Bacteria and their products

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THE CONTEMPORARY
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Bacteri

id their products.

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THE CONTEMPORARY SCIENCE SERIES.
Epitep sy HAVELOCK ELLIS.

BACTERIA AND THEIR PRODUCTS.

BACTERIA

AND THEIR PRODUCTS.

EOPT TI
PPIVT REIT Y
bRLES EY

BY

GERMAN SIMS WOODHEAD, M.D. (EDIN.),
Director of the Laboratories of the Conjoint Board of the Royal Colleges of Physicians

(Lond.) and Surgeons (Eng.); Formerly Research Scholar of the
Honourable Grocers’ Company.

WITH 20 PHOTO-MICROGRAPHS

AND AN APPENDIX GIVING A SHORT ACCOUNT OF
BACTERIOLOGICAL METHODS, AND A DIAGNOSTIC DESCRIPTION
OF THE COMMONER BACTERIA.

LONDON:
WALTER SCOTT, Lrp. 24 WARWICK LANE.
CHARLES SCRIBNER’S SONS,
743 & 745 BROADWAY, NEW YORK.

PREFACE.

-_———

N the following pages an attempt is made to give some
account of the main facts in Bacteriology, and of the
life-history of Bacteria and closely allied organisms, and also
to discuss the more important theories as to the part played
by them in Nature’s Economy ; especially in their relation
to the commoner fermentative, putrefactive, and disease pro-
cesses.

It may be held by some of my readers that too much pro-
minence is given to certain questions, whilst others have
been relegated to a comparatively obscure position, and that
some points have been accentuated, perhaps at the expense
of others; but to such criticism it may be answered that
whilst some facts carry conviction along with them, others
can only be properly appreciated when seen by stage lights.
On the other hand, many points have of set purpose been
lightly touched upon because experience is constantly
bringing home the fact that what is new to-day may be
out of date to-morrow. I have, therefore, thought it better,
in most instances, to confine myself to an exposition of well-
accredited facts and to a discussion of some of the more
stable theories.

Being privileged to hold a Sanitary Research Scholarship
of the Honourable Grocers’ Company for some years, I
was enabled to devote considerable time to the study of the
relations of Bacteria to Disease, especially in the case of
tuberculosis, and many of the interpretations of facts here
mentioned are based on observations then made; the re-

vi PREFACE.

mainder are drawn chiefly from the works enumerated at
the end of each section. These lists, however, represent but
a small part of the enormous mass of literature dealing with
Bacteria and their Products, which has accumulated during
the last decade.

To Professor Léffler’s admirable work, ‘ Vorlesungen
tiber die geschichtliche Entwickelung der Lehre von den
Bacterien,” and to Mr. Watson Cheyne’s ‘ Antiseptic
Surgery,” I am specially indebted for much information and
guidance in my search for facts and papers dealing with the
earlier history of the subject.

It may be said of the Appendix that it is given, not with
the object of supplying intending workers with every known
method of research, and with full descriptions of every
organism with which they may have to deal, but simply to
enable them to commence work, and to recognize the
commoner forms of Bacteria, of which about .one hundred
and forty are here described.

I take the opportunity of expressing my thanks to Dr.
Cartwright Wood, who, whilst revising the proof sheets,
has made several valuable suggestions; to my former as-
sistant, Mr. Coghill, now of the Royal Veterinary College,
and to Mr. Andrew Pringle, both of whom have made for
me beautiful photo-micrographs, which, however, can be but
imperfectly reproduced by any photo-mechanical process

now available.
G. S. W.

CONTENTS.

CHAPTER I.
PAGE

INTRODUCTION - - ° 1-23

Growth of Subject—Place of Bacteria in Nature—Pure Cuttures
—Morphology—Physiology—Protoplasm governed by same
laws whether in higher or lower Plants or Animals—Relation
of Bacteria to everyday Processes—Fermentations, Butyric,
Lactic, Colour, &c.—Brewing—Baking—Kephir making—
Flax preparation — Digestion — Putrefaction — Nitrification,
Mineralization—Relation of Bacteria to Water Supply—Fil-
tration—Sewage—Modes of Transference of Pathogenic Bac-
teria from Patient to Patient, or from Water or Earth to
Patient.

CHAPTER II.

WHAT ARE BACTERIA? - : 24-48

Structure — Myco-protein — Limiting Membrane — Gelatinous
Capsule—Special Cell Contents—Oxide of Iron—Sulphur—
Colouring Material—Flagella—Modes of Multiplication and
Development—Division—Rate of Vegetative Multiplication
—Endospores—Arthrospores—Classifications of Cohn, Van
Tieghem, Zopf, Winter and Rabenhorst, De Bary and
Hueppe, Fliigge, Baumgarten, &c.

CHAPTER IIL

Tue HisToRY OF BACTERIOLOGY - 49-74

Earliest Workers—Kircher’s Contagium Animatum—Bacteria in
Fermentation, Putrefaction, and Disease—Early Classifica-
tions — Miiller — Abiogenesis — Needham — Biogenesis —
Bonnet—Spallanzani—Schultz’s Experiments— Schwann—
Later Experiments— Pasteur — Bastian—Colour and Fer-
mentation — Cohn and Naegeli’s Classification — Henle’s
Researches and Postulates—Pasteur’s Researches on Fer-

vill CONTENTS.

PAGE
mentation and Putrefaction—‘* Flower of Wine ”—‘ Flower

‘of Vinegar ”—Bacteria as Scavengers—Pasteur on Silkworm

Disease, and Wine Disease—Germs killed by Carbolic Acid—

Origin of Antiseptic Treatment.

CHAPTER IV.

BACTERIA AS THE CAUSES OF DISEASE : 75-86

Anthrax — Pollender—-Davaine—Rayer—Laplat and Jaillard’s
Observations controverted by Davaine—Pyzmia and Sep-
ticeemia— Salisbury on Bacteria as the Cause of certain
Fevers — Johanna Liiders’ and Hallier’s Observations on
Pleomorphism or Polymorphism—Burdon Sanderson, Hoff-
man, and others, state that there is no Connection between
Bacteria and the Higher Fungi—Demonstration of Infective
Element in Anthrax Blood—Bacteria found in Organs in
certain Diseases—Sarcina in Stomach—Specific Organisms—
Specific Activities.

CHAPTER V.

FERMENTATION - 87-114

Fermentation, a Key to the Whole Position—Chemical Fer-
mentations — Illustrations — Organic Fermentations — Al-
coholic Fermentation—Result of Activity of Living Proto-
plasm—Lactic and Butyric Fermentations—Cagniard Latour
~——Schwann— Reess—Hansen—Liebig’s Theory of Fermenta-
tioh—High Beer—Low Beer—Methed of obtaining Pure
Yeasts—Hansen’s Classification of the Saccharomyces—Spore
Formation—Film Formation—Character of Species of Sac-
charomyces—Metschnikoff's Monospora—Torulz.

CHAPTER VI.

FERMENTATION (conécnued ) 115-144

Soluble Constituents of Yeast—Action of these: upon Sugar—
Conversion of Glycogen in the Liver and other Tissues—
Growth of Yeast-Cells in Organic and Inorganic Fluids—
Fermentation of Fruit Juice—AZrobic and Anzrobic Fer-
mentations—Effect of Free Oxygen on Yeast-Cells—Fer-
mentation not necessarily equal to Growth of Yeast —
Enzyme or Unorganized Ferment a Secondary Function—
Function depends partly on Organism, partly on Medium in
which it is Growing—Peptonizing Function usually requires
presence of Oxygen—Various kinds of Fermentation: Lactic,
Urea, Butyric, Ammoniacal, Acetic—Formation of Fatty
Acids—Mycoderma Aceti—General Processes of Fermenta-
tion —Hoppe-Seyler’s Classification.

CONTENTS.

CHAPTER VII.

PARASITES AND SAPROPHYTES

Putrefactive Bacteria — Pigment-forming Bacteria — Enzyme-
forming Bacteria—Non-pathogenic, Saprophytic, Parasitic,
Pathogenic Bacteria—Sarcina Ventriculi—Leptothrix Buccalis
— Facultative Saprophytes — Facultative Pathogenic Para-
sites—Anthrax Bacillus—‘‘ Pathogenic,” ‘‘ Parasitic,” ‘* Sa-
prophytic,” merely Relative Terms.

CHAPTER VIII.

CHOLERA :

Cholera a Parasitic Disease—Early Observations on the Comma
Bacillus — Characters—Methods of Staining— Methods of
Isolation—Use of Plate Method in Practical Public Health
Work—Tube Cultures—Motility of Cholera Bacilli—Potato,
Blood-serum, and Milk Growths—Behaviour in Water and
Sewage—Infection of Man through Agency of Bacillus—
Early Inoculation Experiments—Koch—Nicati and Rietsch
—Macleod—Difficulties—Cholera in Guinea-pigs—Position
of Bacilli in Tissues—Presence of Spores doubtful.

CHAPTER IX.

CHOLERA (continued ) .

Pettenkofer’s researches —Saprophytic and Parasitic Stages of
Cholera Bacillus — Temperature Conditions — Relation to
Epidemics —Moisture—Ground Water — Flushing—Cholera
in Shanghai Endemic but Intermittent—Cholera Endemic at
the Mouth of the Ganges — Vitality of Cholera in Ohl
Cultures—Relation of this to Quiescent Periods during Parts
of the Year—Gastro-intestinal Irritations prepare for Cholera
—Chinese Vegetables—Cholera Poison formed in the In-
testine, absorbed into Body—Inoculation against Cholera
—Gamaleia’s Experiments—Germicides useful in attacking
the Cholera Bacilli—Water Supply, Pilgrimages, Feasts pre-
disposing to Cholera—Quarantine except in Harbours Use-
less—Time and Place Dispositions.

CHAPTER X.

TYPHOID FEVER - - . i is “

Typhoid Fever a Bacterial Disease—Recklinghausen’s Observa-
tions — Klein — Eberth — Klebs—Coats— The Bacillus —
Method of Staining—Position in Tissues—Gaffky’s Observa-
tions-—Pure Cultures—Excretory Products—Experiments on
Animals—Mixed Infections—Action of Light and Heat on
Typhoid Bacilli—Pseudo-typhoid Bacilli.

PAGE

145-149

150-170

171-193

194-204

x CONTENTS.

CHAPTER XI.

TUBERCULOSIS

Tuberculosis a widespread Disease - The Tubercle Bacillus —
Koch—Baumgarten—Spores seen by Watson Cheyne—Re-
lation of Organisms to Tissues—Bacillus in Tuberculosis of
Animals—Tubercle Bacilli as Saprophytes—Bacilli cultivated
outside the Body by Koch—Methods—Temperature Rela-
tions—Cultivation on Different Media—Channels of In-
fection — Ransome—Williams— Cornet—Conditions of In-
fection—Methods of Disinfection—Tuberculosis at Different
Ages—Tubercle in Milk—Diagnosis of Tuberculosis in Cattle
— Tuberculous Meat — Koch’s Method of Treatment —
Nature of Virus and Mode of Action—Is Immunity con-
ferred ?—Koch’s Method a New Departure—Sterile Products
cause Marasmus ; Maffucci-——Indications for Treatment.

CHAPTER XII.

LEPROSY ~

Distribution of Leprosy—Similarity to Tuberculosis—Description
of Disease—“ Tubercular,” ‘* Anzesthetic,”’ “‘ Mixed "—The
Leprosy Bacillus—Method of Staining — Position—Leprosy
Cells— Bacilli Resistant and Grow Slowly—Cultivation Ex-
periments mostly Negative—Theories of Cause of Leprosy.

CHAPTER XIII.

ACTINOMYCOSIS :

Nature. of the Disease—Differences in Cattle, Man, Pig, and
Horse—Methods of Preparation and Examination of the
Fungus—Microscopic Appearances of Fungus in different
Animals — Nature of Clubs — Cultivation Experiments —
History of Actinomyces—The Actinomyces a Streptothrix.

CHAPTER XIV.

GLANDERS : : 3

Glanders — Farcy — Clinical Appearances of the Disease —
Chauveau’s observations on Glanders Poison—Léffler and
Schiitz—Method of Demonstrating the Glanders Bacillus—
The Bacillus—Methods of Cultivation—Glanders in Various
Animals—Farcy in Man—Temperature relations of Bacillus—
Desiccation—Germicides.

PAGE

205-243

244~252

253-261

262-270

CONTENTS. xi

CHAPTER XV.

PAGE
ANTHRAX - 271-286

The Bacillus Anthracis — Early Observations —- Pollender —
Davaine—Koch—Pasteur— Methods of Examination—Ap-
pearances of Bacillus under Different Conditions — Spore
Formation—Non-spore Bearing Bacilli—The Vitality of the
Bacillus and of the Spores—Cultivation Experiments—Cover-
glass Preparations—Inoculations into Animals—Methods of
Infection—Anatomical Characters of Malignant Pustule—
Animals Affected—Spores not formed in the Living Body—
The Disposal of Anthrax Carcases—Various Disinfectants—
Pathogenic and Saprophytic Anthrax—Buchner’s Experi-
ments on Anthrax Bacillus and Bacillus Subtilis—Hueppe
and Wood’s Experiments.

CHAPTER XVI.

TETANUS - - 286-206

Tetanus a Specific Infective Disease—A Wound Fever—Organism
found by Nicolaier in the Soil taken from Streets and Fields
—Experiments on Animals—Symptoms of Disease—Pure
Cultivations Obtained—Description of Organisms—Charac-
teristic Shape— Spore Formation — Organism Anzrobic —
Cultivations—Kitasato’s Method of Cultivating the Organism
—The Bacillus found only at the Seat of Inoculation—-Wide
Distribution of Spores—Bossano’s Examination of Earth—
Vaillard and Vincent’s Observations—Tetanus Bacillus a
Facultative Saprophyte—Conditions under which Tetanus
is Contracted—Poisoned Arrows.

CHAPTER XVII.

DIPHTHERIA 297-313

Diphtheria an Infective Disease—The Organism found in the False
Membrane in its Deeper Parts — Method of Staining the
Bacillus—Characters of the Bacillus—Involution Forms—
Cultivation Methods — Appearance of Colonies—Nutrient
Media—Results of Inoculation Experiments—Klein’s Bacilli
differ somewhat from Léffler’s — Streptococci found in
Diphtheria Poison — Extreme Virulence—:Resemblance to
snake-bite poison — Toxicity — Predisposing Conditions—
Conditions fatal to the Bacillus—Roux and Yersin’s Observa-
tions—Fraenkel’s Observations—Attenuated Diphtheria Virus
—Increase of Virulence,

CONTENTS.

CHAPTER XVIII.

HYDROPHOBIA - -
Pasteur’s Experiments—Attempts to Demonstrate Micro-organisms

—Hydrophobia does not arise Spontaneously—Disease not
Confined to Man or Canine Animals—Pasteur’s Early Ex-
periments with Saliva Unsuccessful—Successful Experiments
—Symptoms of the Disease—Position in which Virulent
Material is Found—Different Animals Differently Affected—

_ Alteration of Virulence—Method of Preparing Inoculation

Material—Description of Experiments—Joseph Meister the
First Patient Treated—Method of Treatment now Adopted—
Treatment of Wolf Bites—The Time Factor in the Disease—
Rationale of Inoculation Method — Filtered Virus Non-
Virulent—Method of Inoculation—Description of Depart-
oer Apparatus and Methods of Working in the Pasteur
nstitute.

CHAPTER XIX.

BACTERIA OF THE MOUTH -
The Mouth a Good Forcing Ground for Bacteria—Food Material

—Kinds of Bacteria found in the Mouth by Miller and Vignal
—Bacteria in the Teeth in Caries, in Milk Teeth, and in
Abscesses—Combination of Lactic Acid with Lime Salts—
Organisms Attack Decalcified Base Substance — Artificial
Decay—Mode of Invasion of Teeth by Bacteria—Poisonous
Saliva-—Micrococcus of Sputum Septiceemia—Pneumococcus
—Other Organisms found in the Mouth—Septicemia fol-
lowing Slight Operations in the Mouth.

CHAPTER XX.

Tue BACTERIA OF COLOUR AND PHOSPHORESCENCE 3
Colour-Forming Bacteria— Micrococcus Prodigiosus— Magenta

Micrococcus—Beggiotoa Roseopersicina— Bacillus of Blue
Milk — Sulphur Pigments — Iron Pigments — Bacillus
Fluorescens Putidus—Phosphorescent Bacteria—Six Species
— Method of Cultivation— Conditions under which they
produce Light.

CHAPTER XXI.

Poisonous ALKALOIDS AND ALBUMINOIDS - ©
Early Observations—Burrows, Kerner, Panum, &c.—Ptomaines

Leucomaines—Brieger’s Work—The Alkaloids, Poisonous
and Non-Poisonous — General and Local Reactions —
Structure and Composition—Sketch of Chemistry—Cholera

336-346

347-355

356-367

CONTENTS. xiil

Poisons and other Members of this Group—Mussel Poisons :
two Classes, viz., those Without Oxygen and those Con-
taining Oxygen—Ptomaines the Result of the Activity of
Micro-organisms — Léffler’s Experiments on Diphtheria
Poison, an Enzyme—Roux and Yersin’s Diastase—Hankin,
Albumose—Brieger and Fraenkel, Toxalbumen— Method
of Preparation of Albumoses and of Toxalbumens—Near
Relationship of Proteid Poisons and the Ptomaines—Martin’s
Observations.

CHAPTER XXII.

VACCINATION é

Natural Immunity—Ingrafting of Small-pox—Jenner’s Discovery
—Klebs— Pasteur—Chauveau—Grawitz'’s Theory—Buchner’s
Theory — Metschnikoff’s Work—Greenfield’s Observations
on Anthrax —Toussaint’s Vaccinal Fluid—Pasteur’s Classical
Experiments—Other Methods of Diminishing Virulence—
Chauveau’s Soluble Poison Experiments — Protection by
Alkaloids, Albumoses and Toxalbumens — Saprophytic
‘* Anthrax ’—Hueppe and Wood—Antagonism by Blue-pus
Products—Immunity not the same as Antagonism—Sum-
mative Action of Bacteria.

368-380

CHAPTER XXIII.

BACTERIA IN AIR, EARTH, AND WATER 381-396

Spores in the Airin Hospitals—Effects of Currents and Altitude—
Direction of Wind—Nature of Country over which it passes
—Frankland’s, Carnelley’s, Haldane’s and Petri’s Experi-
ments — Few Bacteria in the Air of Sewers — Tyndall’s
Glycerine Chamber—Examination of Air—Koch’s Method—
Miquel’s Method—Hesse’s Apparatus — Modified Hesse’s

Apparatus—Miquel’s Sugar Method—Bacteria in Water—

Elect of Standing—Sluggish Movement—Oxidation of Or-

ganic Matter in Water—Organisms carried by Sewage—

Number of Organisms in Water—Method of Procedure in

Analysing Water — Pfuhl’s Flasks — Petri's Dishes — Pe-

truschky’s Flask—Plate Cultivations—Cooling Apparatus—

Von Esmarch’s Tube Cultures—lffect of Rains, Frosts and

Thaws—Relation of Bacteria to Ammonia—Basis on which

to determine whether Water is fit for Drinking or not—

Filtration—Methcd of Examination of Soil.

APPENDIX 397-442

INDICES - . x . : : - 443-459

BACTERIA AND THEIR PRODUCTS.

CHAPTER I.
INTRODUCTION.

Growth of Subject—Place of Bacteria in Nature—Pure Cultures—Morph-
ology—Physiology—Protoplasm governed by same laws whether in
higher or lower Plants or Animals—Relation of Bacteria to everyday
Processes—Fermentations, Butyric, Lactic, Colour, &c.—Brewing—

' Baking—Kephir making—Flax preparation—Digestion—Putrefaction
—Nitrification, Mineralization—Relation of Bacteria to Water Supply
—Filtration—Sewage—Modes of Transference of Pathogenic Bacteria
from Patient to Patient, or from Water or Earth to Patient.

ITHIN the last decade bacteria have laid a very strong

hold on the thought and imagination of the scientific
world, and have come to be looked upon as playing a most
important part, not only in the production of disease and in
fermentation, but also in many everyday processes hitherto
supposed to be dependent on very different causes. In
consequence of this, bacteriology has been raised to the
dignity of a science, and its ramifications have become
sO numerous and so wide-spreading that many of the
other sciences, and even some of the arts, have been freely
pressed into the service of one or other of its branches.
The evolution of the science for long went on but slowly ;
the study of bacteria remained almost entirely in the
hands of the botanists, although now and again scientific
medical men, whose powers of observation and deduction
were far superior to the methods of experimentation that
had been at their command, made shrewd guesses at the
causal relationship between the growth of certain bacteria

2

2 BACTERIA.

within and without the body and the etiology of certain
infective diseases, and certain processes of putrefaction and
fermentation. At first bacteria were claimed for both the
animal and vegetable kingdoms by the workers in each,
and the fortunes of war seemed for long to favour the
zoologists ; but after a sharp struggle the fission fungi, as
they are called, were relegated to the domain of botany, and
now for many years the morphology of these organisms has
been an object of most careful study by scientific botanists.
Bacteria were, however, so minute that it was impossible to
study them very accurately with the older microscopes, and
for long the group contained very few species, whilst in
addition to the fallacies connected with actual observation of
size, form, and methods of reproduction, there were others
that resulted from the fact that it was impossible, until com-
paratively recent years, to obtain what are now known as
pure cultivations; forms that were utterly dissimilar in
morphological characters were supposed ‘to be stages of the
developmental cycle of the same organism, although they
might be, and probably were, really nothing but different
organisms that were capable of growing in a common
medium. When at length more or less pure cultivations
of certain organisms were obtained, botanists: were able
to study, in some cases very completely, the life-history
and morphology of various forms of bacteria, and great ad-
vances “in bacteriological science were made. Still no very
accurate observations on the functions or physiological
chemical processes associated with the life-history of bacteria
and allied forms were made, and many of the biological
questions, some of which had already been answered as
regards animal and the higher vegetable protoplasm, still
remained as in a sealed book so far as this lowly vegetable
protoplasm was concerned.

In the last ten or twelve years, however, owing to the vast
improvements that have been made in, the methods of culti-
vation of these organisms, and especially of obtaining what
are known as pure cultures, z.¢., cultures that contain a single
species of organism only, most valuable data as to the func-
tions and biological chemistry of these minute ‘specks of
vegetable protoplasm have been rapidly accumulated.

Pasteur’s wonderful observations on yeasts first opened
up the way for future workers. His practically pure cul-

INTRODUCTION. 3

tures were obtained by a kind of physiological separation,
to obtain which he fed his yeasts on food specially suited to
their nutrition—more suited to their wants, in fact, than to
those of any micro-organisms with which they are usually
found associated— and they were thus enabled to develop so
luxuriantly that they prevented the growth of almost all
other organisms, which might have found their way into the
nutrient medium along with them. These cultures were
comparatively pure, the presence of the small number of
other organisms not being sufficient to vitiate the main
general results,

Klebs (1873) tried to obtain pure cultures by his fractional
method, which was simply the removal of those organisms
that vegetated most luxuriantly in any special fluid to another
.flask, making special cultures of these, and so on until com-
paratively pure cultures were obtained. The difficulty, how-
ever, was that only the commoner forms could thus be
separated and even then they could not be relied upon as
being absolutely pure.

This method (the ‘‘maas” method) is even now sometimes employed to
obtain pure cultivations of cholera bacillus. A drop of the discharge from
a cholera patient containing a large number of organisms besides the
specific cholera bacillus, when placed in broth which is so prepared as to
be specially suited to the nourishment of the cholera organism, soon
becomes almost a pure culture, as the cholera bacilli are able to multiply
in an extraordinary degree at their ‘‘optimum” temperature and to far
outnumber all the other organisms present, and it becomes a much easier
matter to obtain from the broth culture a pure culture of the ‘‘comma”
bacillus than directly from the cholera discharge itself. It may be said to
correspond to the passing of a mixed culture through an animal. Certain
organisms—those that are ‘‘ pathogenic ’—can grow luxuriantly whilst the
others make a much weaker struggle for existence.

As soon as it became evident that no further steps of any
very great importance could be taken unless pure cultiva-
tions were used, various workers set themselves to devise
methods by which single organisms might be isolated, and
from which a “pure-bred stock” might be raised. Then
Roberts (1874) and Cohn (1876) obtained pure cultures of
different forms of spore-bearing bacilli, especially the bacillus
subtilis, by subjecting the fluid to the heat of 100°C., by
which all non-spore-bearing forms were killed off. This
method, however, has a very limited application, though
combined with other methods, it enabled Kitasato to obtain

4 BACTERIA,

pure cultures of the tetanus bacillus, Salomonsen (1876)
observing that the dark colour which makes its appearance in
defibrinated ox blood always commences as minute points,
concluded that each of these patches was due to the changes
set up by micro-organisms growing at these points, He
therefore drew such defibrinated blood into a capillary tube,
and by carefully noting the appearances presented in the tube
was able to follow the development of a variety of organisms
which, when few in number, were separated by considerable
intervals of bright red blood in which no organisms could be
seen, though in time these gradually ran together. Lister
(1878) and Naegeli (1879) obtained their pure cultivations by
a kind of fractionating method, in which they distributed a
number of organisms into large quantities of fluid by making
greater and greater dilutions of their organisms in broth,
until the dilution was such that a single drop did not con-
tain more than a single organism, and a series of such
“drops introduced into a number of flasks containing sterilized
broth gave a large proportion of pure cultivations. With
the pure cultivations so obtained the first really accurate ex-
periments in connection with the physiological chemistry of
bacteria were carried on. This method, however, though a
very great advance on any that had hitherto been devised,
was somewhat complicated, and it was open to certain other
objections (though it is still most useful in many investiga-
tions). It was not until Koch, perfecting Klebs’ and Brefeld’s
gelatine method, was able to “ fix” the organisms 2” szfu as
it were, in the nutrient. medium, that the bacteria could be
kept isolated, the resulting colonies studied and then removed
to other media for further examination. The method was
so simple and so reliable that it was eagerly seized upon
by those who were interested in the solution, not of
-botanical problems merely, but of many questions con-
nected with putrefaction, fermentation, and disease that
had cropped up so frequently, but which had to be left
without a satisfactory answer, and which, in consequence
of Davaine’s and Pasteur’s researches, were again assuming
such prominence.

In the first place, experimenters were able to put to the
test the many observations that had been made on both the
structure and the function of micro-organisms. Once a pure
culture became available, it was an easy matter to determine

INTRODUCTION. 5

the conditions under which a special bacterium would grow.
It could be transferred from medium to medium ; it could
be placed under xrobic and anzrobic conditions; or it
could be plied with antiseptic or other special reagents, and
its behaviour under varying conditions or in the presence of
these material could be accurately observed. There was no
longer any danger of one form being mistaken for the de-
velopmental stage of a totally different organism, and much
confusion as regards classification was gradually done away
with, though the effects of the observations made on impure
cultivations still make themselves felt in the obscurity
that enshrouds certain groups which are still met with
even in some of the better known classifications. As
these pure cultivations were obtained it gradually became
more apparent that, for practical purposes, it would be
necessary to base our classification on certain specific cha-
racters of the organism ; and as in all processes in which
bacteria play a part they are usually met with in a single
and special form, it was originally found advisable to take
these special forms as the diagnostic features in making such
classification. The features of form and size, however, were
merely superficial characters, and as a more careful study
of bacteria was made, especially by the French school,
it was found that the biological characters afforded a much
more satisfactory basis of classification, especially, however,
when taken in conjunction with the morphological features ;
so that the media on which they grow, the products to
which they give rise, the methods in which they cause the
breaking down of dead or living protoplasm, the actions and
reactions that take place between them and living animal
and vegetable cells, and the effects upon them of organic
and inorganic antiseptic substances, are now all taken into
account in drawing up any classification. Thus we have
yeasts that cause fermentation and yeasts that do not, whilst
nearly related to them are ascomycetes that are essentially
parasitic in their character ; we have micrococci that set up
urea fermentation, micrococci that form pigment, others
that give rise to suppuration or that apparently assist in the
production of diphtheria ; and so on, throughout the whole
of these low vegetable forms. The appearance under the
microscope can, of course, give us no information on many
of these points, and it is only by a most careful study of the

6 BACTERIA.

life-history of these organisms that any accurate information
has been obtained.

Within quite recent years it has been observed that very
marked modifications of certain of the features may be ob-
tained by subjecting the micro-organisms to conditions
different from those under which they are ordinarily met
with ; and as the outcome of these observations the way
has been opened up for the study of the evolution, perhaps
not of the micro-organisms themselves, but of their specific
functional characteristics. It has been found, for instance,
that under certain conditions, yeasts that ordinarily set up
active alcoholic fermentation are no longer able to do so ; that
by continued cultivation outside the body and in certainspecial
media the anthrax bacillus loses much of its virulence ; whilst
the tubercle bacillus, the cholera bacillus, the organisms met
with in diphtheria and in other diseases, may, under certain
conditions of cultivation, lose their disease-producing power
in a most remarkable degree. On again being placed under
conditions of a more favourable nature these organisms
resume their pathogenetic or disease-producing properties:
The same variations are met with in the. colour-forming
species of bacteria, according as the conditions under which
they exist are favourable or unfavourable to the accentuation
of certain functions of the protoplasm.

It will be evident from a consideration of all these facts that
bacteria must be looked upon as being governed by much the
same laws which govern other plants and animals ; that they
are composed of protoplasm, the functions of which may be
modified in various ways, and the forms of which may become
more fully differentiated, and that where greater differentia-
tion takes place the modification of function is always in the
direction of greater specialization ; that the conditions suit-
able for the existence of the more lowly organized and
functionally less specialized organisms need to be less
specialized than do those that are necessary for the growth
of the more highly developed fungi ; and that, consequently,
the bacteria of disease and putrefaction are, with few excep-
tions, of a comparatively low form, although the functions
of the protoplasm of these less specialized bacteria become
more specialized as they dwell for a longer and longer
period under any special set of conditions, one phase or
power of the protoplasm being drawn out and developed in

INTRODUCTION. ”

excess and frequently at the expense of, some of the others,
sometimes even at the expense of the living or resisting power
itself of the organism. In all cases, however, it should be
remembered that the differences in protoplasm are differences
in degree rather than in kind, and that the laws which govern
animal and other vegetable protoplasm govern the proto-
plasm of bacteria, and that the results of all investigations
that have been carried on in connection with them must
conform as strictly to physiological laws as any work that has
hitherto been done in the domain of animal or vegetable
biology ; and that where discrepancies of any kind, or appa-
rent departures from ordinary physiological laws, are met
with, the facts must be carefully revised, and if the dis-
crepancies still continue, then so much the worse for either
the facts or the laws. The “facts” are still incorrect or the
laws must be revised.

There has grown, then, and still continues to grow, a
science dealing with microbiology in all its morphological
and physiological phases. Facts have gradually been accu-
mulated, observations have succeeded observations, patient
work and powerful concentration have played their part in
elaborating our knowledge of the habits of micro-organisms
without and within the higher plants and animals; the
conditions under which the modes of life of micro-organisms
may be altered have been investigated ; the effects that
they or their secretions exert on living and dead proto-
plasm have been most conscientiously examined ; the
conditions that:are essential in order that these organisms
may stimulate the. activity of living protoplasm, and the
phases through which this protoplasm passes, from the
stimulated condition to degeneration and death, have all been
carefully studied. The organisms that have been found in
certain diseases have been identified and classified, their
modes of propagation and the channels by which they are
conveyed from one host to another, sometimes through
intermediate saprophytic or non-parasitic stages, have been
determined, and the whole subject has been so prepared,
that the great epoch-making minds of such men as Pasteur,
Chauveau, Lister, and Koch, being brought to bear on
these questions have found sufficient material at their
disposal on which to generalize, and have placed before the
world theories that appear to be more like fairy tales

8 BACTERIA.

than deductions made as the results of sober scientific
investigation and thought. These men have also, how-
ever, been able to obtain many new facts, and to point
out the gaps that still remain to be filled in before the
theories so admirably expounded can be proved to demon-
stration. The French can boast of their Davaine and Pasteur,
who, assisted by Chauveau and others, have given to us the
germ theory of disease and the theories of fermentation and
protective inoculation. In Germany, Klebs, Cohn, Koch,
and their followers, have made marvellous contributions to
the study of bacteria, and additions to our knowledge of their
relation to disease. "The Danes have furnished O. F. Miller,
Warming, Panum, Salomonsen, Bang, and Chr. Hansen,
who, seizing most of what was good in both the French and
German schools, have succeeded in making most valuable
contributions to various branches of the subject ; whilst, in
this country, Burdon Sanderson, Greenfield, Klein, Watson
Cheyne, and others, have all contributed their share, though
Lister’s name must always stand pre-eminent for the mag-
nificent work which he has done in the domain of antiseptic
surgery, by the evolution and perfecting of which he has.
done more, both directly and indirectly, to ameliorate the
suffering, and to diminish the mortality in surgical cases,
than has been achieved by the most brilliant operators the
world has produced during the last century.

Many of the- processes of everyday life are intimately
associated with the specific activities of micro-organisms ; we
are constantly meeting with these organisms, and it is now
proved, beyond all dispute, that their presence is not merely
accidental but is absolutely essential to the carrying on of,
one might almost say, the most commonplace operations.
Take, for example, those that are associated with the different
processes of fermentation. Leaving for the moment the
yeasts or saccharine ferments, I may refer first to the
butyric acid ferment—Clostridium butyricum—which plays
a most important part in interfering with the ordinary course
‘of the saccharine fermentation. It decomposes starch and
cellulose without requiring the presence of oxygen ; it, like
other micro-organisms, appears to require for its nutrition,
nitrogenous material, and, especially in the presence of the
lactic acid ferment, it brings about the conversion of sugar
into butyric acid; it is, as might be anticipated, one of the

INTRODUCTION. 9

bacteria most frequently met with in some of the forms of
putrefaction.

