 1 7
Retting of flax, 191
Rheumatic fever, 133
Rhizobium radicicola, 211
Rideal-Walker coefficient, 259
Rinderpest, 199
Ringworm fungi, 171
Roll cultures, 41
Rontgen rays, 9
Roux unit, 119
Roux-Vaillard serum, 81
Russ's apparatus, 10

Sabouraud's medium, 131
Saccharobacillus, 189
Saccharomyces albus, 167

anomalous, 165

apiculatus, 164

cerevisiee, 162

ellipsoideus, 163

exiguus, 164

marxianus, 164

mycoderma, 164

niger, 167

Pastorianus, 163

pyriformis, 165

rosaceus, 167

ruber, 218

Saccharomycetes, 161
Safety change, 226
Sagin bacilli, 90
Salt, 85

agar, 122

Sand filtration, 240
Saprsemia, 14
Saprophytes, 7
Sarcina, 3, 5

rosea, 218

ventriculi, 130
Sarcoma, 195
Sarkodina, 174
Saturated steam, 245
Saturation test, 152
Scab in potatoes, 194
Scarlet fever, 15, 195
Schirrous cord, 157
Schizosaccharomyccs, 161, 165
Sclavo's serum, 77

smash bottles, 227
Sclerotium, 168

276

AIDS TO BACTERIOLOGY

Secondary infections, 15
Sections, treatment of, 54-60
Sedgwick-Tucker apparatus,

216

Sedimentation, 225
Selective media, 6
Sensitiser, 20
Septicaemia, 14
Serum disease, 26, 120

media, 38, 39, 136, 139

therapy, 23, 77, 81

water, 114
Sewage, 202

Seymour- Jones process, 78
Shake cultures, 43
Sheep-pox, 200
Shellfish, 100, 207
Shiga-Kruse bacillus, 107, 108
Siberian plague, 76
Side-chain theory, 18
Siedentopf 's apparatus, 30
Silica jelly, 39
Silver salts, 253
Simulium, 196
Size of bacteria, 2
Sleeping sickness, 175
Slimy milk, 219
Smallpox, 200
Smear preparations, 51
Smegma bacilli, 28, 61, 73
Snake venom, 17, 27
Soap, disinfectant, 251
Soapy milk, 219
Soft sore, 146
Soil, bacteria in, 77, 80, 82, 101,

210

Solid-staining bacilli, 110
Sommerville - Walker coeffi-
cient, 264
Sour milk, 167
Spirilla, 150
Spirillacese, 5

Spirillum cholerse . Sec Cholera
spirillum

Finkler-Prior, 155

Metchnikovi, 155

Obermeieri, 179

rubrum, 156

tyrogenum, 156
Spirocheeta anserina, 179

dentium, 181

Duttoni, 179

pallida, 179

recurrentis, 179

refringens, 180

Splrcclicota Vincenti, 120
Spirocheetes, 179
Spirophyllum ferrugineum, 193
Spirosoma, 5
Splenic fever, 76
Spontaneous combustion, 10
Sporangium, 168
Spore formation, 3

staining, 52
Sporocytes, 184
Sporotrichon Beurmanii, 171
Sporozoa, 174
Spotted distribution, 99

fever, 138

Spray disinfection, 246
Stab cultures, 43
Staining, 48, 58, 59

of blood, 186

of capsules, 53

of flagella, 52

of moulds, 169, 172

of protozoa, 178, 181

of sections, 57

of spores, 52

Standardisation of antitoxin,
118

of media, 34
Staphylococci, 3, 5
Staphylococcus cercus albus,

129
flavus, 129

epidermidis albus, 129, 215

pyogenes albus, 129
aureus, 128, 133
citreus, 129
Steam, 8, 30, 78, 245
Steapsin, 192
Steatolytic ferments, 192
Sterigmata, 169
Sterilisation, 8

of milk, 9

of soil, 213

of water, 9, 241
Sterilisers, 30
Stock vaccines, 23, 92
Storage of water, 99, 225
Strangles, 136
Straus's reaction, 148
Streak ciiltures, 42
Streptobacillus, 122
Streptococci, 3, 4, 131-136, 237