It must be remembered however that this is not the only
organism that gives rise to the butyric acid fermentation ;
several other bacteria, differing most markedly in many of
their features from the Clostridium butyricum, generally,
however, like that organism, carrying on their work in the
absence of free oxygen, have the power of setting up alone,
or in conjunction with the Clostridium butyricum, the
butyric fermentation. In ripened cheese, part of the flavour
at any ‘rate is due to the products formed in the ripening
curd in the presence of this organism.

The lactic acid fermentation so frequently met with
in milk, is also the result of the vital activity of several
organisms, Pasteur and Lister both describing lactic acid
bacteria and micrococci. _Hueppe also describes a special
bacterium which he says has the power of breaking up milk
sugar and saccharose into lactic acid and carbonic acid ;
whilst from material taken from the mouth and teeth he
succeeded in separating two micrococci, both of which had
the power of converting sugar into lactic acid, and bya
series of experiments he also proved that certain of the
pigment-forming bacteria, such as Bacillus prodigiosus—
the organism that gives rise to what is known as bleed-
ing bread—supply so much lactic acid as a result of
their metabolic processes, that in their presence milk is
curdled, the casein being precipitated. Further, even a
pathogenic form of micro-organism—micrococcus osteomye-
litis —is said by Krause to set up the same reaction.
Jérgensen mentions that “ Delbruck found that in a mash
prepared from dry mould and water, lactic acid was first
formed at a temperature of 50° C., and from this he draws the
conclusion that in this case the active lactic acid ferment
has its maximum temperature at this degree of heat.”
This is an exceedingly interesting fact, for as Schéttelius
and Wood have pointed out, as the temperature rises the
Bacillus prodigiosus loses its power of forming a pigment,
and if it is grown on potato or bread paste for example,
in an incubator at blood heat instead of at the temperature
of the room, the colour is gradually lost and the culture no
longer smells of herring brine, but the power of forming lactic
acid from milk sugar, with the accompanying precipitation

To BACTERIA...

of the casein, is frequently considerably increased ; so that
it would appear that the energy required for the building
up of the pigment substance was in this case diverted into
another channel, and lactic acid, and perhaps other sub-
stances, are produced in-place of the usual pigment.

This example may afford some idea of the complexity of
the problems that have to be grappled with by bacterio-
logists, and it may also help to explain how such different
results have been obtained by different observers, and how it
still may be possible to reconcile statements which at first
sight appear to be in direct opposition.

It is a well-known fact that if the lactic acid fermentation
once obtains a footing, in a solution of sugar for example,
many of the other putrefactive bacteria- find it impossible’
to’ develop. On the other hand, whilst the lactic acid
organism cannot grow beyond a certain point, yeasts
are perfectly able to develop where there is a certain
quantity of acid present. This has: been adduced as an
explanation of the fact that comparatively pure yeast
fermentation may go on even in the presence of the lactic
acid organism, when it is unable to make further head-
“way.and when other putrefactive organisms are unable to
. grow at all.

Another fermentation, the results of the careful studies
of which have been utilized in the commercial production of
vinegar; is that due to the acetic acid ferments Mycoderma
aceti and Mycoderma Pastorianum, by the action of either
of which alcohol is converted into acetic acid.

In all the works on brewing, lists of micro-organisms that
give rise to such conditions as bitterness, muddiness, various
colorations — red, yellow, green, etc.—are given, and it is
pointed out at the same time that all appear to give rise
to some form of acid. It is supposed, for example, that
certain species of sarcinze are responsible for the sour and
bitter tastes which are sometimes developed in beer ; a similar
organism is also described as giving rise to the red colour
which is sometimes developed in white beer, though it may
be remarked that in this case a rod-shaped bacterium is also
frequently present, and may be answerable for the presence
of the substances that so seriously alter the taste of this.
beverage. A series of other organisms, which Lindner
speaks of as Pediococci, all produce acid, and give rise to

INTRODUCTION. Il

ansoundness of beer, but as they are readily killed at a
temperature of 60° C., they may easily be got rid of.
In the process of baking, as carried on in this country,
there is a regular conversion of some of the starch of the
flour into sugar by the yeast used —+ this sugar being in
turn converted into alcohol and carbonic acid gas, to
e setting free of which the “rising” of bread is due.
‘he baking that follows serves three purposes ; it kills

the ite Shins Graco starchy matter in posi-
tion, and it drives off the alcohol and the carbonic acid. In
the baking of other kinds of bread certain other organisms
are said to play a part; for instance, the Saccharomyces
minor (Engel) was supposed to be the active fermenting
agent in the manufacture of rye bread, but more recently
this organism has been superseded, and the work of fermen-
tation has been assigned to a bacillus—Bacillus Panificans
(Laurent)—which in pure cultivations was found to be capable
of setting up all the characteristic fermentation changes in the
dough of black bread. This organism is made up of short
motile rods, from which threads may be formed, these threads
(in which spores are sometimes found) interlacing to form a
film, especially when it is grown on the surface of nutrient
liquids. Its spores are almost as resistant as those of the
hay bacillus, and can only be killed by being subjected to
boiling heat for a period of at least ten minutes.

Jorgensen, summing up Laurent’s investigation, says:
“The bacillus usually dissolves the gluten substance of the
dough, grows in starch paste, and in mixtures of saccharose
and mineral substances. It is found in an active state in
large quantities in bread, and according to the author's
researches, it can withstand for twenty hours the action of
an artificial gastric juice. In the excreta it is found still
more abundantly, and it appears to be generally distributed
in plants and in various substances.” More recent researches,
however, have thrown some discredit even on the Bacillus
Panificans ; Dinnenberger maintaining that these bacteria
are merely an impurity, and that in all cases the fermenta-
tion of bread is due to an alcoholic-forming organism—a
saccharomyces in all cases proving the best agent for bring-
ing about this fermentation.

Certain forms of unsoundness of bread are also due to
micro-organisms. In addition to bleeding-bread (caused by:

12 BACTERIA.

the growth of the Bacillus prodigiosus), which has served the
purpose of the miracle-monger before to-day, sticky reddish-
brown patches have been described as occurring in unsound
bread, in which various bacilli, such as the ordinary potato
bacillus, Bacillus liodermos, Bacillus mesentericus vulgatus,
have been found, and on analysis of these patches dex-
trine, dextrose, starch, sugar, and even a small quantity of
peptone, have been separated ; moulds of other fungi are
also found in unsound bread,

In 1882, Kern described the peculiar ferment known as
kephiy grains, by means of which the Caucasians set up a
double alcoholic and acid fermentation in milk.

These kephir grains, says De Bary, in the fresh living state
are “white bodies, usually of irregular roundish form, equal
to or exceeding a walnut in size. They have their surface
roughened with blunted projections, and furrowed like. a
cauliflower ; they are of a firm, tough, gelatinous consistence,
becoming gradually cartilaginous, and are of a yellow colour
when dried ; they are chiefly composed of a rod-shaped bacte-
rium,” many of these being united to form long threads,
arranged in a kind of felt or network, the meshes of which
are filled with a tough gelatinous membrane, which binds
the organisms together into a kind of zoogloea mass. This
rod-shaped organism is known as Dispora caucasica as at
the end of each rod is a rounded spore.

Along with these may usually be found a small proportion
of a, yeast-like fungus which, however, is merely entangled
in the gelatinous mass, although it certainly undergoes
development by sprouting. There is also present the
ordinary Bacterium lactis which, with a number of other
impurities, adheres to the kephir grains; this also occurs in
the milk itself. To prepare the specially fermented milk,
one volume of these kephir grains is moistened and added
to about six or seven volumes of fresh milk, the whole is
protected from the dust, but is exposed to the air for about
twenty-four hours at the ordinary temperature of the room,
and is frequently shaken ; the milk is then poured off and a
fresh quantity added ; the milk that is poured off is mixed
with double its quantity of fresh milk, put into bottles, well
corked, and frequently shaken. This bottled sour milk
soon becomes sparkling and effervescent, and is ready for
use after it has been bottled for a day or two. It then con-

INTRODUCTION, 13

tains lactic acid, a considerable quantity of carbonic acid,
which varies “ according to the temperature and the duration
of the fermentation, but is sometimes sufficient to burst the
bottle or drive out the corks.” The liquid contains about
one per cent. of alcohol.

_ We have already seen that the kephir grains and the
sour milk, together, contain (1) a yeast fungus which is
capable of bringing about the fermentation of grape sugar ;
(2) the bacillus of lactic acid ; and (3) the rod-shaped bacteria
that predominate in the gelatinous mass of the tough grains.
Now, though this yeast fungus can set up the fermentation of
inverted milk sugar, it cannot affect the milk sugar itself, so
that we must look elsewhere for the inverting power.

This appears to be contained in an enzyme, or ferment,
that is produced by a large number of bacteria, amongst
* others by the rod-shaped organisms already referred to,
and by the lactic acid bacillus, and these were supposed
to prepare the milk sugar for the action of the yeast fungus.
This appeared to be a perfectly satisfactory explanation, until
De Bary found that, by violent agitation of the souring milk
to which no kephir grains have been added, alcoholic
fermentation may still be induced ; so that it would appear
that by freely oxygenating the milk during the process of
lactic acid fermentation, when in fact the molecules are being
re-arranged, oxygen is taken up into chemical combina-
tion, and alcohol and carbonic acid are generated; water, in
all probability, being formed as this goes on. This is
adduced as one of the processes of fermentation by free
oxidation, and is an example of a chemical fermentation
giving results similar to those yielded by biological fer-
mentation. Until, however, it can be demonstrated that
De Bary was working with material in which all impurities
were excluded, these results can scarcely be accepted as abso-
lutely reliable.

In the kephir, which is a perfectly fluid mass, we have a
quantity of lactic acid. Now, it is a well-known fact that,
under ordinary circumstances, when milk turns sour, there
is a precipitation of the curd which forms a more or less
solid coagulated mass. What has become of this coagulum
in the kephir? If a piece of meat be exposed to the action
of putrefactive germs, it will be found that after a time it
is reduced to a soft, pulpy, almost liquid mass. In the same

14 BACTERIA.

way, a number of the nitrogenous foods taken into the
stomach are softened and otherwise very considerably
altered,

These materials have in both cases undergone what is
known as a process of peptonization: from coagulable or
colloid albumen they have been converted into a much more
solubleand diffusible albuminoid, and are thus prepared for fur-
ther changes as they are acted on directly by living animal or
vegetable protoplasm. This peptonization appears, however,
to be also brought about by an enzyme which is elaborated
by certain bacteria, especially by such as are associated
with putrefaction and with the digestive processes. In the
case of the kephir it was thought that the peptonization
took place’ as a result of the action of an enzyme, formed
by the rod-shaped organisms of the zoogloea mass of the
kephir grains, and it was argued that this must be a soluble
ferment which could diffuse from the gelatinous mass in
which the grains were embedded into the milk; it could
there act on the casein as it was gradually precipitated, or
possibly even before precipitation took place; for it was
observed that these rod-shaped organisms were never found
outside the gelatinous masses, and therefore could not be
acting directly on the casein.

This is well enough in theory, but as equally good kephir
can be produced by the oxidation effected by free movement
of the sour milk, the peptonization cannot be solely due to
these organisms. It may be urged that other organisms
may, in the presence of a large quantity of oxygen, supply
the peptonizing function in an acid fluid in which, under
ordinary conditions, they would not be able to exist, the
large amount of free oxygen driven into the milk enabling
them to obtain a supply of energy, to live and carry on their
function even in the presence of the special lactic acid bacteria,
which seize with avidity the whole or a part of the oxygen
contained in the milk, according to the quantity present, in
this instance a part only, as there is such a large supply
under the above conditions.

It has already been mentioned that the Clostridium

* For the further description of the kephir grains the reader is referred to
E. Kern, “Bot. Ztg.,” 1882, p. 264; Alexander Levy, ‘‘ Deutsche Medi-
cinal Zeitung,” 1886, p. 783 ; De Bary, ‘‘ Lectures on Bacteria,” translation,
Oxford, 1887.

INTRODUCTION. 15

butyricum or Bacillus amylobacter plays an important part
in determining the butyric acid fermentation of the vegetable
acids, and that it plays an active part in the ripening of
cheese. Because of its action on cellulose, and then of its
further action on dextrine and glucose, it also has much to
do with the decomposition and destruction of the cellulose
of fleshy and juicy plant tissues, and its aid is requisitioned
in the separation of these parts from the tougher fibre of
hemp, flax, and similar materials, as in the process of macera-
tion the enzyme converts the cellulose into butyric acid.

Van Tieghem holds that much the same processes go on
in the stomachs of ruminant animals, and that the Bacillus
amylobacter, which is usually found there, thus does a very
large part of the work which, otherwise, would have to be
performed by the epithelial cells of the stomach itself. This
bacillus, however, acts not only upon cellulose and starch
paste, but it also exerts a most important action on nitro-
genous substances. Fitz and Hueppe have both pointed out
that the casein of milk is first coagulated in the presence of this
organism, is then peptonized and liquefied by the action of its
enzyme, and that the products thus formed are afterwards
converted into certain lower compounds, such as leucine,
tyrosine, and even ammonia, all of which are constantly met
with during the processes of both digestion and decomposition.
Duclaux also showed by experiment that an organism, which
resembles the bacterium amylobacter very closely in many
respects, sets up a series of similar changes in the casein of
milk, and in casein that has been converted into cheese ;
and he showed that the process of “ripening” brought
about in the presence of the Tyrothrix bacillus, is due to
the peptonization and liquefaction of some of the substances
of which unripe cheese is composed ; certain secondary or
ultimate products similar to those above mentioned being
found during the ripening process.

That bacteria are general scavengers is now generally
acknowledged, and almost innumerable observations have
been made with the object of proving that the presence of
certain definite organisms is essential for the perfect breaking
down of dead or effete animal and vegetable matter. It has
already been mentioned that some species have the power
of breaking up cellulose and of converting it into much
simpler substances, both externally and in the alimentary canal.

16 BACTERIA.

Bienstock went so far as to describe a particular bacillus
which, he considered, was able by itself to produce the whole
series of changes that occur in the contents of the intestine
and in putrefying albumen or fibrine. This organism he
describes as being somewhat smaller than the Bacillus subtilis;
it is rod-shaped, but at one end there is usually a small
enlargement, in the centre of which a clear round spore
may be seen. It is from this feature that the organism
derives its name of “Drum-stick” bacillus. Cultivated on
fibrine it disintegrates it and gives rise to the formation of
leucine, tyrosine, carbonic acid, water, and ammonia, and
of traces of other putrefactive products. The process does
not however stop at this point; the bacillus still continues
to act on the leucine and tyrosine, and decomposes them
into still simpler compounds; whilst, if it be introduced
at once into a prepared solution of one of these earlier
decomposition products, tyrosine for example, it continues
the breaking-down process just as if it were still acting
on the tyrosine which had been formed during the process
of ordinary putrefaction. As De Bary points out, however,
this bacillus cannot claim a monopoly of the work connected
with putrefaction. If any putrefying fluid be examined,
countless organisms will be found, and amongst these very
different species may be observed. There are rods of different
sizes both as regards thickness and length, spirals of different
forms, micrococci of different sizes and arranged in different
groupings, one or other organism predominating according to
the nature of the putrefaction process, of the material that
is being broken down and the stage at which the breaking-
down process has arrived. It would appear in fact as
though there were developed special organisms for the setting
up of special fermentations, and also that after the breaking
down has been carried a certain length by one organism, the
aid of another is invoked to complete the process more
thoroughly and more expeditiously. We have in this, as in
the case of the process of digestion, an exemplification of
the fact that nature economizes her resources as much as
possible ; she does not call on the animal cells of the alimen-
tary tract to do work that can be equally well done by
micro-organisms, nor does she demand the exercise of more
than one or two functions from each of the simple proto-
plasmic spécks that we call bacteria. To each one is assigned

INTRODUCTION. 17

its special work and, though it is possible that many of them
started with certain powers in common, it seems that through
the exercise of some of these common powers under special
conditions they have become gradually so differentiated
functionally, that, as amongst organisms more highly
developed, each is able to carry on its own work best at
those special stages of the putrefactive process at which it
is found. It might at first sight appear that all this can
have but little bearing on any practical work in which we
are engaged, or in which we take an interest, but on more
careful consideration it will be found that these putrefac-
tive organisms really keep up the circulation of matter,
utilizing the excretions of living beings and the carcases of
dead animals and plants, after breaking them down into
their simplest constituents, to supply those elements that
are necessary for the nutrition of plants, allowing them to
present themselves in their most assimilable forms, and in the
proportions most suitable for the nutrition of the growing,
highly organized vegetable protoplasm. Bacteria in fact serve
to transform inert organic matter into inorganic substances,
This transformation, or “ Mineralization,” in most cases, com-
mences only after protoplasm has lost its vitality, and most
micro-organisms are capable of attacking this dead protoplasm
only ; though, as we shall find later, a certain number of
bacteria have acquired the faculty of being able to attack
even living protoplasm. The processes of decomposition
may be divided into two kinds : first, those going on as the
result of the activity of organisms that are capable of taking
up their oxygen from the air, and, second, those the result
of the activity of organisms that so break up and rearrange
the organic molecules containing oxygen, that not only do
they, the bacteria, take up oxygen themselves but they allow
of its being handed on to the products to which in their
processes of metabolism they give rise. It is probable that
here we have to do, not only with nascent oxygen, but that
we have certain products set free during the proces of
decomposition which seize upon oxygen with very great
avidity. This decomposition or rearrangement is spoken
of as a process of nitrification, or a conversion of the nitro-
genous elements into ammonia, nitrous and nitric acids,
carbonic acid and water, or, speaking more generally, it
may be said to be a process of mineralization of the organic
3

18 BACTERIA.

forms of nitrogen, phosphorus, carbon, and hydrogen,
during which they become finally oxidized or mineralized to
nitric acid (HNO,), phosphoric acid (H,PO,), carbonic acid,
CO,, and water, H,O. In nature this process goes on in
the superficial layers of the earth or in the presence of the
atmosphere. That it takes place much more readily near the
surface of the ground and in porous earth can easily be
understood if what takes place in the oxidation that goes
on in spongy platinum is borne in mind. In the earth we
have a spongy material, the upper surface of which is well
supplied with air, and usually also with moisture; there is
also a certain amount of organic matter present on which
micro-organisms feed, and any additional organic matter
brought to this spongy mass is rapidly seized upon by the
micro-organisms, is oxidized, and thus prepared for the
nutrition of the plants that are growing in this soil. So
necessary is this whole process of oxidation by micro-
organisms that Duclaux insists that soil rendered sterile (as
regards micro-organisms), and supplied only with sterilized
water and air, is incapable of supplying sufficient nutrient
material to plants to enable them to flourish even moder-
ately well. All the organisms found in these superficial
layers under ordinary conditions are zrobes ; in the deeper
layers of the soil are micro-organisms that give rise to the
second kind of decompositions. These bacteria, which are
anerobic (that is, they can flourish without being supplied
with free oxygen) in character, have a special power of taking
up the oxygen that is contained or combined in the products
which have filtered down from the surface where the decom-
positions by direct oxidation are going on. Living away from
the atmosphere and being unable to obtain or to utilize
free oxygen, these organisms have developed the faculty of
being able to wrest oxygen, by force, as it were, from the
oxygen-containing bodies that come down to them from
nearer the surface, sometimes however using part only of
the combined oxygen and setting free another part by
which further oxidation may go on; in doing this they
carry the process of decomposition a stage further, and
after the altered organic materials have been attacked by
these anzrobic organisms they appear to contain little that
will provide nutrition for micro-organisms of any kind ; so
that, after we come to a certain depth, bacteria are not

INTRODUCTION. 19

to be found in the soil. This depth, usually reckoned at
about twelve feet, varies of course according to the nature
of the soil, its moisture, porosity and temperature, and
also according to the amount of organic matter that lies on
the surface; cultivated ground always containing more
organisms and to a greater depth than fallow ground having
the same geological characters.

The relation of all this to our water supply is obviously
one of paramount importance. If water be taken from near
the surface of soil in which there is a large-quantity of
organic matter present, there must necessarily be a large
number of putrefactive organisms in it, especially those of
an erobic nature ; if however we take water directly from
the deeper layer, these putrefactive organisms are usually
absent, but a number of “ water” organisms are now present,
which under special conditions, especially if the water be
kept perfectly quiet and unoxygenated, and a high tempera-
ture be maintained, can develop in the water, from which
they may in turn be cultivated by certain well-known
methods. If water be taken from a much deeper layer,
micro-organisms are found to be almost or altogether absent,
and not only micro-organisms, but organic matter, although
in some cases in which micro-organisms are almost entirely
absent, organic matter may still be present in appreciable
quantities. The superficial layers of earth in this case act,
not only as a mechanical, but also as a biological, filter ;
the water, with its contained organic matter, passes through
the successive layers in which bacteria can grow, and gradu-
ally percolates to those layers of earth where there are no
organisms. It has been demonstrated that even deep natural
water contains facultative zrobic organisms, unless it is
obtained at once after it has undergone natural filtration—
that is water that has not stood in an underground basin—
when it may be almost germfree. The organisms cannot go
down with the water; first, because they are held back
mechanically, the soil acting as a porous filter, by which solid
particles, extremely minute as they are, are kept back ; but, in
addition, and quite apart from this purely mechanical effect,
the bacteria (many of which are unable to develop without
oxygen) cannot leave the surface with impunity, and such as
are carried down by the action of the water die as their
supply of oxygen is gradually cut off ; for, in consequence

20 BACTERIA,

of the rapid oxidation that is going on at the surface, very
little free oxygen is left for the use of bacteria at even a
short distance from the surface. It will, of course, be
objected that this does not apply to the anzrobic bacteria,
but in their case it must be remembered that only a certain
definite proportion of oxidized material can reach them from
above, most of the organic matter having already been
converted into inorganic material and used up by growing
plants ; the supply is very rapidly cut off, the reduction
of what remains after the plants are satisfied being com-
pleted, and the bacteria cannot continue to live, because they
can no longer obtain any material for their nutrition.

All the knowledge that has been gained concerning the pro-
cess of putrefaction has not been collected in a day, and it was
only bya careful marshalling of facts as they were accumulated,
and by filling in,sometimes merely with suggestions, gaps that
still remained, that the theory of the filtration and biological
and chemical purification of water has been built up. But to
the practical hygienist it is not sufficient to know that water
coming from springs deep down in the earth is free from
organisms, and that it may be drawn and consumed with
impunity as it rises from the ground. He has something
more to consider ; he has to consider under what conditions
it can be kept fit for drinking purposes, and by what means
it can be most readily and safely distributed to those who
use it. He has to remember that water at rest containing
organic matter, and exposed to the hot rays of the sun, soon
teems with organisms that may or may not be perfectly
harmless ; he has to bear in mind the nature of the ground
from which water is collected, whether it comes from
cultivated areas or from regions in which there is sewage of
any kind; for upon these factors depend the absence or
presence of bacteria from the water supply. Further, he
may be faced with the problem of how to transform a
supply of biologically impure water into a supply fit for
drinking purposes.

The mere chemical analysis of water will not give suffi-
cient information to guide a sanitary engineer on these
points, though it will indicate to him the lines on which
he will have to work. For example, it is quite possible to
have a water containing a considerable quantity of organic
material which, through its treatment by sand filters, or by

INTRODUCTION, 21

some similar process, may contain no micro-organisms, and
may be perfectly fit to drink so long as it is fresh, although,
if it were allowed to stand in a warm place and exposed to
dust and germs of various kinds, there would soon develop
in it such an enormous number of organisms that it would
be looked upon as totally unfit for drinking purposes. On
the other hand, water containing little or no organic matter
might be so contaminated by certain disease germs that it
would be absolutely dangerous to health, and even life, if
used for domestic purposes.

The relation of bacteria to sewage need not be insisted
upon, as it is evident that with such a large quantity of
organic matter, in which putrefactive changes must be rapidly
going on, bacteria of many kinds must necessarily appear,
and it is easy to conceive that an enormous number of
different species might be present, especially where the
sewage is diluted as it flows into comparatively pure water.

Let us observe how a disease-producing organism may
find its way into water that is used for drinking or other
purposes, and thence may attack a comparatively healthy
individual. In typhoid fever, a germ, which will after-
wards be described, has been found. It has special charac-
teristics, and may be separated asa pure culture. A patient
has typhoid fever ; the bacilli which we find specially in the
walls of the alimentary tract pass out along with the excreta,
and, under ordinary circumstances, would be killed by the
addition of disinfectant fluid to the stool ; but, as frequently
happens, some of the excreta without the addition of a
disinfecting fluid by chance finds its way into the drains, or,
worse still, on to the surface of the soil near a well or some
other source of water supply. The typhoid bacillus con-
tinues to multiply in the water or in the organic matter
of the sewage, from which it ultimately finds its way into
the water, and although such water may appear pure
enough (it is often beautifully clear and sparkling), as soon
as it is taken into a slightly disordered stomach and
intestinal canal, the bacillus gains a foothold, and another
patient is attacked with typhoid fever. This may happen
simply through the rinsing out of a milk pail. A patient
partaking of milk (a most admirable nutrient medium for
the growth of the typhoid bacillus) from such a pail is
struck down with “Typhoid.” Another example: there

22 BACTERIA,

is a small drum-stick shaped bacillus, similar to the putre-
factive bacillus described by Bienstock, which, on making
its way into a surgical wound, sets up a series of changes in
the tissues, accompanied by the production of a most
virulent poison, which, acting apparently on the nervous
system, gives rise to reflex spasms and convulsions, and
a ‘condition known as lock-jaw or tetanus is set up.
This organism is found on the manure heap, in cultivated
soil, and even in water that comes from such soil ; it is also
found in the dust of hay, straw, and even in the harness and
cloths used for equipping the horse. If water containing
these organisms be used for the purpose of washing a con-
tused wound, or if any of the above dirt or dust should obtain
access to such a wound—often merely a most insignificant
bruise—tetanus or lock-jaw is set up with terrible certainty,
and the patient very frequently succumbs to the disease.

Innumerable other instances might be given in proof of
the statement that the knowledge, first, of the forms, and,
secondly, of the biological and physiological characteristics
of the various micro-organisms, is now absolutely necessary
for a thorough understanding of even many everyday pro-
cesses. The colour formed in “ blue milk” is due to the action
of a micro-organism ; as also are the phenomena of bleeding
bread, the Cape meal orange ferment, and the characteristic
appearance of green cheese. Asa result of the knowledge
gained through the study of the life history of septic
organisms, thousands of valuable lives have been saved in
our surgical wards alone ; large industries have, through
Pasteur’s indefatigable exertions, been preserved from almost
absolute ruin; as a result of the observations of numerous
investigators, our knowledge of certain classes of diseases is
gradually becoming more precise and accurate, and the
time has now arrived when we may look forward to a
system of medicine in which, by preventive and curative
inoculation, we shall be able to grapple successfully with
some of the deadliest forms of disease with which we have
at present helplessly and almost hopelessly to contend.

LITERATURE.

The following works may be consulted :
BrensTock.—dZeitschr. f. Klin. Med. Bd. vii. p. 1, 1884.
Coun.—Beitr. z. Biologie der Pflanzen, Bd. 1, 1876.

INTRODUCTION. 23

Duciaux.—Chimie. Biologique Encyclop. Chimique, Paris,
1883.

DUNNENBERGER.—Bakt. chem. Unters. Ueber d. Beim.
Aufgehen des Brodteiges wirkenden Ursachen.—Bot.
Centralbl., 1888.

Firz.—Ber. d. Deutsche chem. Gesellschaft, 1876-1884.

Hansen.—Meddel fra. Carlsberg Laborat., Copenhagen.

Hueppre.—Der Bakt. Forsch., 1889.

JorGENSEN.—The Micro-organisms of Fermentation
(transl.), 1889.

Kuiess.—Beitr. z. Kenntn. d. Mikrokokken.—Arch. f.
Exp. Path., Bd. 1., 1873.

Kocu.—Mitt. a. d. k. Gesundheitsamt, Bd. 1., 1881.

Lister.—Trans. of the Path. Soc. Lond., vol. xxix., p. 425,
1878.

NaEGELI.—Nageli u. Schwendener—Das Mikroskop, 1877 ;
Theorie der Gahrung, 1879.

PastEuR.—Etudes sur le vin, 1866 ; Etudes sur le Vinaigre,

-1868 ; Etudes sur le Biere, 1876.

Rogerts.—Phil. Trans., vol. clxiv., p. 457, 1874.

SALOMONSEN.—Bakteriologisk Teknik., Copenhagen, 1890.

Van TIEGHEM.—Bull de la Soc. Bot. de France, p. 128,

1877 ; p. 25, 1879.

CHAPTER II.
: WHAT ARE BACTERIA?

Structure — Myco-protein — Limiting Membrane—Gelatinous Capsule—
Special Cell Contents-——-Oxide of Iron—Sulphur—Colouring Material
—Flagella—Modes of Multiplication and Development—Division—
Rate of Vegetative Multiplication —Endospores — Arthrospores—
Classifications of Cohn, Van Tieghem, Zopf, Winter and Rabenhorst,
De Bary and Hueppe, Fliigge Baumgarten, &c.

UnpbER the general term Bacteria may be included those
minute, rounded, ellipsoid, rod-shaped, thread-like, or spiral
forms of vegetable organisms sometimes spoken of as be-
longing to the class of lower fungi. ‘They are also known as
Fission fungi (the Schizomycetes of the Germans); a term
applied to them because of their multiplication by a process
of division, transverse to the longitudinal axis in the case
of the rod-like forms, but varying somewhat in the case
of the rounded organisms. Each single organism consists
of a small speck of protoplasm or vegetable albumen, to
which may be given the name of cell, as although these
specks are so minute that they can be seen and studied only
with the aid of the very best optical appliances at our
command, it is found that they are by no means simple in
structure, nor are they in all cases even similar. This proto-
plasmic speck is differentiated into certain well-defined parts.

The round cells or micrococci, the simplest of all forms,
are seldom more than 1-25,oooth part of an inch in diameter ;
the elongated cells have, on.an average, the same diameter,
or they may be a little more, and are from 1-12,000th part
to 1-6,0ooth part of an inch in length, though there are
marked deviations from these dimensions in certain forms.
Accepting the above figures as being accurate, it would
require 25,000 of these spherical cells, placed in a row, or
the same number of the longer ones, placed side by side, to
make up a chain or band one inch in length. The vegetable

WHAT ARE BACTERIA ? 25

albuminoid or protoplasmic substance of bacteria -was first
analysed and described by Nencki, who, because of the
nitrogen contained in it, and because of the similarity of
its structure to animal and vegetable protoplasm, looked
upon it as a proteid material; he gave to it the name of
myco-protein or fungus protein.* During certain periods
of the existence of these micro-organisms, especially in cer-
tain species, this white of egg or jelly-like substance, which
invariably occupies the central part of the cell, is perfectly
transparent and is slightly more refractile than water ; at
other periods, or in other species, it may be finely or
coarsely granular ; under certain conditions still more marked
and characteristic changes may occur, vacuoles or clear spaces
making their appearance. It is usually extremely resistant
to the action of acids and even of alkalies.

That this myco-protein cannot always be of the same
composition is evident from the fact that minute granules of
chlorophyll or of fat may be made out lying in the substance of
the protoplasm in special species, whilst in others small starch
or sulphur granules, or particles of different kinds of pigment
may be observed. This speck of vegetable albumen is really
the active part of the cell, and it is in this that we obtain
those histo-chemical colour or staining reactions that are so
characteristic of the protoplasm of the higher vegetable and
animal cells which, as is well known, seem to have a
peculiar affinity for certain dyes or stains, Carmine, for
example, is taken up most voraciously by the nucleus or
central portion of the cell which then assumes a brilliant
carmine hue, not by any means equal throughout ; in
consequence of this inequality the minute structure of
the nucleus may be readily and even accurately studied.
The surrounding protoplasm, which appears to be less
active, is much less vividly stained, whilst the cell wall,
which is the least active part of the cell and serves as little
more than a boundary wall of formed material, if present
at all, remains, in the majority of cases, quite unstained.
Logwood, the aniline dyes, or iodine may be substituted for
carmine, with the general result that the same staining
reactions as regards the different parts of these cells are
almost invariably obtained.

* Analysis of myco-protein: Water, 84.81; albumen, 13.207; fat, 1.198;
ash, 0.638; extractives, 0.327.

26 BACTERIA,

Exactly the same reactions are given when one of the
above colouring reagents or stains is added to a mass of
micro-organisms. The individual cells or organisms are
brought prominently into view, the central jelly-like speck
of protoplasm is most vividly stained in each, and reasoning
from analogy, certain authors have looked upon this active
part as the nucleus of the micro-organismal cell. Sur-
rounding the central protoplasm is a dense, sometimes
lamellated, thin skin or sheath which acts as a limiting or
protecting membrane. It very frequently contains a sub-
stance known as cellulose, almost, if not quite, identical
in composition with that forming the hard covering of
the vegetable cells that are found in the higher plants. In
other bacteria, in fact in the majority of them, the limiting
membrane seems to consist of a firm layer composed of
gelatinous material, sometimes stiff and rigid, or it may
be more or less elastic or pliable. Between the central
stained portion and the limiting sheath there sometimes
exists a narrow unstained area which by some is said to
be a space produced artificially by the chemical action
on the protoplasm of staining and other reagents ; others,
however, hold that it is a kind of modified protoplasm
which, surrounding the more active nuclear protoplasm,
divides it from the limiting membrane. Outside this limit-
ing membrane, or more strictly speaking, continuous with
it, there may usually be seen a mass of gelatinous or muci-
laginous matter which does not take on any special stain,
and which serves to separate the individual cells, or, to speak
more accurately, to bind them together into little groups. It is
due to the presence of this gelatinous material that we have
those frog-spawn masses or jelly-like lumps that are met
with in certain germ fermentative processes. These masses,
in which the organisms are embedded, as it were, in the
softened jelly-like part of their sheaths, are known as
zoogloea masses or masses of living glue. If the cells
remain isolated when the membrane becomes gelatinous,
capsules or highly refractile areas are seen around each
cell, when examined under the microscope. These may
sometimes be very delicately stained by certain methods
(Gram’s method, see Appendix). As examples, may be taken
the capsule that surrounds the bacillus of pneumonia (Fried-
lander), the false Diplococcus of pneumonia and certain

WHAT ARE BACTERIA ? 27

other organisms such as the Leuconostoc mesenteroides,
which is not pathogenic, and the Actinomyces or Ray
fungus. It is rather a curious fact, as Friedlander pointed
out, that this gelatinous capsule is formed only under certain
conditions, and he noted that in the case of the bacillus
of pneumonia the capsule could not always be demonstrated ;
that it occurred in the bacilli found in the lung tissue or in
the prune juice sputum so characteristic of the disease, but
that it could not be made out in the organisms grown on
such cultivation media as peptonized beef jelly. In the case
of the Ray fungus or Actinomyces, the organism that causes
wooden-tongue or Actinomycosis in cattle, this swelling of
the capsule at one end of the organism and not at the other
gives rise to a very peculiar club-shaped appearance of the
threads, and as these are frequently arranged so that the
thin ends of the rods are grouped together, whilst the
swollen ends are placed at the periphery of the radius, a
most peculiar and characteristic radiate arrangement of
wedge or club-shaped rods is the result. In some cases
bacteria first divide and multiply whilst they are embedded
in this common gelatinous mass, and it is only after they
have undergone a certain development that each organism
becomes invested with its own capsule and is allowed to lead
an independent life.

It appears that in most cases in which the organisms con-
tain natural colouring matter, it is deposited in the capsule
and not in the protoplasm itself, and that the red, magenta,
blue, and yellow colouring particles that are met with in
this position give rise to the naked eye colour that is seen
where large masses of these organisms are growing. The
beautiful brown that is seen in Crenothrix and Cladothrix,
not only in the capsule, but in the surrounding cultivation
medium, is due to the presence of oxide of iron which the
organism is able to separate from water, or from other
media in which that substance is held in suspension or com-
bination.

The composition of the limiting or external membrane,
as already pointed out, varies somewhat in different organ-
isms ; in many cases it appears to consist merely of an
altered myco-protein, but in others, as, for example, in
Bacillus anthracis, it is composed of a material analogous
to the casein of plants, combined with a substance which

28 BACTERIA,

has many points of resemblance to the mucine found in the
tissues, especially the embryonic tissues of animals. This
capsule of the B. anthracis, unlike myco-protein, contains
no sulphur, is soluble in dilute alkalies, but insoluble in
water and acetic acid.

Dallinger some time ago pointed out that at each pole of
an organism which he had studied most thoroughly (the Bac-
terium termo, a small oval organism not now recognized
as a species) there was developed an extremely delicate
flagellum which, he assumed, had something to do with
propelling the organism through the fluid in which it
was growing, as this organism exhibited most active move-
ments in such fluid. As a considerable number of other
organisms also possess this same motion in fluid media, it
was argued that as in the case of the above organism and
of other cells that have an extreme degree of motility,
there would also be found flagella or cilia such as are
met with in the swarm cells of certain alge, where cilia
or flagella are continued directly fror: the protoplasm,
through the cell membrane, if present, and so to the out-
side of the organism. Although the existence of these
flagella was suspected in many bacteria, they could actually
be demonstrated in the case of some of the larger organisms
only. Now, however, the number of bacteria in which flagella
have been seen has been gradually but surely increased, and
lately Léffler has described-and photographed most exquisite
lash or thread-like filaments, single or in little groups, in
Koch's cholera bacillus, in the bacillus associated with the
causation of typhoid fever, and in a considerable number
of other motile bacteria.

Whether these cilia and flagella are developed from the
protoplasm of the organism, or whether they are merely
secondary modifications of the external membrane, remains
as yet a doubtful point. It is quite possible that in many,
even of the motile forms, flagella are entirely absent ; the
organism in such case relying for its motile power on con-
traction of the protoplasm within an elastic or not too
rigid membrane. In a straight rod-like organism an un-
dulating movement may often be observed, whilst’ both
rotary and undulating movements are met with in spiral
organisms, though these latter are very frequently supplied
with well-formed flagella. It is possible that in certain species,

WHAT ARE BACTERIA ? 29

in which motion is very slight, if not entirely absent, the
flagella serve to set up currents round the organism by which
food is brought to, and excretions removed from, the bacillus.
These flagella may be met with as a single pair, or we may
have three or four pairs attached to the same organism. They
stain like the membranes, and appear to develop only in those

Photo-micrograph of Spirillum Undala, with pair of well-marked flagella at each
end. X 1000.

organisms that have special affinity for oxygen, for as soon
as the ciliated forms reach the surface of a fluid, they lose
their cilia or they become much less active. This latter,
however, seems to-be the more probable explanation, for
Léffler has shown that many organisms are provided with
cilia, which at one time were supposed to possess nothing of

30 BACTERIA.

the kind. Up to the present, however, micrococci, the
Bacillus anthracis, and many other organisms, cannot be said
to be supplied with flagella or cilia, and in many organisms
in which there seems to be independent movement, really
nothing but the so-called Brownian movement can be dis-
tinguished when they. are examined in fluid, a movement
that may be observed equally well when particles, of

Photo-micrograph of Cladothrix Dichotoma with pseudo-branching filaments,
with well-marked tranverse divisions. x 1000.

inorganic colouring matter are suspended in a fluid
medium. and examined under the microscope.

The simplest form of division or multiplication met with
in bacteria is that known as vegetative multiplication, in
which, taking as an example a short rod-shaped bacillus,
there is first an increase in the length of the rod, then a zone
of constriction in the middle of this lengthened rod, and then

WHAT ARE BACTERIA ? 31

division, complete or partial, of this lengthened rod into
two shorter daughter-cells. Under suitable conditions these
two again increase in length, each again is divided into two,
and so regular chains or swarms of bacteria are developed.
The first sign of division is a delicate transverse mark or
line across the middle of the bacterium in a plane at right
angles to the longitudinal axis of the cell.

By staining, as De Bary recommends, with an alcoholic
solution of iodine and causing the young protoplasm to
retract, it may be made out that this line is due to the
ingrowth of the cell membrane from the periphery towards
the centre so as to form a septum, more or less com-
plete, between the little rods of protoplasm, which are
thus gradually cut off from one another by the constricting,
and growing in, cell membrane.

In the rod-shaped bacteria this division takes place in one
plane only—at right angles to the longitudinal axis of the
rod—and when it is imperfect or incomplete it gives rise to
chain-bacteria or Strepto-bacteria.?

The individual bacteria of which the chain consists are
held together in series by the constricted but incompletely
divided membranous portion.

Similar divisions take place in the rounded bacteria or
micrococci, and we have then chain-cocci or strepto-cocci
formed, but instead of going on to form or remaining in
long chains, they may be arranged in pairs, and are then
spoken of as diplococci. There is sometimes division taking
place in two dimensions of space, the one at right angles to
the other, a good example of which is seen in the Bacterium
Merismopcedioides described by Zopf, in which the lines of
divisions are placed at right angles to one another, but in
one surface plane, z\e., at the surface of a fluid. In other
cases there appear to be division, multiplication, and
growth in three directions. To obtain an idea of what
occurs, suppose that a two-inch cube is divided into single
cubes, each one inch in diameter : and that these single-inch
cubes then grow until each reaches the size of the original
two-inch cube, when it, in turn, is again divided into one-

= A long spiral may divide into short curved rods, as in the case of the
Cholera bacillus. In Cladothrix, where there is a false branching, the
terminal cell before branching, instead of dividing transversely divides
vertically, and then the division again goes on transversely.

32 BACTERIA.

inch cubes. This mode of division was first demonstrated by
Goodsir in the Sarcina ventriculi.

We have already seen that these minute organisms are
endowed with great power of movement and locomotion,
a fact on which stress has been laid in connection with
their rapid diffusion through fluids; but an equally im-
portant factor in the bringing about of this rapid diffusion
is their extreme prolificness. If they can obtain sufficient
food, and if the food is of exactly the right nature, the rate
at which bacteria grow is marvellous. From actual experi-
ment it has been found that if in a cubic centimetre of
any specimen of water, we find, say, a couple of hundred
organisms ; on standing for twenty-four hours the number
will have risen to about 5,000 per c.c. ; at the end of another
twenty-four hours, to 20,000 ; and on the fourth day they
are uncountable. Cohn calculated that a single germ could
produce, by simple fission, as above described, two of its kind
in one hour ; in the second hour these would be multiplied to
four, and in three day they would, if their surroundings were
ideally favourable, form a mass which can scarcely be reckoned
in numbers—or if reckoned, could scarcely be imagined—
4,772 billions. If we reduce this number to weight, we find
that the mass arising from this single germ would, in three
days, weigh no less than 7,500 tons. Fortunately for us, they
can seldom get food enough to carry on this appalling rate
of development, and a great number die both for want of
food, and because of the presence of other conditions un-
favourable to their existence. Vegetative multiplication
only takes place when the conditions are extremely favour-
able to the growth of the organism. If nutrition is in-
terfered with in any way, or if the removal of excretory
products is obstructed, or if there be a large amount of
oxygen present marked changes may at once be observed in
the appearance of the protoplasm of the micro-organism. It
becomes granular, then a small bright point appears in each
cell ; this point gradually increases in size until its diameter
may be greater than that of the original organism. This
large, clear, rounded, ovoid, or rod-shaped node is known asa
spore, or resting-spore. It is really the seed or egg formed
by the bacterium, by which the species may be continued
although the parent should perish. The shape varies slightly
in different species, but in every case it has a dark limiting out-

WHAT ARE BACTERIA ? 33

line ; it is devoid of colour, and is highly refractile. The dark
outline of the spore is usually surrounded by a pale, soft,
gelatinous envelope, the substance of which may, in some
cases, be accumulated in rather larger quantity near the two
poles of the refractile body. As soon as these glistening

Photo-micrograph of Bacillus subtilis, with well-developed free endospores,
‘Those in focus are seen to have clear centre and dark outline. x 1000.

bodies make their appearance, degeneration of the protoplasm
of the bacteria in which they are found invariably follows,
but the period at which the death of the protoplasm actually
takes place varies in different cases. Where the spores are
small they may lie for some time embedded in the protoplasm

4

34 BACTERIA.

of the cell, which, only as it degenerates or dies, leaves the
resting-spore free to be carried about from place to place ‘by
currents of air or water, to be developed when the conditions
of moisture, temperature, and food supply again become suffi-
ciently favourable. Where the diameter of the spore exceeds
that of the bacterium, it may be situated in the centre, giving
rise to a spindle-shaped organism, or it may be at one end,
when the organism becomes clubbed or pendulum-shaped.
The spore in this case appears. to escape moré readily, and
before the complete disintegration of the bacterium has taken
place ; the side or end of the swollen organism gives way and
allows of the spore being set free. This is only one kind of
spore formation, and is spoken of by De Bary as endospore
formation. It is met with in some varieties of vibriones, in
many of the bacilli and in certain of the spirilla (Cornil
and Babes) ; whilst Zopf describes similar spore formation as
occurring in certain micrococci, and Escherich points out
that he obtained undoubted spores in sarcina pulmonum,
z.e., bodies that admitted of double staining.

A second kind of spore, to. which is given the name of
Arthrospore, is also described by De Bary, Hueppe and
others. In this there is a combination of spore formation
and of fission ; the mother-cell undergoes division into aseries
of daughter-cells, a few of which differ from the rest in very
important and essential points. There appear to be two

kinds of arthrospores ; one form, met with in Leuconostoc,

for example, where simple vegetative division of small round
bacteria goes on regularly, so long as the conditions are
favourable, and a regular chain is formed. In this chain
there appear at intervals, micrococci, which differ from the
remainder of the elements of the chain in the following
points: As soon as the conditions of nutrition are altered
they do not, like the other parts of the chain, die off,
but they become “somewhat larger than the rest, acquire
a more distinct outline, become thicker-walled, and their
protoplasm grows darker. Eventually they become free by
the deliquescence of the gelatinous envelopes, and may
claim the name of spores, because, when placed in the fresh
nutrient solution, they develop into new rows of beads like
those of the mother plant.” Here is a body which has most
of the characteristics of the resting-spore or seed, but it is not
formed within the protoplasm of the vegetative organism,

WHAT ARE BACTERIA ? 35

but by a process of fission, and as a result of the vegetative
division of the organism. It is quite possible, however, that
there is just as much differentiation of the protoplasm in
this case, as there is where the spore is formed within the
cell ; the only distinction being that the separation between
the spore and the vegetative element of the chain takes place
at an earlier stage, and more completely than in endosporous
reproduction. The reverse takes place, however, in the
Bacterium Zopfii, which during the vegetative or fission stage
consists of short rods, then of motionless filaments, and, if
the temperature be lowered from 30° C. to 25° C. of short
motile rods. As soon, however, as the conditions become
unfavourable, especially when the nutrient material in which
they are growing is exhausted, the rods, apparently, by a
simple process of fission are divided up into short roundish
cells, which retain their vitality for a considerable time, and
which, when again placed under favourable conditions, act
as spores, z.¢., they develop into the original characteristic
rod-shaped bacteria.

In organisms in which spores are not found, the con-
ditions for their existence and propagation must always
remain favourable or they die out very rapidly, having no
specially resistant phase to enable them to tide over their
period of adversity; and had we to deal with asporous
organisms only, they could, probably, by an organized
attack, be rapidly and completely exterminated. The vege-
tative organisms, as distinguished from their spores, cannot
survive the prolonged action of heat ;—a comparatively
low temperature, 60° C. or less, being usually sufficient to
kill them, whilst the weaker chemical germicidal reagents
are quite sufficient to render them altogether innocuous
and inactive. The spores, on the other hand, can withstand
the action of a temperature anything short of 100° C..for a
considerable length of time. Cold and dessication have no
effect upon them, for after being submitted to any of these,
spores will, if placed under suitable conditions, develop into
the more characteristic vegetative forms.

It must be borne in mind, however, that certain organisms
may, by careful acclimatization, be accustomed to exist and
even develop at extreme temperatures. Globig hasshown that
certain bacteria found in the soil can only grow at a tempera-
ture of 50° C. or more, and that they can flourish up to

36 BACTERIA.

70° C., whilst certain forms of “Light” bacilli can, accord-
ing to Fischer, grow luxuriantly at a temperature of 0° C.

It should be remembered, however, that arthrospores
are much less resistant to all germicidal reagents than
are endospores; indeed, they are able to withstand a
temperature only about as high as that at which the
vegetative forms succumb ; whilst some of the endospores
are capable of withstanding dry heat of 105°, 110°, and even
130°C, They are certainly more resistant to the action of
some of the weaker germicidal reagents, but they withstand
for a short time only, or not at all, the action of the more
powerful ones.

From the nature of the dense membrane that surrounds
these spores, their staining has always been a matter of
extreme difficulty. They stood out most distinctly as clear
spaces in the deeply stained protoplasm of the cell, but could
not be stained. By subjecting the spore-containing organisms
to the action of dry heat at 110° C. for half an hour or an
hour, as suggested by Buchner, or by exposing them to the
action of concentrated sulphuric acid for fifteen seconds, or
to a longer treatment with concentrated caustic potash, the
membrane is so altered that the staining reagents are enabled
to penetrate into the substance of the spore and act on its
protoplasm, and impart to it a characteristic colour. If this
heating be excessive the protoplasm of the bacillus may be
destroyed when it in turn refuses to take on the stain,
although the spore itself may, under these circumstances, be
stained most beautifully, this fact also indicating that the
spore is more resistant to the action of heat than is the
vegetative cell. Hueppe, Babes, and Neisser have all
described arthrospores as making their appéarance at the
end of Koch’s cholera bacillus, which may become free, says
Hueppe, and from which, he thinks, he has seen the bacillus
being developed.*

t In order to stain endospores, the best fluid to use is probably Ehrlich’s
aniline water fuchsin solution. Sections are left in this for several days,
they are then decolorised with 25°/, solution of nitric acid, washed thoroughly
in water and alcohol, to which a trace of ammonia has been added; a con-
trast stain is obtained by treating for a few minutes with a dilute solution
of methylene blue. In some cases the acid removes the fuchsin from
the spores also; it is then well to wash simply with alchohol and stain
with methylene blue as a contrast stain. Instead of leaving cover glass
preparations for so long a period in the fuchsin they may be heated along

edd

WHAT ARE BACTERIA ? 37

These so-called arthrospores of the cholera bacillus do
not, however, give the ordinary reactions of spores, nor
are they any more resistant to the action of heat and
germicidal agents than the vegetative forms themselves :
they must therefore still be looked upon as pseudo-spores.
True spores, then, appear to be special protoplasmic cells,
which are first developed in the mother cells and are then
surrounded by a very thin, but hard and dense membrane.
It is this dense covering that protects the delicate proto-
plasm within, against the action of the numerous destructive
influences to which the spore is exposed.

If this structure be borne in mind, it becomes evident that
‘dry heat must necessarily be less efficacious than moist, in
determining the destruction of the spore. Dry heat causes no
swelling of the protoplasm within, whilst moist heat causing
swelling brings about rupture of the softened membrane by
pressure from within, and the unprotected protoplasm, exposed
under most unfavourable conditions, is at once rendered inert.
It appears probable that this process of expansion from
within also comes into play whenever spores are placed
in conditions favourable to their development, ze., when
they are placed in a warm medium in which are present both
nutriment and moisture.

The first change then noticed is that the clear, strongly
refractile protoplasm becomes cloudy or granular, the dark
outline is not quite so prominent and the clear boundary
line or limiting membrane appears to swell somewhat,
and the spores gradually assume the form of an ordinary
vegetative cell. In some cases, however, as soon as the
spore begins to swell, the delicate outer sheath may be seen
to split either longitudinally as in the Bacillus amylobacter,
or across the middle as in Bacillus subtilis. In either case
there is a swelling of the softened gelatinous layer which
causes the removal of the firm membrane. This thin, firm,
delicate membrane may come away in the form of two
separate cups, or there may be simply a transverse slit
through which the germinating spore escapes. Having
once made its way from the membrane, vegetative division
at once sets in, the segmentation always taking place at
with the fluid almost to boiling point for ten or fifteen minutes. The

after procedure is as above. The spores most difficult to stain are those
of the bacilli of tuberculosis, and of typhoid fever.

38 BACTERIA.

right angles to the long axis of the young organism, as has
already been described. It was at one time thought that in
the case of Bacillus subtilis division at first went on longitu-
dinally, but it has now been demonstrated that the general
rule is not departed from, the well-known appearance being
due to the fact that after rupture of the membrane the two
halves remaining attached by a kind of hinge are thrown
outwards, the ends of the vegetative spore protoplasm remain-
ing within these little cups, and the body growing rapidly
before division takes place ; a kind of loop is thus formed
which gradually becomes longer and longer, and the two
limbs which were supposed to be the result of a longitudinal
division are nothing more than the two ends of the same
rod. After a time transverse divisions may be seen in the
different parts of the loop, the ends escape from the cups
formed by the halves of the opened-up spore capsule,
the rods straighten out and assume the regular straight
form. There are other modifications of the same process
of development from the spore, but the above are the
essential or most important forms.

CLASSIFICATION.

Our present classification of Bacteria is based uponthat given
by Cohn. He arranged these organisms into four groups,
taking as his characterising feature the form that was most
commonly assumed by each organism. His first group con-
sisted of rounded bacteria or cocci—the Sphzro-bacteria ; the
second group was made up of short rods, or cylinder-shaped
bacteria—Micro-bacteria ; longer rods or thread-like organ-
isms—the Desmo-bacteria—were placed in the third group ;
and to the fourth group were assigned screw-shaped or spiral
bacteria—the Spiro-bacteria. According to this author the
cocci consist of rounded or ellipsoid bodies, .5 to 2? in
diameter, the smaller ones being spoken of as Micrococci,
the larger as Mega- or Macro-cocci. ‘They are found singly,
or may be grouped in pairs or in longer chains. The
micro-bacteria or short rods are more variable in their size,
measuring Ip in diameter and a little more than that in
length ; but when the length of an organism reaches more

1 Iw = 0.001 mm, = zoohoooth part of a metre = z,45gth part of an
inch.

WHAT ARE BACTERIA ? 39

than about twice its diameter, it is usually spoken of as a
Bacillus :—the length of a bacillus may be eight or ten times
the diameter, which ranges from 1 to 2n. In some cases
these bacteria take the shape of short stout spindles or lemons,
especially during the stage of spore formation, when they are

Photo-micrograph of Proteus Vulgaris Bacillus, in the form of shortrods, x 1000.

spoken of as Clostridium forms, of which an exceedingly
good example is the spore-bearing Bacillus butyricus.

Bacilli increase in length, and, becoming more or less
jointed as vegetative division takes place, form the long
delicate jointed threads which are spoken of as Lepto-
thrix forms when there is no apparent branching, but if
pseudo-ramifications, such as are found in Cladothrix dicho-
toma and are due to vertical division taking place in one of
the terminal cells, are present, we have the Cladothrix form.

40 BACTERIA,

The Spiro-bacteria vary very much, not only in size but also
in length and in general appearance. :

In some instances other isolated features have determined a
nomenclature. For example, the appearance of sulphur
in one series of forms has determined a species of Ophido-
monades ; whilst the form and length of curves have also been
used asa distinguishing feature. ‘The organism is spoken of as
a Vibrio when the curves are slightly marked ; if the curves

Photo-micrograph of Bacillus Figuraus in which Leptothrix form is
wellseen, X 150,

are short and slightly pronounced and the organism is thin,

it is known as a Spirocheta ; a ribbon-shaped spiral is a

Spiromonad, and a spindle-shaped spiral, a Spirulina.
COHN’S CLASSIFICATION.

Schrzophytes, Thallophytes that develop by division or by
; endogenous germinating cells, : :

WHAT ARE BACTERIA ? 41

TRIBE I.
A. Free cells arranged in pairs or fours.
Cells round—chroococcus (Naegeli).
Cells cylindrical—synechococcus (Naegeli).
B. Cells united into zoogloea masses by homogeneous gelatinous sub-
stance.
a. Cellular membrane shading off into the intercellular substance.
Cells round—micrecoccus (Hallier).
Cells cylindrical—bacterium (Dujardin).
4. Intercellular substance arranged in concentric layers.
Cells round—glzocapsa.
Cells cylindrical—glzothece.
C. Cells forming circumscribed zooglcea masses with a definite shape.
a. Families arranged in flat layers in a single plane—merismopedia.
b. Cells rounded, arranged in a zoogloea mass forming a network—
clathrocystis.
¢. Cells cylindrical or wedge-shaped, the families divided by con-
strictions—ccelospherium.
d. Cells forming families, dividing in several planes, colourless
cubical cells with a quadrate arrangement—sarcina.
e. A large and indefinite number of colourless cells—ascococcus.

TRIBE 2.
Filamentus forms in which the cells are thread-shaped.
A. Without branching.
1. Colourless cylinders with little sign of division, very delicate, when
short—bacillus, when long—leptothrix.
2. Similar filaments, but thicker and longer—beggiatoa.
3. Filaments deeply divided at intervals with colourless spore-bearing
tissue and a well developed sheath—crenothrix.
4. Spiral filaments. ~ ‘
Short and undulating—vibrio.
Short with rigid spirals—spirillum.
et with flexible spirals and containing phycochrome—spiro-
cheete.
Filaments long and spirals flexible—spirulina.
5. Filaments in chains without phycochrome—streptococcus (strepto-
bacteria).
Zoogloea masses or cylindrical cells.
‘When colourless—myconostoc.
In chains—nostoc.
. Filaments thinner at one end——rivolaria.
B. Filaments with pseudo ramifications—cladothrix.
Cylindrical colourless filaments—streptothrix.

It may be of interest to some to glance over a few of the
classifications suggested by different authors, and to compare
-the bases on which these classifications are made. Cohn, as

we have seen, classifies entirely according to the elementary
form of the.organism, the nature of the membrane and the
mode of division, Wan Tieghem founded his classification

s

42 BACTERIA,

on much the same features, but he also takes into considera-
tion some of the physiological and biological characters, the
nature of the processes set up by them, the resulting products
and the nature of the division.

VAN TIEGHEM’S CLASSIFICATION.
Van Tieghem places all the schizomycetes in a family which

Photo-micrograph of Bacillus Figuraus Leptothrix and rod forms. x 1000.

he calls bacteria, a family closely related to the nostocaceze
and the oscillaria of the Alge; he divides them into micro-
cocci; bacteria or short rods; bacilli or short threads;
longer threads, without any definite sheath, being called
leptothrix ; with a sheath, crenothrix ; with a sheath and
undergoing division, cladothrix.

WHAT ARE BACTERIA ? 43

The genus vibrio consists of spiral filaments which break
up into short fragments.

The spirillum consists of longer filaments that have a
helicoid arrangement.

The spirochete, of greater length, are spirilla with more
numerous turns of the spiral. >

Micrococci, arranged in zoogloea masses and held together
by a thick layer of gelatinous material, he called ascococcus ;
when held together, but without or with less of this gelatinous
material, He gave them the name of punctula.

Bacteria united by this gelatinous material are called
ascobacteria ; without the gelatinous envelope, polybacteria ;
bacteria in the form of spiral threads, and massed together,
-form the myconostoc.

These bacteria are again divided according to their pro-
perties of forming colouring matter, chromogenes ; of setting
up fermentation, the zymogenes; and of giving rise to
disease in animals or plants, the pathogenes.

He gave a further classification based on the mode of
division of the primary cells :

1. The bacteria in which the division takes place in one
axis only. This includes micrococcus, bacterium, bacillus,
leptothrix, crenothrix, cladothrix, vibrio, spirillum, spiro-
cheete, ascococcus, punctula, ascobacteria, polybacteria, and
myconostoc.

2. The meristz, in which a membranous thallus divides
in two directions but on one plane only, giving rise to the
formation of characteristic tetrads.

3.. The sarcinz, in which there is division in three direc-
tions, so that the resulting masses always remain cubical in
form.

Zopf’s classification rests (Fligge, “Etiology of Infective
Diseases,” p. 180) on the doctrine of pleomorphism, which
cannot be accepted as in any way proved except in the case
of a few well-known non-pathogenic forms ; but his classifica-
tion may be accepted asa basis from which to work in bring-
ing proof or disproof of the theory of pleomorphism, al-
though it is not at present, at any rate, founded on a large
number of facts.

Zopf, who has studied most carefully the subject of pleo-
morphism, holds that several forms may occur in the cycle

44 BACTERIA,

of development of any species, and he has determined that
both form and developmental series must be used in drawing
up a classification.

ZOPF’S CLASSIFICATION.

Group 1.—Coccace —These are as yet only known in the coccus form.
To these the following genera belong :

1. Streptococcus (cocci arranged in threads like strings of beads).

2. Merismopedia, tablet cocci (division in two directions, leading to the

formation of tablet-like flat layers of cells).

3. Sarcina, packet cocci (division in three directions, leading to the

formation of bale-like colonies).

4. Micrococcus (the cocci become aggregated in irregular heaps).

5. And ascococcus (the heaps of cocci accompanied by marked formation

of gelatinous material).

Group 2.—BACTERIACE#.—These possess chiefly coccus, rod, and thread
forms; the former may be absent; in the latter there is no distinction
between base and apex. Threads straight or screw-like. Genera:

1. Bacterium, forms cocci and rods, or only rods which are arranged in

rows to form ordinary threads ; spore formation absent or unknown.

2. Spiril/lum, threads screw-like, formed only of rods, or of rods and

cocci; spore formation absent or unknown.
. Vibro, threads screw-like, spore formation in the longer or shorter joints
. Leuconostoc, forms cocci and rods, spore formation in cocci.
. Bacillus, cocci and rods, or only the latter in the form of simple or
twisted threads ; spore formation present.

6. Clostridium, the bacillus form in which the spore formation occurs in

peculiar enlarged rods.

Group 3..—LEPTOTHRICHE.—Coccal, rod, and thread forms; the latter
show a distinction between base and apex; threads straight or screw-like,
spore formation not demonstrated. Genera:

1. Crenothrix, threads jointed and enclosed in a sheath, cells contain no

sulphur granules ; inhabit water. .

2. Beggiatoa, threads thicker than Crenothrix, indistinctly articulated,

cells contain sulphur granules ; inhabitants of water.

3. Phragmidiothrix, threads without sheaths, successive divisions very

numerous ; cells contain no sulphur ; inhabit water.

4. Leptothrix, threads with or without sheaths, divisions not very

numerous or well marked ; cells devoid of sulphur.

Group 4.—CLADOTHRICHEA.—Show coccus, rod, thread, and spirillar
forms. The thread form is provided with a sheath, well-marked segments
and pseudo-branches. Spore formation not yet demonstrated. Genus:
Cladothrix.

Winter and Rabenhorst’s classification, though very con-
venient, is far from scientific, but it serves especially well
for the classification of micro-organisms that are found in
disease, as these, as met with in their host, usually correspond
to one stage only of Zopf’s developmental cycle of any special
organism.

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WHAT ARE BACTERIA. 47

Van Tieghem and De Bary, followed by Hueppe, laying
great stress on the method of reproduction, have made two
great divisions of bacteria—those which form endospores and
those which form arthrospores; but as Fliigge points out,
this division is of little service for practical purposes, as
spore formation is at present so imperfectly understood, that
it is most difficult to follow the exact method of sporogenous
reproduction in a Schizophyte. Winter and Fliigge attempt
to get rid of their objection to Cohn’s classification by speci-
ally modifying it to contain only the pathogenic organisms.
They take no account, as already stated, of any morphological
developmental cycle or of any biological features, but they
give a useful and practical classification.

Quite recently it has been suggested that bacteria should
be classified according to the number and arrangement of
the flagella developed, but as such classification is necessarily
based on a single characteristic, and that one of the least
important, and as it will be some time before a complete
examination can be made in order to determine the number
and arrangement of these flagella, such a classification may
for the present be left out of consideration. The same
holds good as regards Baumgarten’s classification; he
divides bacteria into two groups—those which appear to
assume a single form only (the Monomorphic) and those in
which pleomorphism is a well-developed characteristic. In
each he has three genera. In the first: the coccus, the
bacillus and the spirillum ; and in the second: the spirulina,
the leptothrix and the cladothrix. A similar remark applies
to these, however, that applies to Zopf’s classification. It
may be of some service as a provisional classification,
especially to those who are engaged in the study of the
morphology and life history of bacteria. To the pathologist,
however, these classifications are of comparatively little value
except in so far as by their aid the morphologist is able to
supply him with information as to the life history of bacteria
outside the body.

LITERATURE.

Author already quoted. Hueppe.
Bucuner.—Centralbl. f. Bakt. u. Parasiteuk. Bd. iv., 1888.
Coun.—Beitr. z. Biol. d. Pflanzen. Bd. 1, Heft. 2, 1872 ;
Bd. 1., Heft. 3, 1875.

48 BACTERIA,

*CorNIL AND Basrs.—Les Bactéries. Paris, 1890.

DALLINGER.— Monthly Journ. of Micro. Sct. Sept., 1875.

bE Bary.—Morphologie u. Physiologie der Pilze Handbuch
der physiol. Botanik. Bd. 11, Leipzig, 1866.

Dujarpin.—Histoire Naturelle des Zoophytes. Paris, 1841.

EsRENBERG.—Die Infusionsthierchen als vollk. organismen.
Leipzig, 1838.

FLiiccr.—Die Mikro-organismen. Leipzig, 1886. (Trans.
Watson Cheyne, New Syd. Soc. London, 1890).

Goopsir.— Edin. Med. and Surg. Journ., p. 430, 1842.

*LOFFLER.—Vorlesungen, tiber, die Geschichtliche En-
Bos lung der Lehre von den Bacterien. Leipzig,

1887.

NEISSER.—Virchow’s Archiv. Bd. 84, 1881.

Van TirGHEeM.—Traite de Botanique. 1883.

Zopr.—Die Spaltpilze. Breslau.

Those marked * contain full lists of papers and references.

CHAPTER III.
Tue History or BACTERIOLOGY.

Earliest Workers—Kircher’s Contagium Animatum—Bacteria in Fermenta-
tion, Putrefaction and Disease—Early Classifications—Miiller—Abio-
genesis — Needham — Biogenesis— Bonnet — Spallanzani — Schultz’s
Experiments — Schwann — Later Experiments— Pasteur — Bastian—
Colour and Fermentation—Cohn and Naegeli’s Classification—Henle’s
Researches and Postulates—Pasteur’s Researches on Fermentation and
Putrefaction—‘‘ Flower of Wine ”—“ Flower of Vinegar ”—Bacteria
as Scavengers—Pasteur on Silkworm Disease, and Wine Disease—
Germs killed by Carbolic acid—Origin of Antiseptic Treatment.

Since Athanasius Kircher mistook blood and pus corpuscles
for small worms, and built up on his mistake a new theory
of disease and putrefaction, and since Christian Lange, the
‘professor of Pathological Anatomy in Leipzig, in the preface
to Kircher’s book (1671) expressed his opinion that the
purpura of lying-in-women, measles, and other fevers were the
result of putrefaction caused by worms or animalcule, a
“Pathologia Animata” has, from time to time, been put
forward to explain the causation of disease. Crude as was
his theory, and imperfect as were the observations on which
it was based, it is marvellous that Kircher, with the simple
lenses that he had at his disposal, magnifying only some
thirty-two diameters or one thousand times, was able to
make out as much as he did. The observations that he
made were, naturally enough, not generally credited ; and
the theories he formulated were received with chilling in-
credulity by most of his contemporaries.