in air, 214

in milk, 134, 135, 222, 224

in sewage, 202

in water, 231, 232

INDEX

277

Streptococcus anginosus, 134

brevis, 133, 134, 135

conglomerates, 134, 195

equinus, 134

erysipelatis, 132

fcecalis, 134, 233, 265

Hollandicus, 190, 219

lacticus, 190

lebenis, 189

longus, 133, 134

mastitidis, 134

medius, 134

mucosus, 135

pneumoniee, 142

puerperalis, 133

pyogenes, 131, 134

salivarius, 134

scarlatinee, 134, 195

viridans, 135
Streptothrix, 5, 156

Epplngeri, 159

maduroe, 159
Streptotrichosis, 60, 159
Stringy milk, 219
Sulphur, 250

bacteria, 192
Sulphuric acid, 79
Superheated steam, 245
Suppuration, 127
Surface plates, 237
Surra, 177
Susceptibility, 14
Swine erysipelas, 126

fever, 96, 198

plague, 125

tuberculosis, 65
Symbiosis, 15
Symptomatic anthrax, 83

Taettemoelk, 219

Taint in hams, 209

Tanning, 191

Temporary coefficient, 262

Test tubes, 33

Tetanus bacillus, 16, 79, 214

Texas fever, 187

Theobald Smith phenomenon,

26

Thermal death-point, 8
Thermolabile, 20
Thermophilic organisms, 7, 10,

40, 211, 213
Thermostable, 20, 163
Thiophysa, 192
Thiothrix, 6, 192

Timothy -grass bacilli, 28,

159

Tobacco bacilli, 191
Top yeasts, 162
Torulee, 161, 167
Toxaemia, 116
Toxins, 12, 16, 18, 81, 84, 94,

116, 117
Toxogenin, 26
Toxoids, 18, 117
Toxones, 117
Toxophile group, 19
Toxophore group, 19
Travelling vermicule, 185
Treponema pallidum, 179
Trichomycetes, 156
Tricophyton megalosporon en-

dothrix, 172
ectothrix, 172

Trypanosoma Brucei, 177,
178

dimorphon, 177

equinum, 177

equiperdum, 178

Evansi, 177

Gambiense, 175

Lewisi, 178

Theileri, 177

Transvaaliense, 177
Trypanosomes, 175
Trypsin, 191
Tryptophan medium, 12
Tsetse flies, 175
Tubercle bacillus, 7, 8, 28, 49,

59, 60, 159, 214, 265
Tuberculin, 67
Tuberculosis, 62, 265

avian, 66

bovine, 63

pseudo-, 68, 125
Typhaceee, 88

Typhoid bacillus, 28, 97, 108,
202, 211, 225, 234

carriers, 98, 99. 101. 102

spine, 102
Typhus, 199

chicken, 202
Tyrotoxicon, 12, 87, 196

Ultra-microscopic organisms,

197

Ultra-violet light, 9, 28, 243
Unna's bacillus, 131
Urease, 188
Urine media, 37, 136

278

AIDS TO BACTERIOLOGY

Vaccines, 23, 92, 96, 103
Vaccinia, 200
Variola, 200

carp, 202

Vegetative form, 4
Venom, snake, 17, 27
Vibrio, 5

cholera. See Cholera spi-
rillum

proteus. See Finkler-Prior
spirillum

tyrosinatica, 11
Vincent's angina, 120
Viscid-milk bacillus, 219
Voges-Proskauer reaction, 89
Von Pirquet reaction, 69

Wassermann reaction, 21, 72,
182

Water, 98, 224

aerated, 100, 229
anthrax bacilli in, 77
B. pyocyaneus in, 130
cholera vibrio in, 154, 225
diphtheria bacilli in, 115
dysentery bacilli in, 109
infantile paralysis and, 201
paratyphoid bacilli in, 95
plague bacilli in, 122
spotted distribution in, 99
sterilisation, 9
typhoid bacilli in, 98, 225,
' 227

Weigert's law, 18

Welch-Nuttall test, 86

Werbitzki agar, 238

Wertheim medium, 130

White rot, 194
comb, 173

Whooping-cough, 15, 147
Widal reaction, 104
Wild races, 92
Willson precipitation, 234
Wilt disease, 194
Winogradsky's bacilli, 19.1
Wooden tongue, 158
Woolsorter's disease, 76
Wright vaccine, 24
Wurzel bacillus, 206

Xerosis bacillus, 111, 112, 138

Yeasts, 161

in milk, 167

on meat, 208

pathogenic, 167
Yellow disease of hyacinths,
194

fever, 200

milk, 218

Ziehl carbol-fuchsin, 49

stain for leprosy bacillus,

71

Ziehl-Neelsen method, 58
Zipfel's medium, 12
Zooglcea, 1
Zygometres, 185
Zygotes, 174, 185
Zygotoblasts, 185
Zymase, 162
Zymogens, 192
Zymolysis, 191
Zone reactions, 106

Baill&re, Tmdall <t- Cox, S, Henrietta Street, Covent Garden