Remarkable as were Kircher’s observations, still more
wonderful were those of Anthony van Leeuwenhoek, a
native of Delft in Holland, who in his youth had learned
the art of polishing lenses, and who was able, ultimately, to
produce the first really good microscope that had yet been
constructed. Not only did Leeuwenhoek make his micro-
scope, but he used it to such good purpose that he was able

50 BACTERIA.

to place before the Royal Society of London a series of
most interesting and valuable letters giving the result of his
researches on minute specks of living protoplasm. The
results of these observations are fully detailed in his collected
works.

Towards the end of 1675 he discovered in water, in an
infusion of pepper, in the intestinal canals of horses, flies,
frogs, pigeons, fowls, and even in his own diarrhcea stools,
small moving and living forms of such extreme minuteness
that other observers with the best apparatus they had at
command, even with the accurate and lucid description he
gave, could not for long confirm his results. It was not,
however, until 1683 that he actually described and depicted
minute organisms in material taken from the teeth, that
we can at present recognize from his descriptions and
drawings as bacteria. Describing them, he says: “I saw
with very great astonishment, especially in the material
mentioned, that there were many extremely small animals
which moved about in a most amusing fashion ; the largest
of these” (represented by him in an admirable figure)
“showed the liveliest and most active motion, moving
through rain-water or saliva like a fish of prey darts through
the water; this form, though few in actual numbers, was
met with everywhere. A second form moved round, often
in a circle, or in a kind of curve; these were present in
greater numbers. The form of a third kind I could not
distinguish clearly ; sometimes it appeared oblong, some-
times quite round. They were very tiny, in addition to
which they moved forward so rapidly that they tore through
one another : they presented an appearance like a swarm of
midges and flies buzzing in and out between one another.
I had the impression that I saw several thousands in a
single drop of water or saliva which was mixed with a
small part of the above-named material not larger than a
grain of sand, even when nine parts of water or saliva were
added to one part of the material taken from the incisor or
molar teeth. Further examination of the material showed
that out of a large number which were very different in
length, all were of the same thickness. Some were curved,
some straight, lying irregularly and interlaced.” Since, he
says, “I had seen minute living animalculz of the same shape
in water, I endeavoured most carefully to observe whether

THE HISTORY OF BACTERIOLOGY. $1

these also were living or not, but I was unable to recognize
even the slightest movement as a sign of life.” 7

In the material taken from the teeth of an old man who never cleaned
his teeth, Leeuwenhoek found an inconceivable number of living animalculz
which darted about more quickly than any he had ever seen before ; “‘ the
largest were present in very great numbers, and waved about by the
locomotion of their bodies. Besides these, other animalculz were present
in such large numbers that the whole water seemed to be alive.”

This admirable account really contains the first accurate
description of the rod-shaped bacteria, motile and motionless,
of longer threads or bacilli, of the spiral threads or spirilla,
and of rounded micro-organisms or micrococci. It was con-
siderably improved upon in a letter to the Royal Society,
dated October 1, 1692, in which he speaks of small rounded
animalculz, the diameter of which is a thousand times less than
that of a fine grain of sand ; of organisms having a somewhat
greater diameter than the round ones, and being five or six
times as long as they were broad, equally thick throughout
their whole length, and which moved slowly backwards and
forwards through .a bending of their bodies. Along with
these he describes what are evidently spirilla, with their
characteristic movements, “a few organisms about the same
length or slightly longer, which moved their bodies in
comparatively marked curves, swam forwards or backwards,
or twisted themselves in an extremely lively fashion.” He
also observed still longer and more sluggish organisms,
sometimes straight and sometimes bent.

Although Leeuwenhoek did not attempt to theorize as to
the meaning of the presence of these organisms in the
mouth, we find that, in 1713, after finding similar organisms
in the greenish pellicle formed on the surface of the water
in an aquarium, he came to the conclusion that the organ-
isms seen on the teeth found their way into the mouth
through the medium of the drinking-water that had been
stored in barrels, and that some of these found there a nidus
in which they might multiply.

The world that Leeuwenhoek thus opened up so
thoroughly was rapidly invaded by other observers and
theorists. The thoughtful physicians of the time believed
that at last they had found the fons et origo mai, and
Nicolas Andry, reviewing Kircher’s “Contagium Ani-
matum,” replaced his worms by these newly-described

52 BACTERIA.

animalcule or germs, and pushing the theory to its
legitimate and logical conclusion, he also evolved a germ
theory of putrefaction and fermentation. He maintained
that air, water, vinegar, fermenting wine, old beer, and sour
milk, were all full of germs; that the blood and pustules of
smallpox also contained them, and that other diseases, very
rife about this period, were the result of the activity of these
organisms. Such headway did he make, and such conviction
did his arguments carry with them, that the mercurial
treatment much in vogue at that time was actually based on
the supposition that these organisms, the cause causantes of
disease, were killed by the action of mercury and mercurial
salts.

With a kind of prophetic instinct, and certainly as the
result of keen observation, Varro and Lancisi ascribed the
dangerous character of marsh or swamp air to the action of
invisible animalcule ; in fact the theory was so freely and
forcibly propagated that even where no micro-organisms
could be found their presence was inferred, with the inevi-
table result, as Léfiler points out, that these “inconceivable”
worms became the legitimate butts for the shafts of ridicule ;
and in 1726 there appeared in Paris a satirical work, in
which these small organisms received the name of “fainter,”
“body-pincher,” “ulcerator,” “ weeping fistula,” “ sensual-
ist ;” the whole system was thus laughingly held up to
satire, and the germ theory of disease completely dis-
credited. Linnzus, however, with his wonderful powers of
observation and deduction, considered that it was possible
that there might be rescued from this “chaos” small living
beings which were as yet insufficiently separated and ex-
amined, but in which he firmly believed might lie not only the
actual contagium of certain eruptive diseases, and of acute
fevers, but also the exciting causes of both fermentation and
putrefaction.

The man, however, who of all workers earliest recognized
the importance of Linnzeus’ observations was a Viennese
doctor, Marcus Antonius Plenciz, who with great shrewd-
ness recognized the prime importance of these organisms
in connection with the etiology not only of contagious
diseases, but also of putrefaction. He it was who, at this
time, insisted upon the specific character of the infective
agent in every case of disease: for scarlet fever there was a

THE HISTORY OF BACTERIOLOGY. 53

scarlet fever seed or germ—a seed which could never give
rise to smallpox. He showed that it was possible for this
organism to become disseminated through the air, and for it
to multiply in the body; and he explained the incubation
stage of a febrile disease as dependent on the growth of a
germ within the body during the period after its introduc-
tion, when its presence had not yet been made manifest. He
very rationally explained the differences in the character of
the symptoms and the severity of the same disease by refer-
ring them to differences in the constitutions and surround-
ings of the patients. As regards: putrefaction, having
corroborated Linnzus’ observations and found countless
animalculz in putrefying matter, he came to the conclusion
that this process was the result of the development,
multiplication, and carrying on of the functions of nutri-
tion and excretion by these germs; the products of
fermentation being the volatile salts set free by the organ-
isms, which, multiplying rapidly by forming seeds or eggs,
rendered the fluid in which they developed thick, turbid,
and foul. This theory, admirable as it was, and accurate as
it has since been proved to be, could not then be based on
any very extensive or detailed observation, and we find that
some of the most prominent and brilliant men of the
‘ period did not feel justified in accepting the explanation that
Plenciz had offered as to the causes of disease and fermenta-
tion processes; and it was not until the years following 1831
that any real advance could be made in our knowledge of
the presence of a “ Contagium vivum,” or living contagium
element in the production of disease and fermentation.
Previous to this, however, there was being gradually
accumulated a large mass of facts bearing on these wonder-
fully interesting minute living organisms, and numerous
isolated observations were constantly being made by various
workers, none of whom, however, were sufficiently master
of their subject to enable them to make any systematic
attempt at classification of their accumulated facts, and the
scientific results were consequently comparatively small,
standing in no proportion to the amount of work expended
and the number of observations made.
The first attempt to reduce this chaos to something like
order was made by Otto Friederich Miller, of Copenhagen.
‘He thoroughly appreciated the work he was taking in hand.

54 BACTERIA.

With a well-defined plan he set himself to systematize and
arrange the various organisms that had been described by
previous observers—commencing with Leeuwenhoek and
ending with Spallanzani.

When the nature of the optical apparatus Miiller had at his disposal is
taken into consideration, it must be acknowledged that he succeeded in a
most marvellous manner in classifying, on the Linnzean system, the minute
organisms with which he had to deal. Under the head of Infusoria
he divided them into two classes—those that could be seen with the
naked eye, and those that were invisible except with the assistance
of a microscope. The latter class he again divided into Membranacea,
or those forming thin surface membranes, and Crassuiscula, or those
forming thick membranes ; these latter, including Monas, Proteus, Volvox,
Enchelys, and Vibrio, representing, he maintained, the lowest forms of
animal life. Of Monas he described no fewer than ten species, and of
Vibrio he was able to distinguish, by utilizing the characters of form,
motion, nidus or cultivation medium, and other biological features, thirty-
one species. Relying, however, principally on the form of the organism,
he described rounded and slightly oval forms, shorter and longer rods,
rounded and truncated cork-screw-shaped and snake-like organisms,
ee but not spiral in their movements, and also long threads or

acilli.

Although he did not fully recognize the importance of his
observation he described in certain organisms little shining
points, arranged in series at regular intervals, especially in
the rod-shaped forms, points which we must now conclude
were spores. It is certainly not remarkable that he should
never have understood the full significance of these spores,
as even ninety years later, with all the additional light
that had then been thrown on the subject, these bodies were
still not properly understood. Later observers laid stress on
the rapidity of movement of the vibriones or lineole, which
were gradually separated from the other forms of lower
organisms., The vibriones then described, including lineola,
rugula, bacillus, and spirillum, correspond more or less per-
fectly with our bacteria of the present day.

Many advances were made after Miller’s work was com-
pleted as regarcs the morphology of these organisms, but
the question eas to whence these minute forms came still
remained unanswered. Whether they were the result of
spontaneous generation, or were the progeny of pre-
existing forms, was the question which for over a century
occupied the minds of those engaged in scientific research
and speculation. Some observers, prominent amongst whom

THE HISTORY OF BACTERIOLOGY. 55

were Hartsoeker, Reaumur, and Joblot, considered, though
they had no great amount of evidence to adduce in support
of their theory, that bacteria were the progeny of minute
organisms which were present in myriads in the air, from
which they were deposited on fruits, plants, and other matter,
whence they made their way into the various infusions
prepared from them. In this country a prophet arose in the
person of Dr. Needham, who was really the first to suggest
an attempted solution of the question by a theory of abioge-
nesis, or spontaneous generation. Needham at first thought
that these vibriones, or “plant animals,” as he called them,
arose from plants by special vegetative power, and that from
the plant-animals, by a process of evolutionary accretions,
other organisms again arose. He tried to prove, by boiling
a beef infusion and keeping it and allowing it to putrefy
in a well-stoppered bottle (a most scientific method), that
these zoophytes could not owe their origin to germs which
outside insects or organisms had brought into the infusion,
as he eonsidered that the boiling should have destroyed the
germs originally in the fluid, and as no new germs could, he
thought, make their way into the closely-stoppered vessel,
the resulting organisms must be the result of the action of
a special vegetative force.. This apparently logical and
fascinating theory was accepted by many whose names had
great weight in the scientific world. Needham’s observations
were repeated time after time with the same results, and his
theory met with wide acceptance.

To very critical minds it appeared, however, that these
experiments of Needham’s left loopholes for the inser-
tion of other explanations than those which he gave, and
Bonnet, of Geneva, suggested that the vessels used by
Needham were not hermetically sealed, that an almost
invisible opening would be quite sufficient to serve as a
means of entrance to organisms so minute as those with
which he was dealing, and that on the other hand there was
a possibility that the germs were so far resistant to increase
of temperature that they might live through a short period
(afew minutes only) of treatment with boiling water, Abbot
Spallanzani followed up, by his wonderful experiments, the
theoretical criticism of Bonnet. After convincing himself
that organisms did actually develop in unboiled infusions
even when the outer air was rigorously excluded, he argued

56 BACTERIA.

that the germs of micro-organisms, or eggs, as he termed
them, might exist on the walls of the vessel, on the material
of which the infusion was made, or suspended in the air
within the vessel. To get rid of these germs from the vessels
he heated the latter on the fire, then filling them rapidly
with his infusions he allowed them to cool, and sealed them
hermetically, but he still found that after a few days a
number of organisms made their appearance. Could the
organisms have got in along with the air during the process
of cooling ?

Toset this question at rest he made a number of infusions in
hermetically-sealed flasks, and boiled them for a whole hour,
with the result that in flasks so treated no organisms made
their appearance : if, however, the sealing was in any way
interfered with, organisms soon made their appearance, and
he concluded that living germs were’ necessary for the
development of putrefactive organisms. This fact once
established, the whole question was much simplified, and the
principle on which it rested was soon utilized in Paris agd else-
where in the methods adopted for insuring the preservation
of various food stuffs—methods which, with few modifications,
have been handed down to the present day. It was of course
objected that Spallanzani had shut out air from his vessels,
or, that he had so altered the constitution of the air which still
remained, that it was not possible for these minute organisms
to develop in it. This objection was met in 1836 by F.
Schulze, who put the question to himself, “If the access of
atmosphere, light, and heat, to substances in flasks included
of itself all the conditions for the primary formation of -
animal or vegetable organisms.” To prove that this was not
the case, Spallanzani’s conditions of absolute freedom from
germs capable of development in the infusion must be
obtained, and, secondly, air must be admitted to this infusion
in considerable quantities ; but the air so admitted must be
perfectly free from germs.

Schulze proceeded as follows : he filled a flask half full of distilled water,
to which he added various animal and vegetable substances. He gives the
following description of the further methods of procedure :—‘‘I then closed
it with a good cork, through which I passed two glass tubes bent at right
angles, the whole being air-tight ; it was next placed in a sand bath and
heated until the water boiled, and thus all parts fad reached the temperature
of 212° F, While the watery vapour was escaping by the glass tubes I
fastened, at each end, an apparatus which chemists employ for collecting

THE HISTORY OF BACTERIOLOGY. S7

carbonic acid gas ; that on the left was filled with concentrated sulphuric
acid, and the other with solution of potash. By means of the boiling heat
every living organism and all germs in the flask, or in the tubes, were
destroyed, and all access was cut off by the suphuric acid on the one side
and by the potash on the other. I placed this easily-moved apparatus
before my window, where it was exposed to the action of light, and also,
as I performed my experiments during the summer, to that of heat. At
the same time I placed near it an open vessel, with the same substances
that had been introduced into the flask, having also subjected them to
the boiling temperature. In order now to renew constantly the air within
the flask I sucked with my mouth, several times a day, the open end of the
apparatus filled with a solution of potash, by which process the air entered
my mouth from the flask through the caustic liquid, and the atmosphere
entered the flask from without through the sulphuric acid. The air was of
course not at all altered in its composition by passing through the sulphuric
acid in the flask, but if sufficient time was allowed for the passage the
portions of living matter, or matter capable of becoming animated, were
taken up by the acid and destroyed. From May 28th till the beginning of
August I continued uninterruptedly the renewal of the air in the flask
without being able, without the aid of a microscope, to perceive any living
animal or vegetable substance, although, during the whole of the time, I
made my observations almost daily on the edge of the liquid, and when at
last I separated the different parts of the apparatus I could not find in the
whole liquid the slightest trace of infusoria confervee or of moulds; but all
the three presented themselves in great abundance a few days after I had
left the flask standing open. The vessel which I placed near the apparatus
contained on the following day vibriones and monads, to which were soon
added larger polygastria, infusoria, and afterwards rotatoria.”

Schulze was thus able to prove that the sterility was not
dependent upon any alteration in the air within the flask, or
to the small quantity of air contained in it, and that it was not
due to any alteration brought about in the liquid by the heat-
ing process, as on the one hand a large quantity of air
was passing through the flask, whilst on the other the fluid
that had been boiled, but which was left exposed, rapidly
underwent decomposition, a decomposition that was accom-
panied by the development of micro-organisms in very
large numbers. The objection that some particles of sul-
phuric acid drawn in with the air might affect the growth
of organisms was met by Schulze by further experiments ;
and Schwann, who, instead of using sulphuric acid, used
heat as a means of destroying any particles that might
be present in the air that was drawn into the flask,
corroborated Schulze’s statements. Now came further
objections from the supporters of abiogenesis, who stated,
most definitely and categorically, that these workers were
not dealing with germs at all, but simply with particles of

58 BACTERIA.

albuminoid matter floating in the atmosphere, as a result of
the vegetative power of which, organisms of various kinds,
according to the conditions by which these particles find
themselves surrounded, were caused to be developed. In 1854
Schroeder and Von Dusch made a great advance ; they proved
that simple filtration through a layer of cotton wool was
sufficient to deprive the air of its organisms, and so to render
it unfit to produce decomposition in infusions from which
germs had already been eliminated by heat ; and no longer
than thirty years ago Hoffmann, Chevreul and Pasteur
demonstrated that it was quite sufficient to draw out, and
bend downwards, the neck of a bottle in which the germ-
free infusion was contained, in order to ensure the
continuance of a. non-putrefactive condition, and the
perfect freedom of the fluid contained within the flask
from germs: they argued that germs obey the law of
gravitation, like all other solid particles, when not blown
about by currents, and must settle down upon an upper
surface, so that when the tube was bent downwards the
organisms could not fall into the mouth. Tyndall gave
demonstrative proof of this in his exquisite experiment of
removing all particles from a glass chamber, first proving
their entire absence by passing through the chamber a ray
of light which could only be seen so long as particles
remained suspended in the atmosphere. As soon as the ray
of light disappeared from view he placed vegetable infusions
which had been sterilized by heat within the chamber, with
the result that they remained free from any trace of organic
life for several weeks together. Schwann had already pointed
out that blood, taken with certain precautions and introduced
into a flask in which the air was kept germ free, might be
preserved for a considerable length of time, without the
‘development of micro-organisms, and later (1857) Van der
Broek showed that the juice of grapes, and urine as’ well as
blood, might be kept free from decomposition and the
presence of organisms if the apparatus into which they were
received was first thoroughly sterilized by means of heat,
and if the substances were not allowed to come in contact
with the outside air.

Numerous observers, especially Burdon Sanderson, Roberts, Lister,
Chiene and Ewart, and Watson Cheyne in this country, and Rindfleisch,
Klebs, Cazeneuve and Livon, Leube, Hauser and Marchand abroad, con-

THE HISTORY OF BACTERIOLOGY. 59

firmed these observations and made many new ones in connection with the
behaviour of milk, egg albumen, vegetable substances kept under certain
conditions, and pieces of organs from freshly-killed animals to which
organisms from the external air were not allowed to gain access.

It seemed as though the adherents of abiogenesis had not
a leg left on which to stand; but owing to the fact that
certain organisms, especially when contained in such media
as milk and cheese, withstand the action of very considerable
heat, they still contested every inch of ground, though their
foothold was being gradually but surely cut away from
beneath them. Milk, which was one of the strongholds of
- the abiogenists, was first sterilized, with absolute certainty,
by Schroeder, who attained his end by subjecting the fluid
for a considerable time to a temperature of 100° C., and then
by Pasteur, who heated it to 110° C., for a short time only.
Cheese still remained to them, and as late as 1872 Bastian
placed a small piece of this substance in an infusion of white
turnip which had been filtered and carefully sterilized. This
was then boiled in a flask for ten minutes, and whilst still
boiling was hermetically sealed ; at the end of three days
countless living organisms were produced, as Bastian held,
from non-living albuminoid material ; but Cohn, repeating
the experiment, explained that the resting spores or resistant
germs were enclosed in the substance of the cheese, and that
they were thus able to resist the high temperature to which
the outer surface of the cheese, but not its centre, was
exposed. Duclaux’s later experiments with the Tyrothrix
of cheese, which resists the action of a very high temperature
for a considerable time, also helps to explain Bastian’s results.
The matter has now been set at rest, and it is an accepted
belief that bacteria or microbes, as these lowly organized
forms are now called, may be destroyed by heat and by
certain chemical reagents, and that when once destroyed in
any medium, no other organisms can rise from their ashes,
the medium remaining perfectly free from putrefactive
changes until fresh germs are introduced from without.
Harvey's famous dictum, omne vivum ex ovo, has thus come
to have a far wider meaning than that which he originally
attached to it. The triumphs of surgery, of preventive
inoculation, of hygiene in relation to specific infective
diseases, of preservation of food, have had their origin in
the knowledge gained during the battle which waged round

60 BACTERIA.

the question of Spontaneous Generation or generatio
@guivoca ; and to the disciples of that school every acknow-
ledgment.must be made and due credit assigned for the
attitude of scepticism and free ingenious and honest criticism
which they passed concerning half-formed and inadequately-
supported theories and imperfectly-conducted experiments,
for to. their efforts is certainly due the fact that the
experiments of their opponents became more and more.
perfect, and if to-day we have perfect methods of sterilization
and of making pure cultivations, it is because nothing was
taken for granted, and because able men on both sides of the
controversy were ranged against one another to fight the
matter to the death.

Whilst this battle over the origin and development of these micro-
organisms was going on, intermittent attempts were made to improve on
the classification that had been drawn up by Miiller, but it was only as
the optician supplied observers with better microscopic apparatus that
any further advances could be made. Ehrenberg, however, took up the
question and divided the Monad family into rounded and rod-shaped forms ;
these latter—Vibriones—he described as undergoing transverse division,
as they increased in length. These he sub-divided into Bacteria, or short,
straight, inflexible organisms; Vibriones, longer and more flexible; Spirilla,
or inflexible spiral forms ; and the Spirochzetee, or the flexible spiral organisms.
If to this classification we add the Cocci or rounded forms, we have prac-
tically a rude model of that adopted by authors at the present time. In his
vibriones he had six varieties, of which lineola, rugula, and bacillus, had
already been described by Miiller, but subtilis, tremulans, and prolifer,
were new. In consequence, however, of the want of marks of characteri-
zation, Ehrenberg himself was very doubtful as to the propriety of his
system of division or nomenclature. His spirillum comprises three forms,
the old vibrio undula of Miiller, the ordinary spirillum, and a new kind of
spirillum (Tenue). These three differed from one another only as regards
length and thickness, the Vibrio undula having only from one to one and a
half spiral windings, the others being merely longer or thicker. His genus,
Spirocheeta, contained a single form only, the Spirochzta plicatilis, an
organism of great length, but of very small diameter.

In consequence of the active snake-like and rotary move-
ment of these organisms, Ehrenberg was fully convinced
that he had to deal with animals, and this opinion was uni-
versally accepted down to the time when Davaine gave his
opinion that bacteria must be classified as really belonging
to the vegetable kingdom.

In 1840 colour characteristics were brought into play as a
means of distinguishing certain organisms, and we find that
Fuchs and Ehrenberg describe Vibrio cyanogenus and Bacil-

THE HISTORY OF BACTERIOLOGY. 61

lus xanthogenus, as giving rise during their growth in milk to
characteristic blue and orange colorations. He described the
exciting cause as small chain-like organisms, and considered
that they were the cause of both the colour formation and
the acid fermentive changes. As early as 1819 we find the
first description of bleeding bread, the organismal cause of
which, Ehrenberg, in later years, was able to cultivate on

Photo-micrograph of Bacillus Prodigiosus. Xx 1000.—Organism forms beautiful
red colour of bleeding bread, bloody sweat, &c.

various food media, boiled potatoes, Swiss cheese, and white
bread. He describes the organisms of which the coloured
mass was made up as being exceedingly minute, and as
having a characteristic movement quite distinct from the
so-called Brownian or molecular movement.

Some idea of the size of these organisms may be obtained from Ehren-
berg’s calculation that a cubic inch would contain from 46,656,000,000,000
to 884,736,000,000,000 plants ; this organism he called Monas prodigiosa,

62 BACTERIA,

but it is difficult to associate the characters that he gave with any of the red
pigment forming organisms now known.

Felix Dujardin added very little to the actual classifications given by
Miiller and Ehrenberg, but he brought out several most important facts.
In certain large vibrios he was able to make out distinct bifurcation, the
two new limbs becoming segmented transversely ; he was also able to
recognize an outer membrane or resistant covering on these organisms, and
within this a gelatinous or protoplasmic material. This led him to doubt
whether, after all, he was dealing with an animal form, and several of the
forms described he relegated to the plant kingdom as Algae— Vibrio subtilis
of the Oscillaria. i

As Léffler points out, although Dujardin was not able
to make any further advances on the classifications of Miller
and Ehrenberg, his observations on the chemistry of bacteria
were most important. He describes as specially suitable for
the development of bacteria, fluids containing such substances
as phosphate of soda, hyponitrous acid and oxalate of am-
monia and carbonate of soda, and he points out that the
nitrogen from the oxalate of ammonia is gradually used up
in the presence of organic substances.

With the exception of Dujardin, as we have seen, all observers up to
1852 had looked upon bacteria as belonging to the animal kingdom, but
in this year Perty announced that of these minute organisms, some
belong to the animal and some to the vegetable kingdom, whilst a
certain number appeared to him to stand on the borderland between the
two. These vibriones, he says, are colourless or sometimes blue, yellow,
or reddish, never green, organisms, with scarcely a trace of any differentia-
tion of their substance ; they have a spontaneous or automatic movement ;
they increase in number by transverse division, partial or complete ; when
this is incomplete, chains or threads are formed. He divides them into
Spirilla or spiral threads, Bacteria or winding or straight threads, and he
thinks that the bacteria have not only an active animal life, but that they
also pass through a stage during which they must be looked upon as vege-
table in character: a double existence which he assigned to other forms.

In 1854 Cohn insisted even more strongly on the plant
nature of these micro-organisms. He, too, described zoogloea
masses, and he summed up his researches as follows :—(1)
All vibriones seem to belong to the vegetable kingdom, and
they exhibit a very close relationship to the larger alge. (2)
In respect of their want of chlorophyll, and of their occurrence
in putrefying infusions, the vibriones belong to the group of
water fungi (mycophyceze). (3) Bacterium termo is the
active, moving form of a closely-related species of palmella
and tetraspora zoogloea. (4) Spirochzte plicatilis belongs
to the species spirulina, which we can at once indicate as a

THE HISTORY OF BACTERIOLOGY. 63

particular species. (5) The long non-wavy vibrios (vibrio,
bacillus, &c.), are related to the more delicate beggiatoa
(oscillaria). (6) The shorter vibrios and spirilla, which
correspond in both form and motion to the oscillaria and
spirulinz, he was unable to localize in his system of classifi-
cation.

In 1857 Naegelicollected all the forms then known, which
had certain characteristic physiological features in common,
into a group which he termed Schizomycetes or fission fungi,
a group which is now fully recognized by botanical mor-
phologists and physiologists. He included all those lower
forms of plant-life in which chlorophyll was absent, and
which contained carbon, oxygen, hydrogen, and nitrogen in
definite proportions, which elements they were not able to
assimilate and utilize in building up their own substance
from inorganic materials. They, like the other fungi and
animals, can utilize as food only such material as is presented
to them in the form of living or dead organic matter held
in solution, or combined with a,considerable quantity of
water. The processes going on within their protoplasm are
so intimately connected with oxidization that they usually set
free no uncombined oxygen, and this characteristic feature
along with their want of chlorophyll colouring matter Naegeli
looked upon as the special feature by which they might be dis-
tinguished from ordinary fungi. Amongst his fission fungi
he placed the forms bacterium, vibrio, spirillum, sarcina, the
mother of vinegar, the yeast fungus, the organism associated
with the silkworm disease—a small colourless oval organism,
somewhat resembling a yeast, which he named Nosema
bombycis—most of which were characterized by the features
which are now recognized as belonging to the fission fungus
group. The relation of these to disease and fermentation
Naegeli declined to discuss.

Although Leeuwenhoek had described certain micro-
organisms in the tartar of the teeth and in various secre-
tions and excretions so accurately and minutely, it was not
until 1837 that any definite attempt was made to associate
them with the products of a disease; in that year, how-
ever, Donné described an Infusorian, which he likened
the vibrio lineola of Miller, as occurring in pus in
syphilitic diseases. This he thought at first was simply
a vibrio associated with the putrefaction of the pus,

64 BACTERIA.

but as he was afterwards unable to find this same infu-
sorian in the pus taken from other abscesses, and in putre-
fying material that had been exposed to air, he was led to
inquire whether it was really characteristic of syphilitic
contagion, and whether it played any part in the trans-
mission of syphilitic infection. Although he afterwards
retired from his first position he carried out a series of
most careful and ingenious experiments, through which he
was Jed to believe that very characteristic vibriones were
associated with the causation and transmission of syphilis,
and he thus opened up a series of most interesting questions,
the answers to which, though differing somewhat from those
given by Donné himself, were nevertheless destined ulti-
mately to be very much on the same lines as those that he
had laid down.

It is interesting to notice that the monad forms which at the present
day are again coming prominently forward in connection with the produc-
tion of certain diseased conditions, were early recognized and described by
Donné, and that Rudolph Wagner also described a species of monad as
occurring in cancer of the lip.

As was to be expected, however, the connection of micro-
organisms with fermentation was proved long before a
similar association was made out between micro-organisms
and disease, and in the same year that Donné published his
results, Cagniard-Latour and Schwann, who had been work-
ing independently, announced that the yeast cells (torula
cerevisiz), originally described by Leeuwenhoek, and which
were found to grow in grape juice and malt wort were to be
associated with fermentation—that they were indeed the
cause of this process.

For long, although the intimate connection between the
process of fermentation and specific infective diseases was
widely recognized, the efforts of most scientific observers
were directed towards the elucidation of the causes of fer-
mentation and putrefaction. It was, in fact, suggested that
cholera might be due to the action of some ferment-causing
organism, which might become lodged in and multiply in
the intestine.

In 1837, moreover, Bassi described a kind of yeast fungus,
which he thought must be the cause of a miasmatic con-
tagious disease in silkworms. He found extremely minute
spores, on and within the bodies of the silkworms affected,

THE HISTORY OF BACTERIOLOGY. 65

and although parasites had long been accepted as the cause
of certain diseases in plants, this was really the first fully-
described and well-authenticated instance of a fungus para-
site giving rise to disease in any members of the animal
kingdom.

As the direct outcome of these researches, Henle, in 1840,
was led to believe that the cause of miasmatic, infective and
contagious diseases must be looked for in living fungi or
other minute living organisms, and although he was unable,
experimentally, to satisfy himself of the accuracy of his
position, he was fully convinced that he was working in the
right direction. It would indeed have been difficult, at that
period, to satisfy every condition that he required to be
fulfilled; the methods now in use were then unknown
and have only been perfected by workers as it has been
found necessary, from time to time, to comply in the most
minute detail with Henle’s conditions, and as, one point
being carried, it has been found necessary to advance on
others. The first of these was that a specific organism should
always be associated with the disease under consideration.
As such presence, however, might be accidental, these
organisms were to be found not only in pus but actually in
the living body. As they might be, even then, merely
parasitic, aud not associated directly with the causation of
the disease, it would be necessary to isolate the germs, the
contagium organisms and the contagium fluids, and to
experiment with these separately with special reference to
their power of producing similar disease in other animals.
We now know that it has only been by strict compliance
with all these conditions, again postulated by Koch, that the
most brilliant scientific observers and experimentalists in
England, France, and Germany have been able to determine
the causal connection between micro-organisms and disease.
After Henle’s work appeared a regular fungus fever set in;
many skin diseases were proved to be the result of the
action of fungi, and numerous internal diseases were, on
very imperfect evidence, said to be due to parasitic agency ;
and a large number of diseases which we now consider to be
caused by the action of certain specific organisms in the
system, were deemed, on very imperfect data however, to
be due to these fungi. Cholera and typhoid fever became
subjects of great interest, the stools from patients suffering

6

66 BACTERIA.

from these diseases were carefully examined for organisms,
and bacteria and monads were carefully described, but in
no case could any definite proof be obtained that any of
_these stood in any causal relation to either of these diseases.

It was at this stage that Pasteur took up the work initiated
by Cagniard-Latour and Schwann, who had first noted the
connection between the growth of the yeast organism and
fermentation. He applied to other processes of fermentation,
such as those of lactic acid, butyric acid, and acetic acid, the
same process of experiment and reasoning which they had fol-
lowed, and he was able eventually to prove that the organic
ferment in each case had specific characteristics, not only as
regards its physiological action in setting up acertain definite
form of fermentation, but also as regards the special mor-
phology and mode of growth of the organisms that were
found during and at the end of the process.

As these experiments of Pasteur’s are now classical it
may be well briefly to indicate the lines on which he worked.
He first carefully observed the nature of the organic material
in which certain fermentations took place, studying, both
synthetically and analytically, the best medium for his
purpose ; he then, by careful microscopical study, determined
what organisms developed most rapidly during the special
fermentation process. After making an artificial solution of
the substance to be fermented, he added a small quantity of
albuminoid material, and a trace of the ash of the special
yeast that he wished to grow, in order that there might be
sufficient of the necessary salts for the nutrition of the
organism. This fluid was carefully sterilized by being boiled
in flasks, to which only filtered air afterwards had access.
To the germ-free solution he added a small quantity of his
special yeast, and if he then obtained a characteristic
fermentation with the production of the natural special
fermentation products, accompanied by rapid growth and
multiplication of the organism that he had introduced, he
came to the conclusion that this organism was the cause of
the special fermentation.

In 1857 Pasteur described a new yeast (the cells of which
were much smaller than the ordinary beer yeast), which gave
rise to the formation of lactic acid from sugar, and he pointed
out that the nitrogenous material which was necessary for
the production of lactic acid was really needed for the

THE HISTORY OF BACTERIOLOGY. 67

nutrition of the growing yeast, but that otherwise it did
not exert any influence in transforming the sugar into lactic
acid, as had hitherto been maintained by Liebig.

Some idea of the delicacy of Pasteur’s experiments may be gathered
from his observations on the conversion of racemic or paratartaric acid by
a living ferment into right-tartaric acid (which in turn was capable of
undergoing further fermentation) and into left-tartaric acid, which remained
unaltered. He had already noted that the material in which fermentative
changes took place, determined, in a very marked degree, the nature of the
fermentation process. For instance, on adding dust, which of course con-
tained a considerable number of different organisms, to sterilized urine, an
ammoniacal or putrefactive fermentation took place; whilst on adding the
same dust to sterilized milk an acid fermentation, as evidenced by the
curdling of the milk, ensued, whilst in each case there seemed to be a
special development of one particular organism. He had of course to
contend with the difficulties involved in obtaining isolated organisms or
pure cultivations, and for this reason he was for some time heavily handi-

capped.

All the forms of ferment-producing organisms which
Pasteur had studied up to this point he spoke of as
vegetable or yeast forms, and it was long before he was
able to induce butyric acid fermentation. He at length
found, however, a form which he distinguished as an
infusiorian, in contradistinction to the vegetable or yeast
form. This he describes as occurring in the form of small,
straight, cylindrical rods, somewhat rounded at the ends,
occurring either singly or in jointed chains of three, four,
or more, about 24 broad and from two to ten times as
long as broad. The organism has a slight gliding motion,
and its reproduction takes place apparently by vegetative
growth and transverse division. The physiological or bio-
logical peculiarity of this organism is that it can exist
apparently without a trace of organic nitrogen ; whilst, like
the vegetable ferments with which Pasteur had been working,
it can also live without oxygen, and although in form it is
like a vibrio, it differs from the vibrios in this respect and
also in that it is able to bring about fermentation.

In 1863 he found a second anzrobic “ vibrio,” which he
succeeded in cultivating. He prepared a solution contain-
ing tartarate of lime, ammonia, potassium, and yeast ash,
rendered it sterile or germ-free by boiling, and covered it
with a thick layer of oil. To the fluid thus prepared he
added a minute quantity of the organic deposit resulting
from spontaneous fermentation of tartarate of lime ; the

68 . BACTERIA.

was followed by a typical fermentation in the fluid, from
which air was excluded by the film of oil spread over the
surface. On microscopical examination of both the artificial
and the spontaneous fermentations, he found “ vibriones”
Ip thick and sop long. It was objected that in the
spontaneous fermentation a certain quantity of air must
necessarily remain in the fluid in which the organism was
growing, so that the presence of oxygen could not be
inimical to the growth of this “anzrobic” organism (a
name that Pasteur gave to those organisms that are not
dependent upon the oxygen of the air for their growth and
development). He met this objection with proof that,
grown along with other organisms, such as bacterium
termo, which were dependent upon free oxygen for their
existence, the latter developed and grew for a short time in
the fluid and so used up what oxygen there was present
in solution, at which point they were unable to develop
further, so that other resulting changes must be due to the
growth of the special anerobic organism. Passing from
the butyric acid fermentation, and taking it as an analogy,
Pasteur continued his researches on putrefactive processes
in which nitrogenous substances and acrid and offensive
smelling materials are formed, and he eventually came to
the conclusion, which had already been expressed by Mit-
scherlich in 1843, that as yeasts gave rise to fermentation so
“ vibriones ” must be the cause of putrefaction ; and, going
further, he assumed, what has since been proved to be
erroneous, that the whole of the vibriones of putrefaction
were anzrobic, that is, they could give rise to their specific
products only when they were removed from the influence
of the action of the oxygen of the air.

In connection with the subject of anzrobiosis, Pasteur pointed out that
the mycoderms known as “ flower of wine,” ‘flower of vinegar,” &c.,
were able to produce different forms of fermentation according to the
presence or absence of oxygen. Mycoderma aceti, for instance, bringing
about the splitting up of sugar into alcohol, z.e., setting up an alcoholic
fermentation when there is too little oxygen present, but in presence of
abundance of oxygen giving rise to the formation of acetic acid, then setting
up what is known as the acetic fermentation.

These researches eventually led Pasteur to the conclusion
well stated by Duclaux, that ‘‘ whenever and wherever there
is decomposition of organic matter, whether it be the case

THE HISTORY OF BACTERIOLOGY. 69

of a herb or an oak, of a worm or a whale, the work is
exclusively done by infinitely small organisms.“ They are
the important, almost the only, agents of universal hygiene ;
they clear away more quickly than the dogs of Constantinople
or the wild beasts of the desert, the remains of all that has
had life ; they protect the living against the dead; they do
more : if there are still living beings, if, since the hundreds
of centuries the world has been inhabited, life continues, it
is to them we owe it.” Without them the surface of the
earth would be covered with dead organic matter, the
remains of plant and animal bodies, which, retaining the
elements necessary for the building up of new plant-life
and animal bodies, would soon cut off the food supply of
new plants and animals; life would be impossible because
the work of death would be incomplete, or, as Pasteur puts
it, “because the return to the atmosphere and to the
mineral kingdom of all that which has ceased to live would
be totally suspended.” From his experiments on fermenta-
tion and putrefaction Pasteur, by a very natural transition,
turned his attention to the diseases of wine, and then to
those of the silkworm—diseases that specially affected two
important French industries. After most careful research
he found that the acetic fermentation, viscosity, bitterness
and turning flat of wines, were all due to the action of
certain organized ferments, most of which he was able to
isolate and study. ;

The acid fermentation, he found, was produced by the mycoderma aceti,
which consists of short rod-like forms, about double as long as broad,
slightly constricted in the middle; these individual elements, being joined
together in long chains, which, as they grow on the surface of the wine,
interlace with one another and form a regular film or skin. The bitterness,
of wine he ascribed to the presence and action df branched tortuous
filaments of about 1.54 to 4 in diameter, these chains‘ having a
peculiar knotted appearance. The turning flat of wine was due to the
presence of delicate unbranched filaments about Ip in diameter, which
under certain conditions are broken up into short segments which some-
what resemble the bacilli met with in a lactic acid fermentation. The
cause of the viscosity of wine was an organism made up of cocci about
1.2 in diameter, often arranged in chains of considerable length. He
found, indeed, that the relations of cause and effect were invariable ; wher-
ever certain forms were present in addition to the yeasts, or were intro-
duced after the yeasts, the result was a special fermentation superadded to
the wine fermentation. :

The genius who had shed such a flood of light on the

70 BACTERIA.

causation and prevention of wine disease now turned his
attention to the ravages of the “spot ” disease or “ pebrine”
amongst silkworms, a disease that at one time threatened
to destroy the flourishing silk industries of France. The
organisms found in this disease had already been described
by Naegeli as Vosema Bombyczs, and by Latour as Panhis-
tophyton, small, glistening, oval corpuscles which appeared to
-be endowed with life and to lead a parasitic existence in the
tissue of the silkworm caterpillars. Pasteur was able to
demonstrate their presence not only in the butterflies which
develop from these worms, but also in the eggs they laid,
and he found that where any one of the three forms was
affected, the corpuscles were passed on to the next stage.
He was able to show that they increased in the body, that
they were the cause of the disease, and that by careful ex-
amination and destruction of the affected eggs and the
preservation of the healthy ones, the disease could gradually
be eliminated. He found that another disease, the lethargy
of silkworms, was also probably caused by the presence of
micrococci, arranged in the form of chains, in the intestinal
canal of the worms. Pasteur had thus succeeded in showing
the relationship between certain micro-organisms and certain
wine diseases, but he had also been able to demonstrate a
causal relation between certain lowly-organized, parasitic
organisms and a special disease in animals or insects. He
had, in fact, demonstrated that certain specific organisms,
endowed with definite morphological and physiological
characters, gave rise by their presence to specific and
characteristic diseases ; he had by observation and experi-
ment extricated the theory of a living contagium from a
condition of chaos, and he had assigned to definite organ-
isms, each a special réle in the production of certain forms
of fermentation, of putrefaction, and of disease ; and although
much was still left, and still remains to be done, in the
identification and classification of those organisms, he had
separated a few distinct forms, and instead of assigning to
these a general and common action in the production of the
above processes, he had allotted to each one its own part.
This he was able to do with such clearness and to place his
experiments so lucidly before the scientific world, that there
could be no doubt as to his meaning ; the consequence
being that he soon secured a large following of enthusiastic

THE HISTORY OF BACTERIOLOGY. 71

workers from amongst his contemporaries. On the other
hand, however, he brought forward those who were by their
researches led in opposite directions, or who, with less perfect
methods, could not make their facts fit in with his theory,
or who could not repeat or confirm his experiments.

The most important point that he wished to demonstrate
was that which related to the specific character of the various
ferments in fruit juices. He was attacked most vigorously
‘on this subject by Lemaire, Bechamp, Hoffmann, and others,
each of whom pointed out that in any fermentation experi-
ments that he had made he had never seen a single organism
only. (He had, in fact, never been working with pure cultiva-
tions). This was undoubtedly a very forcible objection, and one
which had to be met, but one which was easily enough over-
come as methods of separation and isolation became perfected.
Bechamp, who had found what he termed granu/es in the
cells of living plants and animals, and even in fossil remains,
held that these microzymas, as he called them, remained
alive ; that they set up various forms of fermentation ; that
under different conditions of food, separation from the cell,
and external influences generally, they ran together, became
altered in shape, and underwent various changes, so giving
rise to the various forms which Pasteur had described ; all
these processes going on concurrently or subsequently to the
various changes that occurred in fermentative and pathogenic
processes. He would, however, have nothing to do with any
specific organism; he considered that all organisms were
merely the result of a new grouping and alteration of these
microzymas separated from the cells, and that they were
specifically affected by the various altered conditions in which
they found themselves when removed from the cell in which
they naturally occur. Both Pasteur’s positions were thus
attacked. His first contention was that germs were the
cause of fermentation in disease, and secondly, that each
fermentation was due to the specific action of a definite
organism. In his first contention his position was materi-
ally strengthened by the observations of Lemaire who, after
proving that the presence of carbolic acid was inimical to
the life of the higher animals and plants, carried his re-
searches a step further, and proved that the lower organ-
isms were similarly affected by the same material, and he
found that the addition of a small quantity of carbolic acid

q2 BACTERIA.

to fluids, in which putrefaction and fermentation would
ordinarily take place, prevented the incidence of these
processes. He found at the same time that the fermen-
tations set up by chemical ferments, such as diastase and
synaptase, remained entirely unaffected by the action of
carbolic acid, and the result of his earlier experiments
led him to believe that the process of fermentation was
due to the action of living organized creatures which, like
the higher plants and animals, could be killed by the
carbolic acid. When they were allowed to develop freely
they brought about fermentation ; when their growth was
stopped, or they were killed, fermentation could not go on.
The same reasoning, he thought, might be applied to
infection and miasma, and he concluded that disease pro-
cesses were the result of fermentations or decompositions
going on within the tissues, and brought about by the
above or similar organisms. Pus formation was the result
of the action of germs falling from the surrounding air
into a wound. By the application of his germicidal re-
agents to wounds, vaccine vesicles, and suppurating
surfaces, he attempted to destroy these organisms out-
side the body whilst they were actually attacking the weak
points.

This was really the first step in the direction of an anti-
septic treatment of wounds. Whilst treating by his method
wounds in the human subject and in the dog he saw “ that
pus remained entirely absent, or was reduced to a minimum,
putrid alterations were absent,” and the wound healed
rapidly. All these results were due, he maintained, to the
destruction of the microzoa or infusoria by his carbolic acid
lotions. The paramount importance of this theory was only
afterwards fully appreciated and worked out by Lister, who
saw that, owing to the difficulty of killing germs after they
had once made their way into the tissues, it was absolutely
necessary that such organisms should be prevented from
gaining access to the wounds at all, and it is upon the
attainment of this end that his well-known antiseptic treat-
ment depends for its success.

Accepting the truth of the statement that germs were the
cause of fermentation, Lister also came to the conclusion,
independently, that germs entering the wounds from outside
might be the cause of suppuration, and since germs were

THE HISTORY OF BACTERIOLOGY. 73

floating in the air, were suspended in water, and were
attached to the instruments and bandages that were used
in the treatment of wounds, he determined that it was
necessary, by using some germicidal reagent, to kill all such
suspended and adherent organisms before the various
materials mentioned were allowed to come in contact with
the wounded tissues. With a combination of experimental
resource, patience, and brilliancy almost unparalleled in the
history of surgical science, he, step by step, built up a theory
and practice of antiseptic surgery, a theory and practice
which rapidly revolutionized the treatment of wounds and
the routine of ward management. He thus introduced a
system which has affected the practice not only of those who
believe in its accuracy, but of those who cannot bring them-
selves to accept all its details, but who have nevertheless
accepted its principles, sometimes even unwittingly.

LITERATURE.

Authors already mentioned. Duclaux, Davaine, Dujardin,
Ehrenberg,* Hueppe,* Loeffler, Naegeli and Roberts.
Anpry, Nicotas.—De la génération des vers dans le corps
de homme. Amsterdam, 1701.

Bonnet.—Considération sur les corps organisés, 2 t.
Amsterdam, 1762.

Bastian.—Beginnings of Life, London, 1872 ; Proc. of the
Roy. Soc., No. 145, 1873; ature, 1873 ; Centralbl.
f. d. Med. Wissensch, p. 521, 1876 ; Linnean Soc. Journ.,
Vol. 14, 1877 ; On Fermentation and the Appearance
of Bacilli, Micrococci and Torulae in boiled fluids. Lond.,

1877.
Basst.—Del mal del segno, calcinaccio o mocardino. Milan,
1837.
CHIENE AND Ewart.—Journ. Anat. and Phys. p. 448.
April, 1878.

*CHEYNE, WATSON.—Antiseptic Surgery. London, 1882.
CAZENEUVE AND Livon.—Revue Mensuelle, p. 733, 1877.
Coun.—Beitr. zur Biol. der Pflanzen. Bd. 1, heft 3, 1875.
CAGNIARD-LATOUR.—Comptes rendus de l’Acad. des. Sc.,
t. 4, p. 905, 1873, sate
HarTsoEKER.—Essai de dioptrique. Paris, p. 226, 1694.
Horrmann.—Botanische Zeit., 5 and 6, 1860.
Henie.—Handbuch der rationnellen Pathologié. 1853.

74 BACTERIA.

Kiess.—Arch. f. Exp. Path. u. Pharm. Bd. 1, p. 206,
1874.

LEEUWENHOEK.—Omnia opera sive arcana nature ope micro-
scopiorum Exactissimorum detecta, 1722.

ListEeR.—Trans. of Roy. Soc. Edin., 1875 ; Mzcro. Journ.,
1878.

Leuse.—Zeitsch fur. Klin. Med. Bd. m1., 1881.

Mo ier. — Animalcula infusoria fluviatilia et marina,

__ Hauniae (1786).

NEEDHAM.—Observations upon the generation, composition,
and decomposition of animals and vegetable substances,
London, 1749 ; Notes sur les Nouvelles découvertes de
Spallanzani, Paris, 1768.

Pasteur.—Etudes sur la biére, 1876 ; Comptes rendus, t. L.,
p. 306, 1860; Comptes rendus, t. LVL, p. 734, 1863 ;
Annal de Chimie et de Phys., 1862-1864; Etudes sur
la Maladie des vers a soie, &c., Paris, 1870.

Louis Pasteur.—His Life and Labours. By his son-in-
law. Paris, 1883. Trans. Lady Claud Hamilton.
London, 1885.

RINDFLEISCH.—Virch. Arch. Bd. Liv., p. 397. 1872.

SPALLANZANI.—Phys. u. Math. Abhand. Leipzig, 1769.

Scuuize.—Gilberts Ann. de Phys. u. Chemie. Bd. xxxix,,

p. 836, 1836.
Scuwann.—Gilberts Ann. de Phys. u. Chemie. Bd. u.
1837.

SCHROEDER AND von Duscu.—Ann. der Chemie und Pharm.,
Bd. vxxxix., 1854; Journ. f. pract. Chemie, t. Lx1., 1854.

SANDERSON, BuRDON.— Quarterly Journ. Micr. Sctence, vol.
XL, p. $23, 1871.

TyNDALL.—Phil. Trans., 1876-77 ; Haze and Dust, ature,
Jan. 27, 1870 ; Essays on the floating matter of the air
= aan to Putrefaction and Infection, London.
1881.

VAN DER Brocx.—Annalen. der Chemie und Pharm., Bd.
CXV., 1860.

Wacner.—Arch. der Heilkunde. Bd. xv., p. 1, 1874.

Those marked * give full lists of references.

CHAPTER IV.
BAcTERIA AS THE CAusES OF DISEASE.

Anthrax—Pollender--Davaine—Rayer—Laplat and Jaillard’s Observations
controverted by Davaine—Pyzmia and Septiceemia—Salisbury on
Bacteria as the Cause of certain Fevers—Johanna Liiders’ and Hallier’s
observations on Pleomorphism or Polymorphism—Burdon Sanderson,
Hoffman, and others, state that there is no Connection between Bacteria
and the Higher Fungi—Demonstration of Infective Element in Anthrax
Blood—Bacteria found in Organs in certain Diseases—Sarcina in
Stomach—Specific Organisms—Specific Activities.

* THE bacterium which, in its relation to the cause of a specific
infective disease in man and the higher animals, has been
most thoroughly studied is the Bacillus anthracis, an organism
which, not only on account of its size but also because of its
powers of adapting itself to conditions both outside and
within the body, has been recognized comparatively easily,
and cultivated in artificial fluids and on other nutrient media
in which it grows luxuriantly, so that it has been possible to
study more or less carefully its morphological and physio-
logical characters. This bacillus was observed by Pollender
as early as 1849 in the blood from the enlarged and pulpy
spleens of cows that had succumbed to anthrax or splenic
fever. He recognizéd that the bacilli were not fragments of
broken-down vessels or coagulated fibrin, as had been sug-
gested, but that they were probably small vegetable organ-
isms similar to those described by Dujardin as “vibrio
bacillus,” and he suggested that it was quite possible that
these organisms were in some way or other associated with
the appearance of anthrax disease.

In the following year, and before Pollender’s description
had been published (his full description was published in
1855), Davaine and Rayer described motionless thread-like
organisms and rods in the blood taken from animals affected
with splenic fever. These observations were confirmed by
other observers, but it was not until 1863 that the micro-organ-

76 BACTERIA.

ism was supposed to have any definite relation to the fever.
Davaine, at that time stimulated by Pasteur’s investigations
on the relation between micro-organisms and the butyric
fermentation, was led to suggest that these rods were, in all
probability, the actual and specific cause of the disease, which
suggestion, along with his observations on certain cases of
malignant pustule that were published in the following
year, may be looked upon as the first real attempt to
demonstrate the connection between the Bacillus anthracis
and the diseases known as malignant pustule and splenic
fever. Although he did not furnish rigorous proof of the
connection he was so far successful that, except for the”
cultivation of organisms outside the body and the pro-
duction of the disease by means of the inoculation of pure
cultivations, work which Koch afterwards completed,
Davaine left no proof wanting. is
In the meantime, however, Delafond had demonstrated the
constant presence of the rodlets in the blood of animals affected
with splenic fever, and had also suggested their plant-like
nature. Davaine found that he was able to transmit the
disease to healthy animals by means of the-inoculation of
blood that contained these rods ; whilst where the rods were
absent, although the blood might be exactly the same in
other respects so far as he was able to observe, he could not
produce the disease. He found that a single drop of diseased
blood contained from eight to ten millions of these forms, and
that by diluting the blood a million times, he was still able to
produce the disease by inoculation. Subsequent observers,
especially Leplat and Jaillard, objecting to his conclusions, said
that similar rods had been found in other diseases. They
pointed out that other elements in the blood—an extremely
complex fluid—might be the cause of the infective disease, and
that the rodlets themselves might be only an accidental factor.
Leaving this safe ground, however, their further criticisms
were based on the supposition that all the rod-shaped bodies
were identical in structure and life history, and that conse-
quently they should have the same effect when introduced
into the body, and they concluded that because similar
organisms taken from vegetable infusions did not produce
anthrax, that therefore the rodlets in the blood could not be
the specific infective agent. Davaine’s answer was that their
contradiction of his statements was due entirely to misconcep-

BACTERIA AS THE CAUSES OF DISEASE. 77

tion. He pointed out the different physiological characters
of organisms taken from different media, and showed that
different conditions were essential to the existence of different
organisms, and, in connection with a number of inoculation
experiments that they had carried on (with material sent
by him, which they allowed to undergo decomposition,
and by means of which rabbits were killed but no rods were
afterwards found in their blood), that they had produced no
anthrax because the incubation period was too short, because
the splenic enlargement was absent, because the animals
underwent much more rapid putrefaction, and because the
disease was capable of being transmitted to birds—a class
which up to that time had not been found to be susceptible
to this disease. During this controversy Davaine pointed out
that Pyeemia and Septiceemia could not be produced by the
inoculation of true anthrax virus. He described the bacteridia
of anthrax as totally devoid of movement in the blood,
and carefully distinguished between them and putrefactive
bacteria ; he demonstrated how these latter could, by their
presence and activity, diminish the activity of the anthrax
bacteria, and could in turn produce a septic condition, which
was in all essential respects absolutely different from splenic
fever. He demonstrated that these vibriones were vegetable
and not animal, and recognized the most important fact that
the environment, mode of nutrition, and products of excre-
tions of the anthrax micro-organism had a most marked
influence in modifying its activity and virulence. He did not
believe in the possibility of infecting the organism of the
foetus in utero, basing his belief on Brauell’s and his own
experiments, and advanced the theory that the immunity
enjoyed by the foetus was due to the filtering action of the
placenta which, he contended, did not allow of the passage
of solid particles either from the maternal to the fcetal
circulation or in the opposite direction.

He concluded also that certain species of animals were
much more susceptible to the disease than others. He was
convinced at that time that malignant pustule, malignant
cedema and anthrax were all due to the presence of the same
organisms in animals or in man, and in fact he initiated the
whole theory of contagion from animals to man and vce
versa, and opened up the immense and fertile field of the
comparative pathology of infective diseases. In 1868 he

78 BACTERIA.

almost completed his proof of the causal relation of the
organism to the disease by the ingenious method of mixing
a drop of virulent blood with a large quantity of water, and
using the bacteria which fell as sediment as the medium
with which to inoculate susceptible animals. With this
sediment he was always successful in producing anthrax,
whilst inoculation with the water taken from near the
surface invariably gave negative results.

It was not a perfect proof, however, and it was left for
Pasteur with his filtration process, and for Koch by his
pure cultivation process on solid media, to complete the
proof that Davaine so ardently desired and worked to obtain.

In view of our latter-day knowledge of bacteria, it is
interesting to note that as late as 1870, or only twenty years
ago, these bacilli of anthrax were declared to be albuminoid
crystals ; whilst within the last ten years they have been
described as being built up from the déérzs of fibrinous
filaments.

About this time a great impetus was given to the theory
of a living contagion—an impetus which unfortunately im-
pelled numerous workers and theorizers to see the cause of
disease in every germ that they found. First, Pasteur had
formulated his germ theory of fermentation and putrefac-
tion ; Davaine, in his descriptive and controversial papers,
had insisted upon the connection between his anthrax rods and
splenic fever, and in the animal parasitic world the etiological
relation between trichina spiralis (a small round worm) and an
acute fever, met with especially in pigs, had been fully demon-
strated. Salisbury, in this country, thought that he had found
the organisms that were the cause of intermittent and remit-
tent fevers, of malaria, and of certain other forms of specific
disease, but he was quite unable to give proof in any single
instance. In Germany, Hallier took up the subject with
eagerness. He was led to investigate the subject of Poly-
morphism of the bacteria or fission fungi—a theory that had
been advanced by Tulasne in 1851, and had been later
worked out by Tulasne and De Bary. It was held, indeed,
that yeast was simply a form or stage in the develop-
ment of certain mould fungi, such as the Penicillia or
Aspergilli.

Pasteur had already pointed out the physiological likeness
between the yeasts and the bacteria in their power of pro-

BACTERIA AS THE CAUSES OF DISEASE. 79

ducing fermentation processes, and Hallier, who was well
acquainted with these researches, concluded that if the yeast
cells were part of a developmental cycle, the bacteria might
also be taken to represent only short resting stages of the
same or similar cycles. This was an especially seductive
theory, as up to this time the origin of these minute forms
had, as we have seen, been enshrouded in mystery, and had
provided matter for the keenest controversy.

A lady, Johanna Liders, was firmly convinced that the
lower bacteria and yeasts developed in some way or other
from the individual parts of the mycelium of certain fungi,
or from their spores. Her observations were repeated, and
there was a general concurrence of opinion that the bacteria
were derived in some way from fungi and from other higher
plant forms.

Hallier, with his isolation apparatus, which consisted
really of Schwann’s apparatus, to which an air-pump and a
cotton wadding filter were added, and his cultivation ap-
paratus, which corresponds practically to the potato-jar of
to-day, with water to take the place of bichloride of mercury
solution, came to the conclusion that the cause of almost
every infective disease was to be looked for in bacteria,
monads, and cocci, which in their turn were nothing but
forms produced during the developmental cycle of one or
other of the fungi aspergillus, penicillium, mucor, &c.

Léffler says, in summing up the results of Hallier’s re-
searches, “ He put forward the hypothesis that all contagia and
miasmata are the products of fungi or algz which alone, on
account of their small size, are able to pass through the fine
capillary vessels, and that it was only necessary, in order to
determine the nature of the original cause, first to find out the
micfococcus and then to trace it back to the fungus to which
it owed its existence.” By such new ideas, propounded with
such an air of conviction and authority, Hallier made a most

rofound impression on both the lay and scientific world.
he whole system was so simple and clear and every part
contributed so easily and naturally to form one harmonious
whole ; every assertion was so definitely supported by micro-
scopic observation and cultivation experiments that no doubt
as to the correctness of the demonstrations seemed to be

possible. : ;
It was a somewhat noteworthy fact, however, that the pent-

80 BACTERIA.

cillium occurred so frequently in his cultivations, and, as
Brefeld and others pointed out, however complete Hallier’s
isolation apparatus might be in itself, he did not take sufficient
care to prevent the entrance of the spores of these various fungi
when he introduced the micrococci and bacteria which he
wished specially to study, so that, although nothing fresh
might be added after he had introduced the seed material,
his seed material itself might be a mixture of various kinds,
and along with it he could not be sure that the spores of the
larger fungi had not entered. It was a case, said Brefeld, of
covering with a waterproof a man already drenched with
rain. So faulty, indeed, were these experiments considered
to be by Burdon Sanderson in this country, by Hoffmann,
Rindfleisch, Manassein, and Ferdinand Cohn abroad, that
these observers undertook various experiments to prove
that not only was there no connection between bacteria and
the higher fungi, but that there were actually cases in which
the micrococci did not develop into the longer rod-shaped
bacteria. Moulds could only be developed in artificially pre-
pared food solutions when the seeds or spores of moulds
were sown; whilst bacteria seeds or germs, when obtained
free from the germs of moulds, would in similar solution
give rise to the development of bacteria only. There
was soon a reaction against the whole of Hallier’s teaching,
and it was now pointed out that he had seldom or never
been able to reproduce any disease by inoculating cultiva-
tions of the organisms that he grew, and the theory of living
contagion fell into discredit, though the fact must not be
ignored that Salisbury’s and Hallier’s work led to further
consideration of many points associated with the relation
of bacteria to disease, and that eventually it exerted a
marked influence on the germ theory of disease. Hallier
undoubtedly laid great stress on the fact that a micro-
coccus was the cause of certain diseases, and he pointed out
that its extreme minuteness was in favour of its being able
to enter readily and retain firmly its position in the body.

In 1868-9 Davaine and Chauveau succeeded in demon-
strating that in all probability the infectious element
in anthrax blood (Davaine), and in glanders pus and
vaccine lymph (Chauveau), and in vaccine lymph (Burdon
Sanderson) was not merely a soluble poison, but some
solid material, such as a leucocyte, and probably a

BACTERIA AS THE CAUSES OF DISEASE. 8i

thicro-organism. ‘The blood or pus was diluted many times
with water, the sediment was washed again and again, each
time being allowed to settle at the bottom, after which the
supernatent fluid was found to have no effect in producing
any disease, whilst the sediment which contained pus organ-
isms and “fine granulations” almost invariably set up the
disease process. The virus must accordingly, these observers
thought, be a solid poison, and must be looked upon as a
particulate body. These observations thus confirmed, to a
certain extent, Hallier’s suggestion that a micrococcus or
a bacterium was the cause of most specific infective diseases.

From this time onwards a large number of observations were made on
various infectious diseases and micro-organisms. Micrococci were found in
diphtheria, in scarlet fever, in rinderpest, septicaemia, and in other specific
infective conditions, though Traube, in 1864, had made what might be con-
sidered the first practical application of what had been discovered to be an
important pathological condition when he demonstrated the fact that, if
bacteria found their way into the bladder by means of a dirty catheter, a
severe attack of inflammation of the bladder followed—an observation which
was supplemented by Klebs, who demonstrated the connection between
small abscesses in the kidney and the introduction of micro-organisms into
the bladder. But with all these records there was very little of definite
value to demonstrate the causal relationship between bacteria and disease,
and even when fragments of diphtheritic membrane and of the wall of
abscesses were introduced under the skin of an animal, and gave rise to
both local and constitutional symptoms, there was no proof Prthooming
that these were due to the micro-organism, and not to such special chemical
products as had already been separated from putrid and diseased materials.
Panum had demonstrated, in 1856, that it was possible to obtain from decay-
ing flesh infusions an extremely poisonous substance, and his results were
confirmed by numerous observers, some of whom succeeded in combining
with acids the ‘‘ basic” substance that Panum had separated ; the sul-
phate of this base when injected into frogs proved fatal, and eventually
Ziilzer and Sonnenschein prepared what they described as a septic alkaloid
which was stable in character, and in its reactions resembled most remark-
ably the vegetable alkaloids, atropin and hyoscyamin. It was natural that
as ihese materials could be separated by chemical means from diseased and
putrefying materials, they should be looked upon as the primary and real
etiological factors inthe transmission of disease. In 1871, however, Reckling-
hausen, turning his attention to bacteria, was able to show that in the organs
of patients affected with various infective diseases (such as blood-poisoning,
and puerperal fever, typhoid fever, acute articular rheumatism, gangrene of
the lung) small accumulations of micrococci were present, and that these
were probably the cause or the agent by which deposits of the abscesses or
gangrenous patches occurred in different parts of the body and in different
organs. These micrococci were described as having an exceedingly sharp
outline, as being extremely resistant to strong acids and alkalies, and, in
fact, as being in most other respects like those that had been described as

7

82 BACTERIA.

occurring in diphtheria and in abscesses of the kidney. . These results, with
some modifications and additions, were almost immediately confirmed by
Waldeyer and Weigert; Recklinghausen and Weigert concluding that these
micrococci were all in the lymphatic vessels, Waldeyer, on the other hand,
holding that some, at any rate, were contained in the blood-vessels.

In 1872 E. Klebs found in pus, organisms which he de-
scribes most graphically as rod-like bodies, the so-called
bacteria, motionless, frequently grouped in short chains, or
in longer threads. He also found numerous micro-spores,
very minute refractile organisms, whose diameter might be
at most .5n, some lying isolated and free, and having
oscillating movements, others linked together in rosary-like
threads. Having found them in the discharges he examined
granulation tissue (raw or “ proud” flesh), in lymph canals, in
spaces in the septa or partitions between muscles, in inflamed
marrow of bones, and in the ulcerating cartilage of diseased
and injured joints, in the walls of blood-vessels and in thrombi
or clots formed in the vessels and attached to their walls.
These organisms were in all cases situated near the primary
wound (the researches were carried on during the Franco-
Prussian war), but having found the micro-organisms in this
position he next traced them to the abscesses that formed
in distant internal organs in cases of pyemia. In fact,
wherever there were points of secondary disease or suppura-
tion, there he was able to demonstrate the presence of these
organisms ; whilst in very severe cases of blood-poisoning he
was actually able to demonstrate their presence in the circu-
lating blood. Inconsequence of this constant presence of the
bacteria and micrococci in these various’ disease areas, he
felt justified in concluding that the organisms he had observed
and described were the cause of the pyemic and septiceemic
conditions, and that also to their action was due the forma-
tion of pus, of abscesses, and even of ulcerative inflamma-
tions of the, vessel walls. There can be little doubt that
the ingenious experiments devised and carried out by him
and his pupils formed a groundwork on which succeeding
investigators found it possible to build up the present mag-
nificent structure. He conceived the idea of separating the
micro-organism from the poison which it produced by
means of baked clay cylinders, and found that fluid so treated,
although it gave rise to constitutional disturbance when in-
jected into the blood or under the skin, did not induce

BACTERIA AS THE CAUSES OF DISEASE. 83

suppuration, nor did it cause death : but if to this a quantity
of the micro-organisms were added, and the fluid was then
injected into dogs, these animals succumbed to a true pyemia,
accompanied by the formation of abscesses, especially near
the points of inoculation. He showed that these organisms
were not present in the normal healthy blood; he also,
by means of ingenious apparatus, which he contrived or
modified, was able to observe the actual multiplication of the
micrococci under the microscope. He introduced the method
of fractional cultivation, but not in the complete form in
which it was afterwards used by Lister and Pasteur, as he
relied on the more vigorous growth of a certain organism
that was placed in a special fluid and under special con-
ditions, rather than on the dilution and isolation of individual
organisms in small drops of fluid. Klebs was, however, able,
by his biological method, to obtain comparatively pure culti-
vations of several bacteria and micrococci, and he was the
first to distinguish the division going on in various planes.

Klebs’ anatomical investigations were confirmed by numerous observers,
Birch-Hirschfeld, Heiberg, Orth, and Hueter, the last of whom tried to
build up a system of pathology on the basis of the researches that had
already been carried out ; these, however, afforded very incomplete data on
which to work. Pyzemia was, he considered, the result of the invasion of
the tissues by micrococci, with the formation of little plugs in the vessels,
around which abscesses were developed ; septicemia was a general poison-
ing by absorption from a localized formation of bacteria in the body ; anda
third form, which he looked upon as putrid poisoning, was the result of the
absorption of poisonous matter formed by vibriones existing outside the
body. The only difficulty in the matter was that the same organism
appeared to produce very different conditions, such as pyzmia, septi-
ceemia, puerperal fever, pyelo-nephritis, typhoid fever, phthisis, small-
pox, diphtheria, cholera, rinderpest, whilst even in the healthy ‘body
organisms were sometimes found, especially in certain positions, a fact
which at that time was not reconcilable with the various theories that had
been advanced. Another objection stated was that as the bodies of patients
who had died of pysemia and septicemia putrefied rapidly, they formed an
especially good medium in which innocuous organisms might grow, and it
was maintained that organisms were found in such bodies after death,
because of the special suitability of the soil for their growth, and that they
were therefore probably rather to be looked upon as accidental concomitants
and consequences than as essential factors in the production of disease.

There now also appeared great activity in the French
school: Pasteur had shown that most of the processes of
putrefaction were due to the presence of motile, anzrobic
vibriones, whilst Davaine had observed only non-motile rod-

84 BACTERIA.

lets in the blood of animals that died from anthrax. The
latter was further able to prove to Jaillard and Laplat that
these non-motile organisms of virulent anthrax were actually
tendered inert by the growth amongst them, or alongside
of them, of motile putrefactive organisms, and that the
action of the anthrax organism was also considerably modified
by the growth of septic organisms, which, in turn, when:
introduced into an animal by inoculation, were capable of
producing a disease which might perfectly easily be distin-
guished from anthrax. The knowledge that the septic con-
dition was not due to ordinary putrefactive organisms was
further augmented by Birch-Hirschfeld, who demonstrated
that pus which when fresh was infective lost much or all of its
specific activity when putrefactive organisms were allowed to
grow in it; so that these putrefactive bacteria at any rate could
not be looked upon as playing any part in the production of
wound infection. Then specific forms began to be associated
with specific disease conditions, or kinds of putrefaction and
fermentation ; the Goodsirs described sarcina in the acid con-
tents of dilated stomachs; Trecul found his Bacillus
amylobacter, with its characteristic tadpole shape, which
gave rise to no wound infection, and numerous others were
observed, none of which could be proved to have produced.
septic conditions. An organism which produced the color-
ation met with in blue milk was described by Fuchs, and
another perfectly distinct micro-organism was described by
the same observer as giving rise to a yellow colour in
milk. That the milk was not absolutely necessary for the
nutrition of these organisms he proved by making artificial
cultivations on other media, and he gave very fully the
conditions under which they could continue to exist ; they
were destroyed by a temperature of 50° R. to 55° R.; freez-
ing did not interfere with their power of propagation when
again thawed, and after being dried and again moistened
they were able to develop in milk and to give rise to the
characteristic coloration.

The way was thus being prepared for the discovery that
specific organisms had, under certain conditions, specific
actions and activities. If a peculiar organism was found
to be associated with the production of a special kind of
colouring matter, and if special fermentation and putre-
faction processes were induced by individual organisms, was

BACTERA AS THE CAUSES OF DISEASE. 8 5

it not probable, in the light of Davaine’s work, that special
diseases would ultimately be found to be associated, not all
with the same organism, but each with a special form ?

LITERATURE.

Authors already mentioned. Cohn, Chauveau, Dujardin,

Hoffmann, Klebs,* Léffler, Schwann.

BrRAvVELL.—Virchow’s Arch. Bd. 11, p. 132, 1857 ; Bd. xiv
Pp. 432, 1858.

BREFELD.—Bot. Unters ii Schimmelpilz. Leipzig, 1074.

BircH-HirscHFELp.—Archiv. der Heilkunde. Bd. xm, p.
389, 1872, and Bd. xiv., p. 193, 1873.

Davaine.—Revue Scientifique. Ser. m1, t. v., p. 78. Comp-
tes rendus, pp. 320, 351, 386. 1865.

Drtaronp.—Recueil de méd. vét. 1860. 3

pE Bary.—Untersuch. i d. Brandpilze. Berlin, 1853.
Bot. Zeitung, 1854.

Fucus.—Gurlt. u. Hertwig’s Magazin f. d. gesammte Thier-
heilkunde, Bd. vi.

Goopsir.—Fadin. Med. Journ., vol. tv. 1842.

HerserG.—Die puerperalen u. pydamischen Processe. Leip-
zig, 1873.

HvetTer.—Die allgemeine Chirurgie. Leipzig, 1873.

JAILLARD AND LapLtat.—Comptes rendus, t. LIx., p. 748,
1864 ; and t. LxI., pp. 368, 523 ; 1865.

Kocu.—Cohn’s Beitr. z. Biol. d. Pflanzen, Bd. u., p. 277,
1876.

Livers, JoHANNA.—Schultze’s Archiv. fur Mikr. Anat.,
Bd. m1., p. 317, 1867.

ManassEin.—Wiessener’s Mikr. Unters. Stuttgart. 1872.

OrtTH.—Virch. Arch. Bd. Lvut., p. 437, 1873.

PasTeuR.—Comtes rendus, t. Lu. 1861.

Panum.—Bibliotek for Laegar, 1856.

PoLLeNDER.—Casper’s Vierteljahrschrift, Bd. vim, p. 432,
1855.

RINDFLEIscH.—Virch. Arch. Bd. Liv., p. 397, 1872.

Saispury.—Amer. Journ. of Med. Sctence, Jan., 1866, and
Jan., 1868.

SanperRsoN, Burpon.—/Vature, vol. vul, p. 478. Bret.
Med. Journ., p. 119, 1878. Quart. Journ. Micro. Se.
1871.

86 BACTERIA.

TRECUL.—Comtes rendus, t. LXI., p. 156, 1865 ; and t. LXV.,
p- 927, 1867.
Puteswn—Comtee rendus. t. XXXII, p. 427-470. March,
1851.
Von RECKLINGHAUSEN.—Virch. Arch. Bd. xxx., p. 366, 1864.
WEIGERT.—Centralbl. f. d. Med. Wiss., No. 39. 1871.
ZUELZER UND SONNENSCHEIN.—Berl. Klin. Wochenschr.
No. 12, p. 121, 1869 and 1872.
* Full list of references given.

CHAPTER V.
FERMENTATION.

Fermentation ; a Key to the Whole Position—Chemical Fermentations—
Illustrations—Organic Fermentations—Alcoholic Fermentation—Re-
sult of Activity of Living Protoplasm—Lactic and Butyric Fermenta-
tions—Cagniard Latour—Schwann—Recss—Hansen—Liebig’s Theory
of Fermentation— High Beer—Low Beer—Method of obtaining Pure
Yeasts—Hansen’s Classification of the Saccharomyces—Spore Forma-
tion—Film Formation—Characters of Species of Saccharomyces—
Metschnikoff’s Monospora—Torulz.

From what has preceded it will be evident that if any light
is to be thrown on the subject of the production of organic
poisons in the course of disease a careful study of the subject
of fermentation is a necessary preliminary ; for, taken in its
widest sense, fermentation includes all those processes in
which there is the formation of special ferments and of
special products as the result of the life-history of certain
vegetable organisms. It is quite as rational to speak of a
putrefactive as of an alcoholic fermentation, and we might
even go beyond this and speak of a colour fermentation or a
disease fermentation, as the organisms by which one or the
other is started, and the conditions under which they are
carried on, have many features in common, and, in fact, do
not differ more than do the causes and conditions that are
already known as associated with true alcoholic fermenta-
tions,

Fermentation consists, essentially, in the breaking up of
chemical compounds, the molecules of which they are com-
posed being separated from one another for a brief period,
and then allowed to combine and form simpler and more
stable compounds. Owing to the setting free of such energy
as has been stored up in the highly complex fermentable

substance which is no longer required to maintain the- -

high level of combination, a certain proportion of this
energy is released in the form of heat, the temperature of

88 BACTERIA,

a fermenting fluid being found to rise without the addition
of any external heat. An object lesson may be used to
illustrate the processes of breaking down and re-combination
that go on in fermentation. Let us suppose that we have
a tall tower built up of two kinds of blocks, placed one on the
top of another ; first a large block and then three small ones,
then another large block and then three more small ones ;
let us consider that there are pulling on these blocks elastic
bands of different strengths, one very strong one between the
two large blocks, another very strong one between each pair
of the small blocks, and two sets of three weaker bands run-
ning from the larger blocks to the smaller ones; these sets
of blocks are so arranged that if left perfectly undisturbed
they will remain piled up one on another forming a tower of
considerable height. The higher the tower the more easily,
as a rule, will it be upset, though this depends on the way in
which the larger and smaller blocks are distributed in the
structure. If, now, some disturbing element comes in, and
if one of the blocks is withdrawn or is even slightly shaken,
the whole structure may collapse, even the movement
of a small top block may bring this about by altering the
tension on one of the elastic bands, so disturbing the
equilibrium of tension on the. whole tower, that the two
large blocks, with their strong elastic band, are set free
and fall to the ground ; the pairs of smaller blocks, with
their strong uniting bands, also fall to the ground, and the
other thin bands of which we have before spoken are now so
far stretched or broken that their influence in holding the
different sets of blocks together may now be left altogether
out of account. But in falling from the top of the tower to
the bottom energy of position has been lost, and in falling
and striking the ground a block may do a certain amount of
work ; if it were in connection with a series of. pulleys it
might be made to lift a certain weight ; if it were to strike
the ground with sufficient force it might produce a flash of
light, or a certain amount of heat. We are, perhaps, not
justified in stating that this is exactly analogous to what
takes place in fermentation, but it is sufficiently so to
explain the nature of the process, if we consider that the
kinds of blocks that are built into our tower are multiplied.
In the most characteristic and best form (for our purposes) of
fermentation that can be taken as an example, we have

FERMENTATION, 89

grape sugar, consisting of three kinds of blocks, six of car-
bon, twelve of hydrogen, and six of oxygen, all built into a
high tower of twenty-four blocks (sugar, C,H,,O,).- In the
process of fermentation the equilibrium is disturbed, and we
have the tall tower replaced by four smaller ones, two con-
sisting of three blocks each (2CO,, carbonic acid), and two
consisting of nine blocks each (2C,H,O, alcohol), and the
heat that is evolved in the process may be represented by
the sum of the figures representing the distances which these
various blocks have had to fall from the high tower into the
lower ones, multiplied by the figures representing the mass
of the volumes. Other features have to be taken into
account, but the above example will serve to explain part of
our meaning. But this is not all. The children’s line of
“soldiers” may be used to explain another feature: a
child arranges its wooden “bricks” in a long line, placing
_ them on end; if they are all of equal length the distances be-
tween them are also made equal ; if they are of unequal length,
they are so arranged that one block falling will just touch the
one next to it. If the end block be now so disturbed that it
loses its unstable equilibrium, and so falls as to acquire a
stable equilibrium, it not only loses its own unstable equili-
brium, but it upsets that of the brick next to it, which in
turn acts on its neighbour, and so on till the whole line is
brought to the ground. If, instead of upsetting the first brick
at once, you simply give it a slight tap and only just move it,
instead of falling over, it sways backwards and forwards fora
little but then settles into its original position ; you give
it another tap, it sways a little further, but still returns to
its erect position ; whilst if now you give it a good strong
push, or if two of you tap it at the same time, over goes the
brick, bringing along with it the whole line to a condition
of stable equilibrium. The towers or the rows of erect
bricks are fermentable substances, the disturbing forces are
those agents that bring about fermentation, and the smaller
towers or the bricks laid down in rows instead of on end are
the products of fermentation. The larger number of smaller
towers contain exactly the same elements as did the smaller
number of larger ones. They had energy of position, which
is converted into kinetic energy, exactly in proportion to the
distance that the elements have to fall; some of the energy
appearing in the form of heat, other parts being used up in

90 BACTERIA.

the rearrangement that takes place when the smaller towers
are formed. It may be asked, How does the tilting over of
the bricks bear on’all this? The first tap that you give
them may be said to represent the action of some ferment ;
the tap that your friend gives at the same time may be held
to represent one of.the conditions essential for the production
of a ferment, say the presence of a certain amount of light ;
whilst the tap that a second friend gives may be looked upon
as representing a certain degree of temperature ; it may be
a forcible tap, representing 40° C., or it may be a slighter one,
representing a temperature of 20°C. only, in which case you
do not get sufficient energy out of the ferment to start the
process. One or two only of your taps may upset certain
towers or certain lines of bricks, but other towers that have a
somewhat broader basis or a’ more definite arrangement
require the whole three forces to be applied, and these have
to be applied in certain definite proportions if the same
results are to be obtained in every case. Thus, for example,
by graduating the power of the disturbing force, the equili-
brium of your tower may be simply upset and the elastic
bands drawing in certain directions cause a rearrangement
of the blocks according to the strength with which these
bands pull; if, however, instead of simply just distributing
the equilibrium to allow of the new arrangements taking
place, your tower is struck with a sledge hammer, or over-
turned by a pistol shot, or by the explosion of a charge of
dynamite near it, the conditions are so altered that you
cannot rely upon any plan of rearrangement being adhered
to. Asaresult you simply obtain a series of disconnected
blocks or of much smaller and irregular towers. It will be
said that this regular breaking up is exactly what takes place
in the breaking up of chemical combinations. This is perfectly
true, for that is exactly what fermentation is. It is the upset-
ting of the equilibrium of unstable compounds by most deli+
cately adjusted forces which are so accurate, so constant, and
so delicate, that the stronger affinities of certain elements for
one another are allowed to act to their full extent, and regular
stable combinations are formed always in a definite manner.
Let us take first such a substance as nitrogen trichloride or
nitrogen teriodide. If a sharp blow be given toa small quantity
of either of these materials, or if either be heated to a certain
temperature, it will immediately break down into nitrogen

FERMENTATION. 9g!

and chlorine, or into nitrogen and iodine. Thus a tower
made of 2NCI,, or of two large and six small blocks, is trans-
formed into two towers, one composed of two large blocks
and one of six small ones, or rather into four towers, one
composed of the two large blocks and three each composed
of two small ones (2NCl,=N,+3 Cl,), and along with the
falling of these molecules into their lower positions there is
a setting free of energy which manifests itself in the form of
heat and light. Consider now that in your tower or in your
row of bricks you have continual oscillation going on; the
elastic bands are being continually pulled upon by certain
forces which we may say are light and heat ; there is continual
motion in every part of the tower, or the bricks standing on
end are continually oscillating backwards and forwards, but
never sufficiently far to disturb the equilibrium completely,
when suddenly a third element of disturbance sets in, a small
organism comes near and wishes to take out one of the
blocks from the tower for its own use; it seizes the time
when the oscillation is greatest, and giving a little extra pull
it removes the block, seizes on it firmly and immediately, and
the rest of the tower collapses ; or in the case of the swaying
bricks, although it has no power alone to upset the first brick
in the row, by striking it just when its oscillation is at one
extreme phase it assists light or heat, pushes this first sway-
ing brick a little further and causes the collapse of the whole
line. Bunge gives a series of examples of the breaking down
of such chemical substances into simpler materials, and shows
how certain ordinary chemical substances along with heat, or
even heat alone, may act as fermentation exciters. Thus he
points out how a blow can initiate the breaking up of nitro-
glycerine into carbonic acid, water, nitrogen, and oxygen.
Nitro-glycerine is highly unstable, not so much from the
elements which it contains as from the method of arrange-
ment of the atoms of the elements. Some oxygen has been
induced to unite with nitrogen,*a substance for-which under
ordinary circumstances it has little affinity, it having at the
same time a much stronger affinity for both carbon and
hydrogen than these have for one another ; rapid and exten-
sive oscillations are constantly going on, the slightest increase
of which must be followed by a new arrangement of mole-
cules.’ A sharp tap so increases these oscillations that the
equilibrium of the tower is disturbed, the weak bands between

92 BACTERIA.

the oxygenand the nitrogen are severed, and the free oxygen is
immediately pounced upon by the carbon and the hydrogen,
which are set free from one another, each of these elements
taking up a certain quantity of the freed oxygen; the
atoms of nitrogen having, of course, a strong affinity for one
another, combine, and a small portion of oxygen is set free.
The amount of energy released here is very great indeed,
and it is the more readily observed, and even measured,
from the fact that the process goes on rapidly and violently,
as it usually does where the resolution is that of a very
complex body, into extremely simple substances. Other
examples given by Bunge are the resolution of nitrogen
trichloride into its constituent elements by “contact with
various substances, such as phosphorus, phosphorus com-
pounds free from oxygen, selenium, arsenic, some resins
(other kinds being inert), non-volatile oils, &c.;’? and he
instances chlorate of potash, which splits up into chloride
of potash and oxygen at a certain temperature, and, in
the presence of binoxide of manganese, ferric oxide or
oxide of copper at a much lower temperature ; he says:
“The presence of this substance probably so modifies the
heat-wave, that the atoms of the chlorate of potash are more
easily thrown into responsive vibrations, and thus decom-
posed. In the same way peroxide of hydrogen decomposes
on being brought into contact with platinum, gold, silver,
binoxide of manganese, &c. In these cases it is called an
effect of contact, or a catalytic effect. We can form the
following hypothesis of the process which goes on here, as
in the cases above cited: The substance which acts ‘cata-
lytically’ exercises an attraction on one of the atoms in the
unstable molecule. It does not necessarily always unite
with the atom, but the unstable arrangement of the atoms
in the molecule is invariably altered to a stable one.”

. Now let us see what actually takes place when grape sugar,
of which we have already spoken, is being split up into
alcohol and carbonic acid. We have seen that there is a
rise of temperature quite distinct from any heat that is
applied to bring about the fermentation. It has been proved
that in some way or other the presence of the yeast-plant
has a very definite effect in starting a process of fermenta-
tion, and there are theories as to the ré/e that this yeast-
plant plays in starting the fermentation, In the first place,

FERMENTATION. 93

it can only bring about the detachment of the molecules or
bricks of a substance when the motion of the molecules of
that substance is started or extended by the action of a
certain degree of heat ; thus fermentation will not take place
unless the material to be fermented is kept at a temperature
of from 10° to 40°. This increased temperature acts, pro-
bably, in two ways: first, by increasing the motion of the
molecules as above stated, and secondly, by enabling the
protoplasm to act more energetically, by increasing the rate
and extent of molecular motion within the organism itself.
The determining motion, according to Bunge, “ might pro-
ceed from the vital functions of the cell. But it is likewise
conceivable that certain substances occur in the cell, and
that these substances act in a similar manner to the catalytic
bodies in the examples adduced above.” Heat and moisture
are both necessary factors in all processes of fermentation,
but neither of these alone can give rise to it.

Pasteur, who was really the first to understand this sub-
‘ject so far as to be able to throw light upon it for others,
looked upon “ fermentation, properly so called, as a chemical
phenomenon, co-relative with physiological actions of a pecu-
liar nature,” the elements in which the peculiar physiological
actions were manifested being spoken of as ferments which
were not dead albuminoid matter, as held by Liebig and his
school, but actually living organisms “ of a peculiar nature
in this sense, that they have the property of exercising all
the functions of their life, not excepting ‘ vegetative multi-
plication,’ without necessarily employing the oxygen of the
atmospheric air ;’’ and he thus generalizes his results :
“Guided by all these facts, I have been gradually led to
look upon fermentation as a necessary consequence or mani-
festation of life when that life takes place without the direct
combustion due to free oxygen.” This opened up exceed-
ingly wide and important questions: Was it possible that
all living plant-cells might have the power of inducing
fermentation in a more or less marked degree? and did
yeasts differ from other living cells only in the fact that
they had more marked powers of acting on certain carbo-
hydrates, and of exciting alcoholic fermentation? Experi-
ments on fruit, on barley, on leaves, all went to prove that the
elementary cells of plants possessed within themselves this
power of inducing fermentation of sugar that was already

94. BACTERIA. !

present or of sugar that was artificially introduced. By a
natural transition from the observation of vegetable cells to
a study of animal cells, we are led to consider how impor-
tant may be the part played by these latter in the digestive
tract and in the tissues of the body, especially when they are
called upon to act in conjunction with vegetable ferments,
either in’ normal or in abnormal positions. Fermentation,
then, may be looked upon as an ordinary chemical trans-
formation of certain substances taking place as the result of
the action of “ living ” cells, the nature of the fermentation and
of the substances ultimately resulting being due, firstly, to the
nature of the fermented body ; secondly, to the nature of
the organism which induces the fermentation ; and thirdly,
to the physical conditions under which the fermentation takes
place. ‘Thus the results may be extremely complicated if a
. mixture of ferments, say an alcoholic, a lactic, and a butyric, be
sowed in a single nutrient material ; but if we sow only one,
say the alcoholic, the sugar will split into alcohol and carbon
dioxide ; if we sow the butyric fermentation, butyric acid
will be formed as a result of the splitting up of the sugar ;
and soon. As will later be seen, the special name of any
fermentation serves to indicate merely that some special
product predominates. Inthe alcoholic fermentation, alcohol
is the chief product, but there are also formed as bye pro-
ducts, glycerine, succinic acid and a number of other
substances, the amount and nature of these bye products:
varying, first, with the yeast, and second, with the condi-
tions under which it is allowed to grow. The character and
aroma of beer and wine indeed depend essentially on the
formation of such bye products—compound ethers. It is of
course possible, nay, even probable, that what bacteriologically
may be termed impurities, may effect the same result, and
that special aroma and flavour may depend upon the presence
of small quantities of other organisms than the special yeast |
used. Further, the activity of the process is dependent
in a very marked degree upon the nature of the fermenting
substance, and a medium which may afford ample material
for the carrying on of one kind of fermentation is absolutely
valueless as a medium for other fermentations. The only
real difference that exists between a pure alcoholic or butyric
fermentation and the complicated fermentations which take
place in the animal body or in putrefactive processes is,

FERMENTATION, 95

that in the one we have a single ferment only, playing its
part, acting on comparatively simple and non-complicated
media, whilst in the other we have a complex substratum
for the growth of the organisms and a considerable variety
of organized ferment cells.

. Fermentation may be considered from two points of view—
first, as merely a chemical process which is started by the
products of micro-organisms or yeasts, in which it resembles
many other chemical reactions which are initiated by light,
heat, a blow, or some other molecular activity unassociated
directly with organic life ; whilst, under the second heading,
it may be looked upon as due to the action of living proto-
plasm or cells, special fermentations being induced by special
organic forms. The.soluble products of these organisms,
however, appear to play a secondary part in the process of
fermentation, some accelerating, others interfering with it.
The process appears to be associated with the necessity which
there is for the organized ferments to obtain certain elements
for their growth and development—elements which can only
be obtained under special conditions, and which, if obtained
otherwise, do not lead to the breaking down of the substance
which should be fermented in the usual fashion.

As we have already seen, the early history of bacteriology
was almost entirely associated with the work that was done in
connection with fermentation, and it was not till Cagniard-
Latour demonstrated that his yeast was made up of small cells
which appeared to be capable of reproducing themselves by
budding, that the inevitable conclusion was drawn that these
globules of yeast were really composed of vegetable proto-
plasm, and that it was in consequence of their growth and
proliferation that sugar solutions underwent the process of
fermentation with the evolution of carbonic acid gas and
the production of alcohol. Schwann and Kiitzing, indepen-
dently, arrived at the came conclusions after obtaining the
same results, and other observers? soon corroborated the
observations made by these pioneers, although the whoie
facts were not discovered at once, and the knowledge ot the
structure and life-history of the yeast-cell that we now

* Kieser, 1814 (Schweigger’s Journal, No. 12, p. 229) described spheri-
cal corpuscles, all of nearly the same size, which were transparent and
motionless, and Desmaziéres depicted yeast globules in 1826 (‘‘ Ann. des
Sciences Naturelles,” p. 4).

96 BACTERIA.

possess has only gradually been accumulated. These cells
are composed of a granular protoplasm surrounded by a
definite envelope. When these vesicles or cells are watched
during their development, growth, and multiplication, there
may be seen, at or near one or other extremity of each,
small protoplasmic bodies, which are projected beyond the
general outline of the cell, and which gradually but surely
increase in size. Ultimately there is a constriction, more
or less marked, between the original cell and the bud, and
the bud grows to the size of the parent cell; the same
process is repeated time after time, until there is formed
a chain or series of ellipsoidal or rounded yeast-cells. At
one time it was supposed that there was no development
either of spores or of mycelial chains, but, thanks to the
researches of Reess, by whom the presence of spores within
the cells of certain forms of yeast was demonstrated, and to
those of Hansen, who was able to confirm their observations
as regards spore formation, and also to demonstrate the
presence of typical chain mycelia as well as of the budding
form of mycelium, these organisms have been put into a
separate family by botanists, who have given them the name
of Saccharomyces, sugar fungi, or yeasts. These saccharo-
myces are indeed to be looked upon as fungi, for although
they are closely related to the alge in many respects they
contain no chlorophyll.

Many of the later observers made very definite statements,
founded apparently on very accurate data, that the process of
alcoholic fermentation is closely bound up in the question of
organized ferments ; nevertheless Liebig continued to defend
his doctrine of unorganized* ferments with great ingenuity
and vigour. His theory was that fermentation was the result
of “internal molecular motion which a body in the course
of decomposition communicates to other matter in which the
elements are connected by a very feeble affinity ” (Schitzen-
berger, on Fermentation, p. 40). ‘Yeasts, and in general
all animal and vegetable matter in a state of putrefaction,
will communicate to other bodies the condition of decom-
position in which they are themselves placed. The motion
which is given to their own elements by the disturbance
of equilibrium is also communicated to the elements of

* The term unorganized is not here used in its modern signification,
which will be mentioned in the next chapter.

FERMENTATION. 97

bodies which come into contact with them.” This
was nothing more than an extension of Willis’ and Stahl’'s
view of fermentation ; they held that a ferment is a body
which has a peculiar internal motion which is capable of
-being transmitted from the ferment to a fermentable matter.
So fascinating and plausible a theory, of course, received wide
recognition, and until Pasteur’s admirable demonstrations of
his theory of fermentation were made, had received very
general acceptance, especially amongst German chemists
and biologists. ‘The mechanical theory and the theory of
catalytic forces as used in the old sense have now been
laid aside, and the vitalist theory—expressed in the following
words by Turpin : “ Fermentation as effect, and vegetation
as cause, are two things inseparable in an act of decom-
position of sugar "—has taken the field against all opponents.
This theory is that living organisms build up structures
and develop energy from the materials in which they
live, and break up by their vital activity, either directly
or through a soluble ferment, the sugar in which they
grow. In this theory albuminoid material is considered
to be necessary for the process of fermentation or decom-
position only in so far as it is required for the nutrition
of the micro-organism, it being denied that nitrogeneous
elements play any such part, as that ascribed to them by
Liebig, of producing the molecular motion, which brings
about the splitting up of the sugar, by undergoing a spon-
taneous decomposition. Albuminoid material, in fact, is
merely an accompaniment of the process of fermentation—
a necessary one, no doubt, but one not in any way playing
the part of causal factor. ;
What takes place in brewing, a process which, though until
recently incompletely understood, has long been carried on
on an enormous scale in most northern countries ? Malt is
barley in which-a certain proportion of the starch of the
grain has. been converted into sugar by the process known
as “malting.” This consists essentially in moistening the
grain several times, keeping it at a temperature high enough
to promote its sprouting, during which a substance called
diastase is developed as the result of the vital activity of
the cells in the germinating grain which acting on the starch
converts it into sugar. As soon as this takes place the
sprouting is stopped by ee the temperature and then by

98 BACTERIA,

drying the grain to kill the young plant and so prevent
further sprouting. To obtain a fermentable liquid, a solution
of the sugar and of the other soluble constituents of the
malt is made in hot water; this is allowed to cool to a
temperature of about 16°C. A certain quantity of “ high”
yeast is then added to the solution, and during the pro-
cess of fermentation the temperature may run up to 18° or
20°C, After a time little bubbles of carbonic acid gas are
seen to rise, the yeast increases in quantity and gradually
‘rises to the surface, whence it is skimmed off, and may be
again used to set up fermentation, if still pure. The fluid
becomes bright, clear, and sparkling (from the presence of
carbonic acid), and contains a certain proportion of alcohol ;
whilst the sugar, if the fermentation has been properly
carried on, has almost entirely disappeared. This is what
is known as “high yeast”? fermentation. It goes on most
readily at a comparatively high temperature, and the yeast
rises to the surface as it is formed, bringing up with it
a certain proportion of the impurities contained in the
liquid, the heavier particles falling to the bottom. The
process goes on rapidly, but unless great care is taken it
is said that there is a danger that impurities may get in,
and that secondary fermentations may be set up, though this
is a position now scarcely tenable in these days of India Pale
Ales.

The “low” fermentation is brought about by a ferment
which acts more slowly, at a much lower temperature, and
through the agency of yeast-cells that sink to the bottom as
they areformed. This fermentation of beer must be allowed
to go on at a temperature of from 4° to 5° C., and the fluid
is not completely ripened until the end of about fourteen
days. This low temperature is maintained in the small
breweries by inverted cones of metal, containing ice, which
are allowed to float in the fermenting liquid ; they are kept
constantly supplied with ice, and the number used is regulated
according to the temperature of the external air. In the larger
breweries the same results are obtained by passing currents
of purified cool air over the surface of the fermenting tanks,
which, as a rule, are underground, so as to allow of the
temperature being maintained at an extremely equable
level. Formerly all beer was made by the high fermenta-
tion process, a system that still prevails in this country, but

FERMENTATION. 99.

in Germany, Austria, and Scandanavia, and also in France,
the low fermentation has now almost entirely ousted the high
form. There can be little doubt that this is due, in part at
any rate, to the impetus that Pasteur’s studies gave to the
subject of the careful examination of yeast ; he pointed out
that at the higher temperature there was greater danger of
contamination by organisms which produce other fermenta-
tions than the alcoholic, and that these micro-organisms
unable to flourish at the lower temperature, would at the
higher temperature grow most luxuriantly. This of course is
undoubtedly true, but it should be remembered that where
the brewing is in the hands of men in a smail way of business
the conditions are very different from those where all the
apparatus and skill that capital can command are at the
disposal of the brewer. If it were once known which was the
best kind of yeast for high beer fermentation it would be
possible to obtain pure cultivations, and with it so to conduct
the beer fermentation by attending most carefully to the
cleanliness of the vessels in which beer is made and stored as
to obtain a very pure beer, having excluded all organisms that
by their fermentation might render itunsound. ‘The low beer
may be brewed in smaller quantities and under less favourable
conditions as regards the possibility of keeping the yeast pure
than at higher temperatures. The yeast then used causes the
fermentation to go on at such a low temperature, that a large
number of the organisms which usually giverise to impurity in
the beer cannot multiply. Such beer, however, can only be
stored when the temperature is kept very low, for as soon as
this begins to rise the dormant spores of other organisms
begin to develop, set up various acid fermentations and spoil
the beer. The different methods of brewing are also deter-
mined in part at any rate by the climates of the different
countries the cool, light beer apparently being more palatable
in warm continental countries; the heavier, with its own
peculiar flavour, being usually more sought after in this
country.

Given pure yeasts, thorough cleanliness, and means of
keeping out other organisms, both beers may be kept sound
even though the temperature be comparatively high. To
-obtain such pure yeast it is necessary first to take a single
cell, and from this to grow a series of buds and chains in a
sterilized wort, to break this up into separate portions of seed

100 BACTERIA,

material, to produce other crops from this, and so on until a
sufficient quantity of pure yeast is obtained to bring about
the special fermentation in the large mass of wort. This
method has another very great advantage—it enables an
investigator to take a single yeast-cell and to follow it in its
life-history ; in doing this Hansen completed the work that
Pasteur had commenced.

As the process of obtaining pure cultures is of great interest not only to
scientific investigators but also to practical men, we may here give it in brief.*
In a Pasteur flask containing wort, a cultivation of the yeast to be experi-
mented on is started and carried on as vigorously as possible. A quantity
of water, that has previously been sterilized by boiling, is added to the
growth ; the yeast-cells in a drop of given size are then counted under the
microscope. Let us suppose that ten cells are found ; a drop of similar size
is then transferred to a flask containing a known volume, say 20 c.c. of
sterilized water, so that we have one yeast-cell for each 2 c.c. of water.
The flask containing the 20 c.c. of water with the added 10 yeast-cells,
is now thoroughly shaken and then divided equally, 1 c.c. being placed
in each of twenty flasks containing sterilized wort.? If the separation has
been complete ten out of twenty flasks should contain one organism each ;
but this of course cannot be absolutely depended upon. After carrying
the process to this stage Hansen shakes the flasks very vigorously, by
which he separates the cells as far as possible from one another, puts them
in an incubator, and allows them to remain perfectly still, so that the cells
may sink to the bottom or become attached to the walls of the flask. If
there are more cells than one in any flask, they should have been completely
separated by the final shaking, and each will probably take up a separate
position on the wall or bottom of the flask, and at the end of several days
“the flasks are carefully lifted and examined, and it is noted whether one
or more white specks have been formed on the walls of the glass ; if only one
such speck is found we have thena pure culture.” This method is especially
useful in the case where the yeast-plants are at all weakly, as all yeasts grow
much more luxuriantly and strongly in a fluid medium than they do on gelatine
plates, even when wort is added to the gelatine in order to render it more
suitable for the nutrition of the yeast. Where the species are mixed, but
where the individuals that we desire to separate are vigorous, the gelatine
method may be used with advantage, especially as the individual colonies
can then be examined from time to time, or continuously observed under
the microscope as they pass through their various phases of development.

The characteristic appearances that were said at one time
to belong to the yeast-plants have been proved by Hansen
to be really non-existent, except in a very limited sense.
He maintains that the shape and relative size of the cells
and the appearance of the protoplasm of a yeast-plant are

* For the method of carrying on the process on a large scale see Jérgen-

sen’s work. : . ~
2 1 c.c. or one cubic centimetre = about 16 minims.

FERMENTATION. IoI

not really specific characteristics. He finds that within
certain limits the same species, under different external
conditions, may exhibit very different appearances, but he
also holds, and brings forward very strong proof in support
of his position, that there are limits to the influence which
can be exerted on the cells of a species, and that different
species exhibit very different characteristics when placed
under similar conditions ; thus, for example, Saccharomyces

/

Thotomicrograph of Saccharomyces Cerevisiz. x 500. Mother cells with small
buds coming off. Yeast from an Edinburgh Brewery.
cerevisiz, when developed in the ordinary manner and then
grown vigorously for twenty-four hours at a temperature of
27° C., exhibits the ordinary appearance of rounded or ovoid
cells with a formation of septa in some cases, and with more
or less well-developed cells in others; whilst this same
organism, when cultivated at 7.5° C., occurs in the form of
dense colonies with beautiful mycelium-like branchings.

102 BACTERIA.

As Hansen’s work is the mostrecent and most complete that
has yet been carried out, we may give a short account of
the species that he describes in his classification as quoted by
Zopf. He divides the yeasts into three groups : Saccharomyces
cerevisiz, 11-5# most frequently 8-6 in diameter, which
contains a single species only ; Sacch. Pastorianus, 17-2.5p
average diameter 8—7p, in which he describes three species,
all of them oblong or sausage-shaped ; whilst the third group,
Sacch. Ellipsoideus, 13-2.5# in diameter, the most usual size
being about 8-74, in which most of the cells are oval or
rounded. These forms so overlap one another that by
mere microscopic examination it is impossible to determine
whether we are dealing with absolutely pure cultures or not.

Hansen’s researches were carried on practically, and on a
most extensive scale, in connection with a large brewing
industry at Old Carlsberg in Copenhagen, and he very
naturally turned his attention to the classification of beer
yeasts for the purpose of obtaining pure and healthy yeasts
for the production of beer.

Taking up the subject where Pasteur nad left off he,
however, again went over some of the old ground, and
studied the-various yeasts with the greatest care ; cultivated
them in all kinds of media, noted their behaviour under
different conditions as regards temperature, moisture, presence
or absence of oxygen and the like; and as the result of
prolonged and ingenious experimental work he was able to
dispel much of the inaccuracy that had grown up around the
subject, and to give detailed accounts of the life histories of
the saccharomyces. He found that the older observers had
not understood the conditions under which spore formation
occurs ; that it was not possible, as Reess had stated, to classify
the saccharomyce by a mere microscopic examination, in
which the characters relied upon were necessarily merely the
form and size of the cells and spores. He also pointed out
that even Reess’ observations on the conditions under which
spores were formed were not to be relied upon. That they
were not the result of the yeast being attacked by mould
fungi or bacteria, as Reess and Van Tieghem had, maintained,
both of whom believed that spores were evidence of disease‘
in the yeast-cells, he was soon convinced. Nor could he
believe that Wiesner and Brefeld, who made very careful
study of yeasts, without, however, enjoying the advantages of

FERMENTATION. 103

methods for obtaining pure cultivations, were right. These
observers had ascribed to certain forms of yeasts the power
of forming spores; whilst in others the same power was
denied, Wiesner holding that spores could not develop from
pressed yeast, although they could from beer yeast, and
Brefeld maintaining that cultivated yeast had lost its power
of forming spores, whilst the wild yeast still retained this
faculty. To evolve some kind of order from this confusion
was the task that Hansen set himself ; he wished to determine
the conditions under which ascospores could be formed.

For this purpose he used the method of complete aeration that is obtained
by the use of Engel’s gypsum blocks. To well-baked plaster of Paris add
distilled water until the plaster is nearly liquid; pour this on to a sterilized -
glass plate, on which rests a small mould of thin metal or paper, made rather
less than the vessel in which the experiment is to be carried on. These
blocks are first thoroughly sterilized by means of heat, after which a small
particle of yeast is placed on the upper surface of one of them, an air
chamber is sterilized, and in this a small vessel containing water, in
which the block rests, is kept. The whole vessel may be placed in an
incubator if any special temperature is required, or it may be left at the
ordinary temperature of the room.

He found that the following conditions were necessary for
the perfect formation of spores: a plentiful supply of air
(oxygen) and moisture; a certain temperature—the most
suitable for the six forms that he examined being about 25°
C.; a young condition of the protoplasm of the yeast-cells,
the older cells with their thickened walls appearing seldom,
if ever, to give rise to spores. As regards temperature, he
found that the extremes, at which the individuals of the
different species grow, vary somewhat, the lowest tempera-
ture at which they are developed being from .5° to 3° C. ;
whilst at the other extreme he found that they could still
grow at a temperature of 37.5° C. He found that spores
became visible as irregular bodies formed from the cell
contents in a period of about thirty hours from the com-
mencement of the sowing of the yeast-cells when the
temperature was kept near the higher extreme, or as low
down as 25° C.; but working with lower temperatures,
differences occur in the different species, though in none of
‘them does the development of spores take place so rapidly
as at the higher temperatures ; for example, he found that at
11.5° C. the Saccharomyces cerevisize does not form spores
until a period of ten days has elapsed ; whilst, on the other

104 BACTERIA.

hand, Saccharomyces Pastorianus II., kept under exactly the
same conditions, gives evidence of the commencement of
spore formation after seventy-seven hours ; z.e., supposing that
the previous conditions have been the same for the species
experimented upon. ;

This single observation proved to be of enormous impor-
tance to brewers, for Hansen’s pupils, Holm and Poulsen,
determined that it was possible by Hansen’s method to show
that pure cultivated yeasts produced spores at a much later
date—under similar conditions—than did those saccha-
romyces that were capable of producing certain diseases in
beer when present in proportions of jth of the yeasts intro-
duced ; and as it was found possible to determine by this
method the presence of wild yeasts when they occurred in
the proportion of 44th of the whole of the yeast used, it was
an easy matter to determine within a very short time whether
a yeast was fit to be used for brewing purposes or not; in the
case in point, forty hours at the temperature of 25° C. was
sufficient for the purpose. It was found, indeed, that all

‘the bottom yeasts might be separated from one another by
ae cultivations at a temperature of 15° C. and at
25°C,

Having obtained these pure cultivations, and being so
successful with the study of the spores, Hansen turned his
attention to the other characters, by the use of which he was
able to separate in a still more definite manner the various
species of saccharomyces one from another. He subjected
to a most careful examination the films which appeared on
the surface of liquids that were undergoing fermentation.
It had been supposed that the Sacch. mycoderma was the
result of a yeast fermentation; but Hansen found that the
saccharomyces film was something quite different from the
film formed by the Sacch. mycoderma, and he came to the
conclusion that film formation must be looked upon as a
phenomenon that may be met with under certain conditions
wherever micro-organisms are or have been growing. Here
again the presence of a plentiful supply of air is an absolute
necessity ; another essential condition is a state of perfect
rest of the surface of the fluid in which the process of yeast
growth and fermentation is going on. It is interesting to note
that when carbonic acid gas is passed through the fluid and is
allowed to accumulate on the surface these films arenot formed,

FERMENTATION. 105

so that in fermenting tanks in which, of course, large quantities
of carbonic acid gas accumulate the films are not usually met
with. From this one may gather how laboratory experiments
may go wrong, or give untranslatable results, when the exact
conditions met with in nature, or on a large scale, are not
adhered to; it also explains the different results that
have been obtained by various workers, the conditions in
their experiments not always being the same. For instance,
in Chamberland or ordinary flasks covered with filter paper
the film forms will develop rapidly as soon as the primary
fermentation is completed ; whilst in the closed Pasteur
flasks, in which the air cannot be changed rapidly, the
films grow comparatively slowly.

To produce these films Hansen proceeded as follows:
Having obtained his pure cultivations, drop cultures were
made into carefully sterilized four-ounce flasks, half filled with
sterilized wort and protected from falling particles by being
covered with sterilized filter paper. As soon as the films be-
came apparent to the naked eye they were examined. They
appear, according to Hansen, as small opaque points, which
gradually increase in size and then run together, forming
irregular patches floating on the upper surface, with a con-
vexity on the side resting on the fluid. The film at length
overspreads the whole surface and becomes adherent to the
wall of the flask at the margins; on shaking the flasks much
of the film can be made to sink, but a new one forms to fill
up the gaps that are left.

These young films he divided into two groups, the first
consisting of Sacch. cerevisiz, Sacch. Pastorianus II., Sacch.
ellipsoideus IT., in none of which could he find mycelium-like
colonies (at one time it seemed that Hansen’s researches had
demonstrated this fact) ; whilst in the second group, which
included Sacch. Pastorianus I. and III. and Sacch. ellipsoideus
I., such mycelial colonies were early developed. Later
researches have tended to show that this division is very
much a matter of time, and that it cannot now be looked
upon as having any real scientific value.

As regards the temperature at which these films are
developed Sacch. cerevisie and Sacch. ellipsoideus I. are
developed between 38° and 6° C., the three Pastorianus
varieties between 34° and 3° C.; Sacch. ellipsoideus II., 38°
to 40° C., and 3° C. The film of this latter appears to be the

106 BACTERIA.

most vigorous at all temperatures, at 13° C. it develops so
quickly and vigorously that it out-distances all the others,
giving at 23° C. a film covering the whole of the surface in from
six to twelve days ; whilst the others at the same tempera-
ture only gave a much more delicate film in from three to
five weeks. The next to this as regards rapidity of the forma-
tion of the film is Sacch. Pastorianus JII., which at the
temperature of a warm room forms a film much more rapidly
than any except Sacch. ellipsoideus IT.

It may be noted, in connection with the limits of tempera-
ture at which this film formation takes place in the three
Pastorian varieties, that they cease to develop and form
spores at a temperature of 36° to 38° C., if this be con-
tinued for ten or eleven days ; whilst the other three species
all continue to grow for a much longer period.

In addition to the forms studied by Hansen others may
be mentioned. Below is appended a short classification of
the more important forms of yeasts, with some of their more
characteristic features.

The Saccharomycetes or yeast fungi are a family of
ascomycetes, divided into two very unequal groups—the
Saccharomyces of Reess and the Monaspores of Metschnikoff ;
the mode of spore formation is somewhat similar in the
two, the only difference being that in the saccharomyces the
spores are rounded and are sometimes multiple in the same
sporangium, whilst in monaspora there is in each cell a
single needle-shaped spore developed. In the first genus are
included the following forms :

1. Saccharomyces cerevist#, may be looked upon as a
typical English high yeast, some of the characteristics
of which have already been described. It grows as rounded
or slightly ellipsoidal cells, which give off small cells by
budding ; in the earlier stages of film formation there are
formed delicate mycelial-like threads, but as the film becomes
older longer and more regular threads are formed. In the
yeast-cells nuclei are frequently to be made out, especially on
being stained with osmic acid or hematoxyline. These nuclei
are very distinctly seen in old cultivations.

The development of ascospores takes place most rapidly (after twenty hours)
at 30°C., most slowly (after ten days) at from 11° to 12” C., and stops alto-
gether below this. The spores may be very distinctly seen, as they are highly
refractile, and their walls are well defined. They are usually from 2.5 to 64

FERMENTATION. 107

in diameter. Film formation takes place most rapidly (seven to ten days)

at a temperature of from 20° to 22°, most slowly (two to three months) at

6° to 7°C., and ceases altogether at 38° C., and at 5°C., at the other extreme.

Between 20° and 30° C., the cells are frequently sausage-like and irregular ;

from 6° to 15° C. the cells are usually like the parent cells in younger

cule op but in older cultivations the forms are like those already
escribed.

This species, like all those investigated by Hansen, secretes
a peculiar substance, which, acting on saccharose or crude
cane sugar, inverts it to ‘invert " sugar ; the yeast then brings
about the fermentation of this latter substance, and also of
dextrose and maltose, giving rise to the formation of alcohol
and carbonic acid gas, with an evolution of heat and great
multiplication of the yeast-cells. It does not seem to exert
any action on lactose or milk sugar. In this respect these
ferments resemble mucor racemosus, which first brings about
the inversion of cane-sugar ; it also secretes invertase, which
causes inversion of saccharose, the products of which it
ferments; it also sets up a weak fermentation in beer wort
of the maltose and dextrose present in that liquor.

Reess’ genus of Saccharomyces ellipsotdeus Hansen divides
into two—l. and II,

2. Saccharomyces ellipsotdeus I. is really a ‘‘ wild” species
of wine ferment ; in beer wort it grows as a low yeast. It is
usually rounded or ellipsoidal in shape, though it sometimes
assumes the sausage form. ‘The spores, of which two to four
are usually found in a single ascus, are from 2 to 4p in
diameter.

These spores develop most rapidly (in twenty-one hours) at 25° C., most
slowly (eleven days) at 7.5° C., and are not formed at all at 32.5°C. at the
one extreme, and at 4° C. at the other. Grown on the surface of beer wort
gelatine, its colonies form a peculiar net-work along the line of the inocula-
tion streak. The surface membrane is formed rapidly (eight to twelve
days) at a temperature of from 33° to 34° C., most slowly (sixty to ninety
days) at 6° to 7°C. ; it is always a delicate membrane, and cannot be grown
at 5° C. on the one hand and at 38° C. on the other.

The most characteristic growth takes place at from 13° to
15° C., when it occurs as a complicated branching mass,
with elongated cells, or threads, arranged in rows, with
several lateral processes coming off at the points of junction.
Secondary branches are formed at the constrictions of the
primary branches. '

It appears to exert as powerful and rapid a fermenta-

108 BACTERIA.

tion process as does the Saccharomyces cerevisize on the
various carbo-hydrates on which that ferment acts. .

3. Saccharomyces ellipsotdeus II. is also a “ wild” or wine
fermentation yeast which gives rise to the muddiness of beer.

It is essentially a low yeast, the film that forms is exceedingly
delicate ; it makes its appearance in from three to four days at a tempera-
ture of 33° to 34° C., but not for five or six months at 3°to 5°C. At
2° and at 40° C. no film forms. Young cultivations at 15° C. are
usually somewhat rounded or egg-shaped, whilst the older cultures show
longer mycelial rods, with forked transverse shoots given off at the joints.
Asci containing from two to four spores may be egg-shaped, slightly
irregular or elongated. The spores measure from 2 to 5m in diameter;
they are developed most rapidly at 29° C., most slowly at 8° C., and are
not formed at above 35° or below 4° C.

Hansen does not look upon Saccharomyces Pastortanus as
a pure species ; he divides it into three.

4. The first of these, Sacch. Pastordanus I. (Hansen), is a
wild yeast, spores of which frequently occur in the atmosphere
of breweries. It gives an unpleasant bitter taste to beer ; it,
also, is a bottom ferment, occurring as elongated ellipsoidal
or pear-shaped cells, from which small. apical or lateral
branches may be given off.

The asci are usually elongated or rounded; they may contain two
spores or multiples of two, up to eight or even more. The spores vary
very much in size from 1.5 up to 54; are developed most readily
(twenty-four hours) at a temperature of 27.5° C.; most slowly (fourteen
days) at a temperature of 3° to 4°C. The spore formation ceases at .5° C.,
and at 31.5° C. The films are usually very delicate, are developed most
readily (seven to ten days) at from 26° to 28° C., most slowly (five to six
months) at from 3° to 5° C., development ceases altogether at 34° and 2° C.
At from 3° C, to 15° C. mycelial-like threads are developed pretty freely in
this film and most irregular forms make their appearance ; many irregular
club and skittle-shaped and other forms are formed in the older films, but
fewer in the younger ones ; in these films the cells are usually smaller.

5. Saccharomyces Pastorianus YI. (Hansen) was also
separated from the air of the brewery. In gelatine made
with yeast water it grows along the line of the inoculation
streak (at 15°C. at the end of sixteen days) in the form of
colonies with smooth edges. It is a feeble top fermentation
yeast when grown in beer wort.

The sedimentary cells of this yeast are mostly elongated, but they may
be slightly rounded, varying considerably in size. " The cells found in the film
are rounded, egg-shaped, or somewhat elongated. The spores are from 2
to 5u in diameter; the asci are usually elongated and the spores occur in
multiples of two. They are formed most rapidly (twenty-seven hours)

FERMENTATION, 109g

at a temperature of 23° C., most slowly (seventeen days) at 3° to 4°C.,
and cease to be formed at 29° C. and at .5° C.

This yeast gives rise to neither cloudiness nor to any
unpleasant bitter taste. It secretes an invertase and causés
fermentation of all the carbo-hydrates that are fermented by
the other yeasts of this group. In old cultures of the films
the cells are small, very irregular in shape, and thread-like,
like the preceding.

6. Saccharomyces Pastortanus III. (Hansen) is, according
to Hansen, one of the causes of turbidity in beer. Grown on
yeast water gelatine at a temperature of 15° C., at the end of
sixteen days the colonies present peculiarly fringed edges ;
grown in wort it gives rise to a top fermentation, and causes
considerable turbidity with a production of alcohol and
carbonic acid gas.

The spore formation is very much like that in the preceding species: it takes
place most rapidly (twenty-eight hours) at 25° C., most slowly (nine days)
at 8.5° C., and ceases at 29°andat 4° C. The film appears in the form of
small flakes most rapidly (seven to ten days) at 26° to 28° C., most slowly
(five to six months) at 3° to 5°, and ceases altogether at 34° and 2°. Here
again the elongated or sausage form predominates, but large and small
rounded and oval cells are also present in the sedimentary forms in the
films at from 20° to 28° C. The cells are of much the same shape as are those
of the sedimentary yeast, but at a temperature of from 15° down to 3° C.
there are elongated mycelial-like threads which in old cultures become still
more characteristic. These mycelial-like threads are developed at the
above temperature, which is much lower than in the case of the threads in
Saccharomyces Pastorianus I., where they are most characteristic at a
temperature of 13° to 15° C. At the same temperature, 15° to 3° C., the
cells in Saccharomyces Pastorianus II. are oval and rounded.

Hansen describes in less detail a number of other ferments
which produce alcohol from sugar.

7. Saccharomyces Ludwzgiz, though found in the sap of
oaks, grows freely in yeast water, when it appears as a peculiar
caseous mass or as fungus-like specks which float in the
fluid. One great peculiarity of this form is that it may be
so modified by cultivating it in beer wort through several
generations at a temperature of 25° C. that it does not form
spores, or that it forms them but slowly. The spores,
when formed, are usually from one to four in number, but
there may be more; the cells of the film are usually con-
siderably elongated. The film formation goes on most
rapidly at about-25° C.; at the ordinary temperature of

IIo BACTERIA,

the room it goes on very slowly, taking a whole month to

form a comparatively delicate membrane. In very old: -

cultures well-marked mycelium formation may be met with
in which the cells are ellipsoidal, elongated, or sausage-
shaped, or somewhat club-shaped. It is capable of acting
on grape sugar; it inverts cane sugar and ferments it ; but
‘has no action on maltose, lactose, or dextrine in yeast water,
nor does it attack starch.

8. Saccharomyces Marxianus, named after its describer,
was first found in wine. Hansen studied it most closely.
He found that in beer it develops as small ellipsoidal
and egg-shaped cells, with here and there sausage-shaped
cells, which are often combined into colonies. There
are developed on a quiescent fluid small viscid masses, some
of which remain on the surface whilst others ‘sink to the
bottom. The film develops exceedingly slowly, but in it
are found cells which resemble very closely those of the films
of the first six species of Saccharomyces described. The
true film contains oval and short sausage-shaped cells.
The spores are not freely developed. When it is grown on
solid nutrient media spores are more frequently formed, in
which case they are usually oval or kidney-shaped. In beer
wort this yeast is not very active, nor is it able to ferment mal-
tose, but it acts vigorously on saccharose, inverting it and then
fermenting it with great activity ; it also acts upon dextrose.

9. Saccharomyces exiguus, found by Hansen in German
yeast, differs from the preceding saccharomyces in the fact
that it forms no mycelial threads on beer wort, or on solid
nutrient media. It forms spores, but sparsely, and the film
is exceedingly delicate, the cells of which this is made up
being short rod-shaped or ovoid. It acts on the sugars exactly
as does the Saccharomyces Marxianus.

10, A somewhat peculiar saccharomyces belonging to this
group is the Saccharomyces membranefactens, which forms
on beer wort a bright yellow tough scum, composed of long
and sausage-like cells, which may be closely packed together
or may occur singly. It forms spores rapidly, liquefies nutrient
gelatine, and is peculiar from the fact that it does not cause
fermentation of any of the ordinary carbo-hydrates, nor has
it any effect in inverting cane sugar.

11. Saccharomyces minor (Engel), who describes it as
spherical cells 6# in diameter, arranged in chains of 6~9

FERMENTATION. Ill

elements. The spore-forming cells are larger 7-8.5y, and
contain from 2-4 spores 3.5# in diameter. He ascribes to
this ferment the action of fermentation in bread, a notion
that has since been scouted and again accepted.

12. Saccharomyces conglomeratus (Reess), Hansen thinks
is simply a form that may be met with in old films of all the
six species that he specially investigated, and in recent works
this species has been dropped.

13. Saccharomyces apiculatus, described by Reess, can
scarcely be said to be a true saccharomyces, although
it is included amongst them by Zopf as a doubtful
member of the group; it has not yet been ascertained
to have any spore formation, and is therefore retained by
Hansen in this group only provisionally. It occurs in fer-
mented wine and spontaneously fermented beer, and, in the
hot seasons, on sweet succulent fruits, such as cherries, goose-
berries, plums, or grapes ; whilst in the winter it is found in
the soil beneath the trees that bear these summer fruits. It
occurs in cultivation fluids as lemon-shaped cells—hence the
name—though under certain conditions it assumes elongated,
crescent-shaped, and rod-shaped forms. It gives off buds of
two kinds : one oval, the other lemon-shaped. It isa bottom
yeast giving rise to a feeble alcoholic fermentation ; it does
not invert cane sugar, but acts on dextrose in yeast water,
but does not ferment it completely. Mixed with Saccharo-
myces cerivisize it retards the action of the latter.

An organism that was long classified with the true yeasts
is the Rosahefe or Pink yeast of the Germans. Accord-
ing to Hansen, however, no spores are formed during any
phase of its development and for the present he excludes it
from the Saccharomyces or true yeasts. It belongs rather to
the Torule.

Genus II. (1) Monospora (Metschnikoff). The single mem-
ber of this group, Monospora cuspidata, is of interest princi-
pally because of the elaborate researches that have been made
by Metschnikoff on its relation to a peculiar disease of the
Daphnia, a small fresh-water crustacean. It occurs as a bud-
ding mycelial thread made up of elongated cells which before
spore formation become elongated ; there then appears a long,
thin needle-like body situated in the centre of the cell in its
long axis. This spore is taken into the alimentary canal of
the Daphnia, whence it is driven through the walls by the

Ilz2 BACTERIA.

peristaltic action of the muscles of the intestine. It thus
passes into the body cavity or into other tissues, where it
is immediately attacked by the white blood corpuscles, or
by the connective tissue corpuscles ; or it may first become
developed into the rod-shaped vegetative form. When it
has once commenced to divide it is no longer attacked by the
above cells (which Metschnikoff speaks of as the phagocytes) of
the insect. If the Daphnia is in moderately good health the

Photomicrograph of Rosahefe. x 1000. Rose-coloured yeast (?) No spores
have been found, and Hansen does not classify it with the yeasts.

monospora is gradually overcome, but if the insect is feeble,
or if the monospora is ingested in very large quantities, it
multiplies so rapidly that the animal may eventually succumb
to its attacks. This form is specially interesting from the
fact that it afforded some of the strongest proofs of Metschni-
koff’s phagocyte theory that he was able to obtain.

We have already seen that the yeast-cell is usually
observed in a fermenting liquid as a rounded or ovoid body,

FERMENTATION. 113

that it gives rise to buds by sending out small processes from
its wall, and that these latter then become detached from the
mother cell. This cell consists of a distinct membrane and
of protoplasm, the former of which may be thicker or thinner
according to the age of the cell, whilst the latter may vary
very considerably. Whilst the cell is merely growing actively,
the plasma or cell contents are homogeneous and highly re-
fractile, but when it is placed in beer wort or other highly
nutritive media, it multiplies rapidly and gives rise to the
fermentation of the sugar and maltose, and the protoplasm
becomes differentiated and undergoes certain changes. Large
clear spaces, which are supposed to contain the more fluid
part of the protoplasm (vacuoles), are formed; cloudy
granular change occurs in the other protoplasm ; and larger
fat globules also make their appearance. As the cell gets
older, and consequently less active, the finely granular proto-
plasm accumulates as a thin layer inside the cell wall, whilst
the centre is occupied by clear fluid, in which are floating a
number of fatty granules and globules.

It would appear that this last is a somewhat degenerated
condition, and that it is due to imperfect nutrition, for if a
few of these cells are transferred to a fresh rich nutrient
medium, the protoplasm becomes again modified, the cloudy
granules disappear, small shoots of the clear plasma pass into
the large central cavity, small rounded vacuoles are formed
in place of this large central cavity ; these in turn become
subdivided, and eventually the whole cell is again occupied
by clear protoplasm. By appropriate staining, especially of
an older cell, a nucleus may be distinguished, whilst under
certain conditions, to be afterwards mentioned, spores or
ascospores are formed.

Other organisms which in certain respects resemble the
yeast fungi are the Torulz, which Pasteur described as being
somewhat of the nature of yeasts, but different in the fact
that they were unable to give rise to such marked alcoholic
fermentation. Hansen, however, was able to show that this
was not a sufficiently distinct characteristic, as some of the
saccharomyces give rise to the formation of little or no
alcohol, whilst, on the other hand, some of the Torulz set
up very marked alcoholic fermentation. The great point
of distinction is, that none of the Torulz, so far as has
yet been observed, are capable of producing endospores ; all

114 BACTERIA.

of them multiplying by budding, some of them also giving
rise to the formation of mycelia. It is of course quite possible
that such a classification may not hold good, as it has been
suggested that some of the torule could not be definitely
brought within it, as far as non-sporulation is concerned.
Hansen describes seven species varying in size from 1.5 to
8; some invert cane sugar; some give rise to scarcely
a trace of alcohol, whilst others produce as much as 6.2 per
cent. of alcohol. They occur as spherical or elongated cells,
and cannot be distinguished by the microscope alone from
the round cells of the different species of saccharomyces.
As a group they have not much action on maltose, and only
some of them affect dextrose.

CHAPTER VI.
FERMENTATION (continued).

Soluble Constituents of Yeast—Action of these upon Sugar—Conversion of
Glycogen inthe Liverand other tissues—Growth of Yeast-Cellsin Organic
and Inorganic Fluids—Fermentation of Fruit Juice—£robic and Anz-
robic Fermentations—Effect of Free Oxygen on Yeast-Cells—Fermen-
tation not necessarily equal toGrowth of Yeast—Enzyme or Unorganized
Ferment a Secondary Function—Function depends partly on Organism,
partly on Medium in which it is Growing—Peptonizing Function
usually requires presence of Oxygen—Various kinds of Fermentation :
Lactic, Urea, Butyric, Ammoniacal, Acetic—Formation of Fatty
Acids—Mycoderma Aceti—General Processes of Fermentation—
Hoppe-Seyler’s Classification.

On approaching the subject of fermentation by yeast we find
at the very outset that Pasteur and others had noticed a
very remarkable peculiarity of the water in which yeast had
been mixed, and from which it had again been separated.

It was observed that a certain substance, perfectly soluble in
water but precipitable by alcohol, had the peculiar property
of inverting saccharose into equal quantities of dextrose and
levulose, and it was at once assumed that this invertin,
invertase, or some similar substance, produced through the
vital activity of the yeast-cells, was necessary to bring about
conversion of starch into sugar (diastase), or of saccharose into
invert sugar (invertin), before the cells could bring about a true
fermentation, or splitting up and hydration into alcohol
and carbonic acid gas, with certain other products to be
mentioned immediately. It was noticed by Bertholot and
Hoppe-Seyler, moreover, that, even if the living organisms
were first killed by the addition of ether, the invertase or
invertin still continued to act, and was able to invert a
quantity of saccharose altogether out of proportion to the-
amount of inverting material present. In consequence of
the latter part of this observation, many physiologists and
chemists maintain that the action of the invertin is essenti-
ally due to the setting up of a certain rate and length of

116 BACTERIA.

molecular vibration, such wave rate and iength being trans-
mitted through the whole body of material to be inverted.
In fact, that the addition of the invertin is simply the light-
ing of the spark that fires the whole train. There is some-
thing very fascinating in this theory, and it certainly explains
many, otherwise, obscure chemico-physical questions con-
nected with these two subjects—inversion and fermentation.
We see at any rate that part of the process takes place
entirely outside the yeast-cells,and may go on even when the
organisms that produce the inverting material have been killed
or completely removed. There are other similar examples
of conversion by purely chemical means, as, for instance,
where, on the addition of a dilute acid, such as sulphuric
acid at a certain temperature, starch is converted into glucose,
the heat and the acid setting up such molecular vibration
amongst the molecules composing starch, that in the dilute
acid there is a rearrangement of molecules, water is taken up,
and by hydration of the starch glucose is produced. In
this case the conversion takes place much more rapidly and
completely at a temperature of 130° C. than at 100° C.

In a similar manner glycogen may be converted into
a sugar that will reduce Fehling’s solution. This process
goes on in the liver and other organs and tissues of man and
animals, in which, probably, the secretions of the cells take
the place of the sulphuric acid and the high temperature,
or of the protoplasm of yeast and other vegetable cells.
Let us now, however, see what relation the yeast-cells
themselves (as apart from their products) are supposed to
bear to the real process of fermentation. If it were possible
to obtain an absolutely pure solution of sugar, z.e., a solution
containing no nitrogenous elements of any kind, and if we
were to place in this a minute quantity of yeast, we should
find that a very slight fermentation might take place—z.e.,
there would be an almost inappreciable diminution in the
quantity of sugar present ; a small quantity of alcohol and
carbonic acid gas would be developed, but very shortly the
process would stop, and there would be no marked increase
in the number of yeast-cells found in the whole solution. If
now we were to add a smalt quantity of nitrogen in the form
of an albuminoid substance and a certain quantity of ex-
tractives and salts, such as are found in the ashes of burnt
yeast, there would very quickly be observed a very different

FERMENTATION. 117

state of matters ; first there would be very marked turbidity
of the fluid. If we were using a high yeast this turbidity
would rapidly become more and more marked, and there
would rise to the surface a yellow scum ; bubbles of car-
bonic acid gas would be seen rising in the liquid, and a
spirituous or alcoholic taste would soon become pronounced.
If from the first fluid, z.e., the fluid in which there was
nothing but pure sugar, we were to examine the very
minute quantity of yeast, we should find that, although
there was apparently an attempt at budding in a few of the
cells, in most cases there are a series of clear globules with-
in the yeast-cells, that there is a very large proportion of thick
walled granular cells (which have certain other peculiarities),
that, in fact, throughout the whole we have evidence of very
little proliferative activity, and that young, vigorous, healthy,
yeast-cells, budding and giving rise to other cells by vegeta-
tive activity, are conspicuous by their absence. A micro-
scopic examination, in the case of the yeast growing in the
sugar solution containing a small quantity of albuminoid
material and extractives, reveals a very different state of
matters ; here the yeast-cells are in a state of extraordinary
activity, buds are being thrown off from the extremities,
terminally or laterally,the protoplasm is evidently exceedingly
active, vacuolation is conspicuous by its absence, except in
certain cells, and we are at once struck by the extraordinarily
large proportion of young vigorous cells—the more active the
process the greater the number of the new cells, and the
‘larger the quantity of yeast formed. In one case the
yeast-cells die of starvation, although large quantities of
sugar are present; and as the yeast-cells have not been
able to grow and reproduce by the exertion of their vege-
tative activity, they have not been able to resolve the
sugar into alcohol and carbonic acid ; badly nourished or
dead yeast-cells, therefore, exert little or no influence in
bringing about an alcoholic fermentation. In the other case
the cells have supplied to them all the elements necessary
for the nutrition of their protoplasm. The carbon, the
hydrogen, and the water, may all be obtained from the sugar
solution, albuminoid material of course supplies the requisite
nitrogen, whilst the ash of yeast, which has been added, con-
tains all the other elements necessary for the nutrition of
the yeast-cells,

118 BACTERIA,

Under these conditions, the cells, as we- have seen, may
undergo rapid development, growth, and multiplication, and
we have an increase in the amount of yeast, and at the same
time in the amount of alcohol and carbonic acid gas gene-
rated. These two experiments afford most exact evidence
that Liebig’s theory was essentially incorrect, and that
Pasteur's theory that true fermentation was the result of the
action of the living protoplasm, gives us the key to the whole
situation.

It was, naturally enough, objected that as the fermen-
tation process could not go on except in the presence of an
albuminoid material, this might really be the cause of the
whole process, and many most elaborate experiments were
brought forward to prove that it was this organized but
dead material that was the real and primary factor in the
process.

Pasteur, however, equal to the occasion, was able to
demonstrate in a most convincing manner, that the nitrogen
might be supplied to the organism in the form of inorganic
salts, instead of being presented as albuminoid material.
He utilized for his purposes a mixture containing 150c.c. of a
io per cent. solution of sugar candy, .5 grammes of the ash
of yeast, .2 grammes of bitartaric of ammonia, and .2
grammes of sulphate of ammonia. He found, on introducing
Saccharomyces Pastorianus into this solution; that a some-
what slow but very complete transformation of the sugar
took place, and that the nitrogen from the ammonia was used
up by the growing. yeast-cells, which at the same time
increased enormously in number. It was thus evident that
these mineral salts could take the place, in fermentable liquids,
of “ media of natural composition.” He found, however, that
the process went on more slowly, that somewhat peculiar
forms of yeast showed themselves, and that an essential factor
for the success of the experiment was that no other organisms
should be allowed to make their way into the fermenting
solutions—z.e., the absolute purity of the various materials of
which the nutrient solution was composed, and of the ferment
itself, must be guaranteed, and any relaxation of the strict
‘conditions of extreme purity was invariably followed by an
interference with the vital manifestations and physiological
actions of the yeast organisms.

It would appear, indeed, that although the fermentation

FERMENTATION. 119g

is, under such conditions, very complete if sufficient time is
allowed for the process, the yeast-cells experience a certain
difficulty in wresting the nitrogenous elements from the
inorganic ammonia salts, and that all other conditions must
be extremely favourable, in order to allow of their taking up,
and utilizing for their own use, inorganic nitrogenous material.
The presence of other organisms, for example, that have a
stronger affinity for nitrogen in this form, z.e., organisms
which are better adapted to exist under such conditions,
and which can, by their vital activity, interfere with the
growth of the yeast-cells, remove from the sphere of action
of the yeast-cells material that is absolutely necessary for their
rapid and perfect morphological and biological development,
as a result of which fermentation and hydration of the sugar
do not take place in the ordinary way ; other substances or
bye products are formed, and the decomposition of the sugar
is incomplete, or is irregularly carried out.

What, then, are the conditions necessary for the growth of
the fermenting organism in a fermentable fluid ? In the pro-
cess of wine-making it isa well-known fact that the fermenta-
tion is set up by some organism, which, though present
either as young cells or as spores on the outside of the grape,
cannot attack the juice so long as the skin remains
unbroken—a fact brought out by Davaine at a very early
stage of his researches. When, however, the grapes are
plucked, the skins are bruised and the juice is set free, the
fermenting organism,—the wine yeast or Saccharomyces
ellipsoideus, or some similar variety—utilizing the grape
juice, which contains not only sugar but also all the ele-
mentary constituents necessary for its nutrition,—grows
vigorously and sets up the vinous fermentation. It is
a remarkable fact, but one well known to wine pressers and
fermenters, that for the commencement of this vinous fer-
mentation there must be an access to free oxygen, or oxygen
mixed merely mechanically with nitrogen of the air to the
fluid that has to be fermented, as without this the yeast
spores and old yeast-cells are utterly unable to develop or to
give rise to active and vigorous yeast-cells. Pasteur insists
that we have evidence of the necessity for the presence of
such free oxygen in the fact that the fermentation of grapes
takes place much more rapidly and completely when the
grapes are left attached to the stalks of the bunches, by

120 BACTERIA,

which means there is a freer circulation of air allowed to
take place through the fermenting mass than when they are
plucked from the stalks and pressed and fermented. Of
course, it might be objected that the air is useful merely in
carrying the yeast cells into contact with the fermentable
fluid, but numerous experiments have been carried on to
prove that the presence of oxygen is absolutely necessary
for the resuscitation of old and spore-bearing yeast-cells.
This is in itself a most remarkable circumstance, and a very
significant one when it is borne in mind how very different
the conditions are under which the later stages of fermenta-
tion are best carried on. Jt must be remembered, however,
that the conditions most favourable for the multiplication
and production of a sufficient number of active cells are not
by any means the conditions most favourable for the decom-
position by the cells of the largest amounts of sugar

In our large breweries (as is sometimes brought home to us
only too closely by the reports of the death from suffocation by
carbonic acid gas, of men who go down into vats to clean them
out) there is always, as the fermentation process goes on, a very
great accumulation of carbonic acid gas on the surface of the
fermenting liquid ; so dense and so deep is this layer that it is
at once seen how impossible it is for much free oxygen from
the atmosphere to obtain access to the yeast-cells. Any
oxygen they utilize for the building up of their protoplasm
must be derived either from air held in solution in the fer-
menting liquid (which can only be a very small amount, as
the boiling of the wort must have driven out a very large
proportion of such air from the fluid), from the small amount
of oxygen that can pass through the layer by diffusion, or it
must be derived from the breaking down of those substances
rich in oxygen that are contained in the malt solution.

In the same way in the later stages of wine-making the fer-
mentation is allowed to go on in large casks ; carbonic acid gas
here also rises to the surface, fills the cask up to the bung-hole
and gradually flows over, and, as the carbonic acid gas comes
in minute bubbles to the surface from all parts of the fluid, it
can scarcely be imagined that any large amount of free oxygen
can be left in suspension in the fermenting grape juice ; and
certainly very little can find its way from without, as the car-
bonic acid gas is pouring out from the bung hole in such
considerable quantities. The fermentation at this stage, then,

FERMENTATION. 121

is going on very rapidly; there is a growth and multi-
plication of the yeast-cells, a transformation of the sugar into
alcohol and carbonic acid gas—all without the presence of free
oxygen. Pasteur set himself to reconcile these two apparently
contradictory facts, and as a result of his observations he
made one of the most important of his discoveries, important
in its relation to the life history of both fermentative and
pathogenic organisms ; and specially important because of
the parts that the zrobic and anzrobic conditions under
which organisms exist play in determining the growth of these
organisms and the spread of certain specific infective diseases.
He found that old cells with thick membranes, even con-
taining spores, were not able to multiply in media other-
wise suitable unless oxygen was present ; and that the vigour
of the rejuvenating process depended to a very great extent
on the amount of free oxygen that was contained within the
fermenting fluid. He found, in fact, that with these cells, as:
in the case of fungi and of other animal and vegetable cells,
a sudden and complete cutting off of the supply of free
oxygen immediately proved fatal to them, so that the cells
which had been exposed to the air for some time—that is, which
had acquired an zrobic habit—were rendered inactive by the
cutting off of the supply of oxygen. “Along with this he
found that the yeast organisms could multiply in liquids
containing nutrient materials for a considerable length of
time before there was any appearance of alcohol in the fluid.

After a time, however, the free oxygen being used up, it
would naturally be expected that the growth of the yeast-cells
would also come to an end, but such was not found to be the
case. The yeast-cells continued to multiply, the oxygen in the
fluid was used up and replaced by carbonic acid gas, and car-
bonic-acid gas was found on the surface, cutting off any further
oxygen supply from without. How was it that these cells
went on multiplying? Pasteur answered the question as
follows : During the gradual elimination of oxygen the yeast-
cells, having once become vigorous from the presence of a
good supply of oxygen, and of other nutrient requisites,
had acquired an activity that they did not before possess ;
they had at the same time become gradually acclimatized,
and their protoplasm had become so far altered that, as the
free oxygen was partially cut off, it was able to wrest from the
sugar what oxygen it required for the building up of its own

122 BACTERIA.

substance, and as the free supply was more and more cut off it
became gradually more able to take what it required from the
sugar solution—that is, it was gradually acclimatized. In
reading this if instead of oxygen the word energy be used it
would appear that we should have a more accurate
physiological statement. It has already been stated that, in
the presence of a free supply of oxygen, relatively less alcohol
is formed than under conditions of anzrobiosis ; it appears as
though the oxidation is too complete ; there is enough oxygen
to supply not only the wants of the living organism but also
those open bonds of combination in the various molecules that
are unsatisfied when the yeast takes for its own use certain
constituent atoms from these molecules, and as a conse-
quence very perfect oxidation is brought about and alcohol
is transformed into carbonic acid gas and.water. In the
anzrobic condition that is brought about when the oxygen
in the fluid is used up, and as the carbonic acid gas accumu-
lates on the surface in the flask or in the vat, the vigorous
yeast-cells are able to separate sufficient oxygen for their own
use from the sugar, or.sugar and water, disturbing the com-
positions of the fluid, and necessitating further rearrange-
ment of the remaining molecules ; additional oxygen can-
not be brought from without to satisfy the vacant bonds, and
other very definite products are formed. These products are
comparatively stable even when afterwards exposed to free
oxygen but not to oxygen in a nascent condition, and we have
as aresult the alcoholic fermentation. Here, in fact, are two
essentially different metabolic processes at work in the proto-
plasm of the yeast-cells. When oxygen is freely supplied
the anabolic or building-up processes predominate, a fact
evidenced by the rapid division of the yeast-cells under these
conditions ; whilst, when oxygen is excluded, the catabolic
or breaking-down processes are in the ascendant, as shown
by the larger amount of the medium decomposed, accom-
panied, however, by a slower multiplication of the yeast-cells.
Summing up, Pasteur says :—

‘ This being so, it is evident, we repeat, that to multiply in a fermentable
medium, quite out of contact with oxygen, the cells of yeasts must be
extremely young, full of life and health, and still under the influence of the
vital activity which they owe to the free oxygen which has served to form
them and which they have perhaps stored up for a time. When older they
reproduce themselves with much difficulty when deprived of air, and

FERMENTATION. 123

gradually become more languid, and if they do multiply it is in strange and
monstrous forms. A little older still, they remain absolutely inert in a
medium deprived of free oxygen. This is not because they are dead ; for in
general they may be revived in a marvellous manner in the same liquid if it
has first been aerated before they are sown. . . . At this point we must
observe—for it is a matter of great importance—that in the operations of
the brewer there is always a time when the yeasts are in this state of
vigorous youth of which we have been speaking, acquired under the in-
fluence of free oxygen, since all the worts and all the yeasts of commerce
are necessarily manipulated in contact with air, and so impregnated more or
less with oxygen. The yeast immediately seizes upon this gas and acquires
a state of freshness and activity which permits it to live afterwards out of
contact with air, and to act asa ferment. Thus, in ordinary brewery prac-
tice, we-find the yeast already formed in abundance even before the earliest
signs of external fermentation have made their appearance. In this first
phase of its existence yeast lives chiefly like an ordinary fungus.”

But as soon as the process of fermentation ends, and some-
times even before the whole of the sugar has been converted,
‘the yeast if originally not sufficiently rejuvenated gradually
loses its power of living by deriving its oxygen from its
nutrient medium, and the cells revert to their original con-
dition of senescence. They become dormant, and until again
supplied with oxygen can bring about no further fermenta-
tion. It is for this reason that to obtain the hest fermenting
yeasts, cultivations must always be made in the presence of
free oxygen, and although this was done before Pasteur
explained the reasons for-its necessity, it is now carried on in
a much more systematic manner. Brewers knew perfectly
well that they had to clear out their vats from time to time,
not only to get rid of foreign organisms, but also that they
might obtain oxidation or more perfect aeration in the early
stages of the process of fermentation.

It is very interesting to note that Pasteur, although laying
such stress on the connection between vital processes in the
cell and the process of fermentation, at the same time holds a
very definite opinion that the vegetative activities of the yeast-
cells are independent of their characters as ferments; and
he maintains that the presence of oxygen, although increas-
ing the activity of the cells as regards their subsequent
power of setting up a rapid fermentation, may, nevertheless
during its presence, give rise to weakening of their
fermenting action. He says :—

“ Free oxygen imparts to the yeast an increasing vital activity, but at the
same time gud oxygen, impairs rapidly its power as yeast inasmuch as under

124 BACTERIA.

this condition yeast approaches the state in which it can carry on its vital
process after the manner of an ordinary fungus; the mode of life, ze, in
which the ratio between the weight of sugar decomposed, and the weight of
the new cells produced, will be the same as holds generally among organ-
isms which are not ferments. In short, varying the form of expression a
little, it may be concluded, from the sum total of observed facts, that the yeast
which lives in the presence of oxygen, and can assimilate as much of that
gas as is necessary for its perfect nutrition, ceases to be a ferment at all.
Nevertheless, yeast formed under these conditions, and subsequently brought
into the presence of sugar, away front the influence of air, would decom-
pose more zz @ given time than in any other series of conditions under
which it could be placed. The reason is, that yeast which has formed in
contact with air, having the maximum of free oxygen that it can assimilate,
is fresher and possessed of greater vitality than that which has been formed
without air or with an insufficiency of air.”

In other words, when the ordinary respiratory power which
it has in common with the fungi is reduced to its lowest
point, after the protoplasm of the cell has been thoroughly
rejuvenated, its fermentive power in the absence of oxygen
reaches its maximum.

It is thus evident that the processes which go on in fermen-
tation are similar in kind to those chemical metamorphoses
that are constantly being brought about in the animal and
vegetable organisms ; the only difference being, first, in the
nature of the material that is broken up; second, in the
nature of the cells that bring about the metamorphoses ; and
third, in the exact nature of the ultimate products of the
various processes; the difference in all cases being not so
much in kind as in degree. As to the exact nature of the
process, there is much difference of opinion, some authorities
holding that it is necessary for the whole of the sugar which
is altered by the living cell of beer-yeast to penetrate or
pass, by a process of endosmosis, through the membranous
envelope of the cell and become an integral part of the
cell protoplasm, and that, unless this takes place, the
resolution of sugar into alcohol, glycerine, carbonic acid
gas, succinic acid, &c., cannot take place. It is necessary,
in fact, that the whole of the sugar should be, as it
were, digested by the yeast-cells, and combined in great
part into protoplasm before it can be converted into the
various substances above mentioned, which are, on this
assumption, merely the excretory products of the vegetable
cells feeding on a definite kind of nutrient material. We
have seen, however, that the cells of yeast secrete a definite

FERMENTATION. 125

substance, invertin, which has the power of materially
altering the carbo-hydrates to which it is added, and from
our knowledge of the functions of other cells we should be
led to expect that these cells may exert some influence on the
fermenting fluid without the whole of the sugar actually be-
coming part of the cell; or perhaps that, on the other
hand, the protoplasm may set up such molecular motion in
its immediate neighbourhood, especially at certain tempera-
tures, that a certain area of the sugar present is so acted
upon that some of its molecules are set free for the use of
the yeast-cell, and that the others can only rearrange them-
selves in a definite manner ; and that, as a result, we have
hydration and the formation of alcohol, carbonic acid gas,
and water, the most stable elements that can be formed
under the existing conditions, and out of the molecules of
oxygen, hydrogen, and carbon that are available. There
may be slight modifications giving rise to the formation of
succinic acid or other bye products, as a greater or less
number of accidental or superfluous molecules are set free
to become converted into the superfluous or additional
substances.

It is a well-known fact, that when yeast is placed under
conditions of moisture and warmth suitable for its develop-
ment, if there be sufficient nutriment present, but sugar
be withheld, or even if nitrogenous elements be kept from
from it, it becomes soft, and certain marked changes go on in
the substance of the cell.

Bechamp found under such conditions leucine and tyrosine,
both of them products of protoplasmic metamorphoses, a
soluble albuminous substance coagulable by heat, an enzyme,
a peculiar gummy substance, phosphates and acetic acid,
along with which there was of course the production of a
certain amount of alcohol, some carbonic acid gas, and pure
nitrogen.

Schitzenberger, repeating the experiments, found other
products, such as xanthine, hypoxanthine, carnine, and
guaine, that pointed most distinctly to a process of meta-
morphoses of the protoplasm, and by a series of most inge-
nious experiments he found that yeast in distilled water
lost in five days about 9 per cent. ofits protoplasm. That is,
in yeast there are certain soluble elements that can be re-
moved by washing with distilled water, leaving the insoluble

126 BACTERIA.

protoplasm, which can be filtered, calcined, and weighed ;
after the process of self-digestion, which goes on when the
yeasts are deprived of saccharine fluids, a further quantity of
9 per cent. is transformed from insoluble protoplasm into
the soluble elements above mentioned. he protoplasm
has, in fact, been living on itself, and, showing how indis-.
solubly these metabolic changes are associated with the
process of fermentation, both alcohol and carbonic acid gas
are formed in the process. Yeast, then, has the power of
disassimilating its substance by a series of steps into simpler
bodies, but under favourable conditions it may be said to
exert its metabolic power only in breaking down in a very
superficial way large quantities of the medium on which it is
grown—sugar. It is specifically adapted to break down this
sugar as far as the stage of alcohol formation, but most yeasts
cannot carry disintegration further. Other organisms, how-
ever, have the power of carrying on the process as far as the
formation of acetic acid for example, at which point another
organism may again intervene and take up the work. We
have thus the yeast forming alcohol and then dying out as
it were, then the Mycoderma aceti comes in and does its
work, and later various putrefactive organisms may continue
the breaking-down process. On the other hand it must be
remembered that whilst yeast sets up the alcoholic fermenta-
tion, Hueppe’s lactic acid bacillus gives rise to the production
from sugar of lactic acid, whilst the Bacillus amylobacter or
the bacillus of the butyric fermentation causes the sugar
to be split up directly.into butyric acid. We have here
an example of three specific actions or fermentations of the
same substance by the intervention of three different sets of
organisms, and it is quite possible that other organisms effect
an even further transformation into H,O and CO, at one
step, the organism being specially adapted at each stage for
the work that it has to carry on, or perhaps it would be
better to say that the conditions are adapted to the organism.

As might be expected, such self-digesting yeast is materially
weakened ; it is no longer in a position to absorb much
oxygen, and, if the process be long enough prolonged, both.
the power of absorbing oxygen and the power of inducing
fermentation are lost. But if the water that has been used
to wash yeast, z\e., water containing the soluble products
necessary for the perfect nutrition of the yeast cells, be

FERMENTATION. 127

added to the exhausted yeast, the conditions of nutrition
are rendered so favourable that the yeast cells again acquire
in a most marked degree their original and characteristic
power of absorbing oxygen, of vegetative proliferation, and
of setting free alcohol and carbonic acid gas.

We may briefly give in his own words the position that Schiitzenberger
holds, so that it may be compared with Pasteur’s position, previously stated.
‘* The cell ferment is not developed in the absence of free oxygen, even in
its most favourable medium, the must of grapes. The ferment, in process of
development, continues to increase in suitable media, even in the absence
of all trace of oxygen, as M. Pasteur had already shown. The contrary
assertions of Brefeld are erroneous. M. Pasteur’s theory, according to
which yeast, in the absence of air, takes from the sugar the oxygen neces-
sary for its development, is not well founded; in fact, this development
stops long before the greater part of the sugar is decomposed. Is it from
the albuminoid matter that the ferment takes its oxygen in the absence of
air? Yeast sets up alcoholic fermentation in a solution of pure sugar in
the absence of all trace of oxygen, but without developing. This is
contrary to the affirmation of M. Pasteur that fermentation is bound up
with the organization of the yeast, or is a phenomenon correlative to the
vital activity of the cells.” ‘

We find that in nature there is, in protoplasm, not only an
extreme adaptability to surrounding circumstances, but an
attempt to utilize, as far as possible, the whole of the
energy set free from cells. We find that in this matter
the protoplasm of yeast cells differs in no essential respect
from other kinds of protoplasm, and we have already seen
that in the process of fermentation of saccharose there is a
preliminary change brought about by the soluble products
of the yeast-cells (invertin), by which we obtain dextrose and
levulose, both of which materials may, under certain con-
ditions, be split up into alcohol and carbonic acid gas.

It may now, further, be noticed that if the fermentation
be stopped at a comparatively early stage, there is found in
the fermenting solution a larger quantity of levulose than
of dextrose ; z.¢., the dextrose is more readily converted into
the characteristic products of fermentation than is the levu-
lose. It has also been proved that if the yeast-cells be heated
to a temperature of about 60° C., they are destroyed, as is
evidenced by the fact that they are no longer able to
multiply, and they never give further evidence of vitality.
But there is left in the fluid a substance known as enzyme
(é» Sun, leaven—yeast), which, added to the mixture of dextrose
and levulose, does not affect the levulose in the slightest..

128 BACTERIA.

(This may also be done by using the Pasteur-Chamberland
filter, which keeps back the yeast-cells, but allows the enzyme
to pass.) It is only on the addition of living yeast, or that
yeast in which there is active protoplasm capable of actually
digesting and transforming food materials, that the levulose
becomes transformed into dextrose.

We have here three very significant statements, all
of them attested by eminent authorities, which seem to
explain many of the anomalies and disagreements in indi-
vidual statements. The process of inversion is reduced to a
purely chemical one, in which the chemical reagents bring-
ing it about are manufactured by the living protoplasm ;
apart from which, however, it is able to exist and act so that
saccharose is converted into dextrose and levulose merely
by the action upon it of the chemical products or enzymes
contained in yeast water; whilst the conversion, and cer-
tainly the fermentation of levulose, can only take place when
this material comes into actual contact with the living pro-
toplasm, from which it seems, necessarily, to be more closely
associated with the actual process of digestion.

This is a decided step in advance in assisting to explain
the process of fermentation, of digestion in the higher
animals, and of those changes that take place in putrefactive
and pathogenic processes. It also appears to throw light on
some of the points in dispute on fermentation..

For example, we can easily understand how different the
results would be in cases where experimenters use yeast as
the fermenting agent, and crude saccharose as the fermentable
substance in one case, and yeast with dextrose in another.

The process of inversion of the saccharose by the invertin
into dextrose and levulose is carried out easily enough,
whether oxygen be present or not, and this dextrose is easily
broken up by the yeast. When we come to the levulose,
however, it is a different matter, as this substance can
apparently only be converted into dextrose when it is in
direct contact with the protoplasm of the cell, and so long as
food materials and oxygen can be obtained from other sources
there is not the same tendency for the protoplasm to take up
this levulose that there is when other sources of food supply
are cut off. The active and rejuvenated cells are still able to
utilize the levulose, whereas older cells, which, as we have
seen, after self-digestion lose even their power of transforming

FERMENTATION. 129°

dextrose, lose their power of transforming levulose at a still
earlier period.

This enzyme function, although so intimately associated
with the vital activity of protoplasm and so usually possessed .
by cells, is considered by Hueppe to be a secondarily derived
function of the protoplasm, and to be really a modification
or development of the primary power of digestion possessed by
all protoplasm independent of the action of enzymes, only
to be met with when the conditions for the growth of the
organism and the development of its functions are most per-
fectly combined. It is, as Cartwright Wood says, a function
developed in such a high degree, that it may actually be
temporarily separated from the protoplasm which develops it.
It must be looked upon as something separate, and as some-
thing that can act, under favourable circumstances, apart
from protoplasm, economizing the energy of the proto-:
plasm, and utilizing the most suitable products for the nutri-
tion of the organism and for the manifestation of its special
functions. Under less favourable conditions, where the pro-
toplasm has to struggle for existence, as it were, this enzyme
function is withdrawn ; and although the protoplasm can
still exert its characteristic powers of digestion and of forma-
tion of special products, these processes take place within
the cell, they are of a less specialized nature, and are, in fact,
the primary and inherent functions of the cell protoplasm,
which are capable of doing their work within the protoplasm,
but are not so highly differentiated that they can act exter-
nally to it. The primary function is, then, not separable
from the protoplasm. This formation of enzymes is of special
interest in relation to the processes of disease; for it is found
that, by modifying the conditions under which an organism
grows, or by varying its food, special ferments come into
operation. For instance, Lauder Brunton and M’Fadyean,
experimenting with a certain organism, found that if it were
grown on peptonized beef jelly, it would give rise to an
enzyme which was capable of liquefying the gelatine. If this
organism were now introduced into a solution of starch, it
would continue to grow, but it could give rise to no diastatic
ferment—z.e., it was incapable of transforming, except within
its own protoplasm, the starch material into sugar, or into
material that it could utilize for its own nutrition. If, how-
ever, successive generations of this same organism were cul-

10

130 BACTERIA.

tivated on starch, there came a period at which it generated
a diastatic ferment, which could be separated from the solu-
tion, and which alone, and without any direct intervention
of the protoplasm, could bring about a transformation of
starch into sugar—z.e., a secondary separable diastatic func-
tion had been developed, in addition to the primary digestive
function of the organism. When taken back to the gelatine
from the starch, the organism had lost its power of liquefy-
ing gelatine ; but this again it regained, after being again
cultivated, through a series of generations, on gelatine.

It is quite possible that this may be a somewhat too strong
statement of Lauder Brunton’s and M’Fadyean’s position. It
may be that they maintain only that the organism when
grown on starch produces the diastatic ferment, whilst when
on albumen it forms a peptonizing substance. It may be
merely a temporary modification just as under analagous
conditions, the cholera bacillus when grown on sugar produces
butyric acid, whilst when grown on albumen it forms
ptomaines.

It is thus evident that special forms of fermentation require
that, in addition to a special kind of protoplasm, there shall
be a special nutrient material—a fact that explains some of
the different results that have, from time to time, been
recorded. The yeasts, as we have seen, are capable of fer-
menting dextrose and of producing a substance, invertin, by
which the saccharose, or cane sugar, is converted into dex-
trose and levulose—both fermentable substances. Wood
points out that only three yeasts are known that are able to
ferment milk sugar directly, although many of -the bacteria
are able to invert and bring about its fermentation. He says
that—

“* Of still greater interest is the varying manner in which the same organism
condicts itself towards different albuminoids. . . . Asa general rule, those
organisms which liquefy gelatine are able to coagulate milk, and then
peptonize casein which has been separated; but some organisms which
peptonized gelatine are without action on the milk, and some that are
Inoperative on gelatine peptonize milk, though this is exceptional ” ; and he
further says that even the manner in which “ milk is peptonized is subject to
considerable variations, and that, although the vast majority first coagulate
the casein and then dissolve it, certain microbes seem to peptonize the
casein directly. . . . Very striking is the way in which the same organism
conducts itself to the different albuminoids, gelatine, fibrin, blood-serum,

and egg albumen. One organism is unable to liquefy gelatine, but pep-
tonizes fibrin; another liquefies the gelatine, but cannot peptonize the

FERMENTATION, 131

egg albumen. . . . They must, accordingly, be regarded as specific in their
nature, depending on the specific nature of the protoplasm of which they
are merely further differentiations.”

Micro-organisms, when grown under such conditions that
oxygen cannot gain access to them—under conditions of
anzrobiosis—usually appear incapable of developing their
separable peptonizing enzyme function, as they are no longer
able to liquefy the gelatine in which they are growing. The
fact that this separable enzyme function could be kept in
temporary abeyance was utilized by Chantemesse and Widal,
to prevent the liquefaction that takes place in plate cultiva-
tions where a certain specific organism requires a considerable
time for its complete development. By adding dilute carbolic
acid to their nutrient medium they found that the peptonizing
power of the organisms was interfered with ; whilst if the
carbolic acid solution was sufficiently weak, even the more
delicate organisms still retained sufficient vitality to enable
them to grow on the surface of the carbolized gelatine.

Having corroborated the above observations, Hueppe and
Wood sought to obtain the same results while avoiding the
dangers resulting from the use of such a substance as carbolic
acid, by offering for the nutrition of the organism a certain
preparation of a material which would enable it to develop
the diastatic, instead of the peptonizing ferment ; and, by
adding glycerine to the gelatine, they found, as they expected,
that Lauder Brunton’s and M’Fadyean’s experiments were
practically repeated. The organisms feeding on the glycerine
did not in most cases attack the gelatine, and the peptonizing
became exchanged for a diastatic ferment ; plate cultivations
were not so rapidly liquefied, and bacilli of very slow growth
could thus be separated from impure cultivations, in which
organisms that ordinarily exert a liquetying or peptonizing
function were present in considerable numbers.

Our knowledge of most other fermentations, although
being gradually increased, is still in a somewhat nebulous
condition, for the reason that no one has as yet (with
the exception of those who have worked at the matter
from the industrial point of view) taken up the matter
thoroughly since it became possible to obtain pure cultiva-
tions of any single organism, the whole energy of bacterio-
logists having been diverted to the consideration of the very
important questions that have come. up to be answered in

132 BACTERIA.

connection with the production of specific infective diseases
in man and animals by micro-organisms.

There has, indeed, been so much work to be done in
separating out the causal bacterial agents in these diseases,
that there has been little time or energy left to be devoted
to anything more than a most cursory study of the
chemical and biological processes of the non-pathogenic
forms. We do know, however, something about the man-
nitic fermentation of sugar, the lactic, the butyric, the
ammoniacal, and the acetic fermentation ; and, speaking
generally, these processes are essentially the same as those
which take place in ordinary alcoholic fermentation, the
differences being that the protoplasm cells of the ferment
differ from those of yeast both in morphological and bio-
logical characteristics, and that the composition of the
end - products is also different. The food material acted
upon by different protoplasm, and giving rise to different
results, may be the same ; the fermentation is still brought
about by a process of hydration or some other specific
process, and is intimately associated with the vital activity
of the special ferment. In the mannitic fermentation
the sugar is converted by hydration into dextrose and
levulose, after which comes the conversion of some of the
sugar into gum, and of some into mannite, during which
second part of the process there is also an evolution of carbon
dioxide and the formation of water. Mannite is a sugar to
which a molecule of hydrogen has been added; gum, a
cane sugar from which one molecule of water has been sub-
tracted. The viscous change that takes place as the fermen-
tation proceeds is due to the formation of this gummy
material in the fluid. Pasteur described the special man-
nitic ferment as consisting of chains of small cocci, each
coccus having a diameter of 1.2 to 1.5@ of an inch.
These, like the yeasts, require nitrogenous material, in
addition to salts, extractives, and sugar, in order that they
may develop. This form of fermentation is specially in-
teresting, from the fact that it is to it that the ropiness of
the white wines is due—a ropiness which, as Pasteur pointed
out, might be prevented by heating the wine for a few
minutes at a temperature of 60°C., or, as suggested by
Frangois, by the addition of a certain proportion of tannin, a
substance which appears to interfere most markedly with
the development of the viscous fermentation organism.

FERMENTATION. 133

In the lactic acid fermentation it does not appear to be
necessary that any process of hydration should take place,
as there is here merely an exact division of one molecule of
sugar into two molecules of lactic acid, there being neither
addition nor loss of either oxygen or hydrogen. It must,
however, be borne in mind that the lactic acid fermentation
is frequently accompanied by the formation of CO,, in which
case, of course, the process is not nearly so simple.

By fermenting lager beer at from 30° to 34° C., Chr. Hansen found that
there was developed a form of bacterium made up of long chains, of dumb-
bell or hour-glass shaped organisms, of bacteria as long curved or straight
threads ; whilst at irregular intervals were formed spindle-shaped or club-
shaped bacteria, even when the nutrient conditions still continue good at a
late stage of the growth. That these organisms, however, were not all
alike, Hansen proved by the fact that some of them are stained yellow by
iodine, whilst with others a blue reaction was obtained. The conditions
necessary for the development of this organism, and the production of acetic
acid, are a high temperature—from 30° to 34° C.—and a plentiful supply
of oxygen. So necessary is this latter that a film forming on the surface
is all that there is to denote the presence of an organism, the fluid
beneath, from which free oxygen is cut off, remaining perfectly clear; and
Hansen even gives this as a diagnostic feature to enable an observer to
determine whether he is dealing with pure cultivations of these mycodermi
or not, as, wherever other bacteria are present, roughly speaking, turbidity
is set up.

It is a curious fact that the pure lactic fermentation cannot
go on when the medium is too acid, and it is only by remov-
ing the lactic acid as it is formed that a complete trans-
formation even of milk sugar into lactic acid can be obtained.
This fact was observed long before the exact nature of the .
process was understood; and in all the earlier methods
devised for the preparation of this substance the daily
neutralization of the fluid with chalk or carbonate of soda
played'a most important part. That the process is essen-
tially the same as the others, in so far as it is the result
of the activity of micro-organisms, was proved by Pasteur,
who found that by sowing the lactic acid ferment, which
he described as composed of small globules or short
joints, either isolated or in mass, in a fluid which he
had found specially suitable for alcoholic fermentation,
active lactic fermentation was coincidentally set up, and
lactic acid was found present along with the resulting
alcohol ; whilst, as we already know, if no lactic acid fer-
ment be introduced, either accidentally or intentionally, no

134 BACTERIA,

lactic acid fermentation ever takes place. Although this is
true in a certain degree, it is found, as has already been
stated, that there are actual differences in degree as to the
readiness with which the various sugars undergo the alco-
holic and lactic fermentations. For instance, although the
glucoses are readily converted into lactic acid, the saccharoses,
which are specially fermentable by the saccharomyces, are
not very easily transformed into lactic acid; whilst, on the
other hand, sugar of milk, which does not readily yield
alcohol, may be comparatively easily converted into lactic
acid. Muscle sugar and mannite, neither of which can be
converted into alcohol, may both be broken up into lactic
acid, as may all those substances that are specially affected
by the butyric fermentation.

It was for some time thought that the fermentation of
urea, through which carbonate of ammonia is frequently met
with in the urine, was the result of a single micrococcus, the
micrococcus urez, a small organism about i in diameter,
which may occur in pairs, in tetrads, or in longer chains, and
which, in the presence of suitable nutrient materials, and of
urea, either in artificial solution or in urine, determines a
regular hydration of these substances and their conversion
into carbonate of ammonium.

Since the time of Pasteur’s researches, however, Leube
has found an organism about double the size of the above
and arranged in chains, which rapidly decomposes solutions
of urea into carbonate of ammonia. It differs, too, from
Pasteur’s organism in that it peptonizes or digests gelatine,
causing it to liquefy, and it appears that, unlike some
other bacilli and sarcine that can set up a feeble urea
fermentation, it is quite as powerful an ammoniacal ferment
as the small micrococcus ure described by Pasteur, as in
both of them the fermentation can go on most vigorously, even
in the medium that has become distinctly alkaline, ze., the
carbonate of ammonia accumulates until about 13 per cent.
of this substance has been formed ; these two microbes may
be said to bear the same relation to the other urea-forming
bacteria that the Saccharomyces cerivisie and the other
more powerful alcohol-forming yeasts have to the weak
saccharomyces and other sugar fermenting organisms.
That this power is not confined to a single organism is not
remarkable, since it is quite possible by boiling the urea in

FERMENTATION. 135

water, or, better still, in slightly alkaline solution, to bring
about direct hydration of the urea into ammonium car-
bonate.

When it is asked what value the study of fermentation has been to the
cause of medicine, it is not necessary to go further for an example than
this urea fermentation. In the operations of the older surgeons it
was very frequently recorded that the introduction of instruments into the
bladder (in which, of course, was a solution of urea) was followed by a
regular fermentation, which too frequently led to the death of the patient ;
and it having been determined that micro-organisms were the cause of this
fermentation, it became an easy matter to prevent their introduction by
careful sterilization of the instruments; and, as a result, such ammoniacal
fermentation within the bladder is now a matter of very infrequent occur-
rence, and is, in those cases in which it does occur, the result not so much
of accident as of carelessness on the part either of the patient or of the
surgeon. So marked, indeed, has this been, that even those who sneer at
bacteriology and antiseptics have been compelled to accept new methods
of procedure in regard to the cleanliness of their instruments, a cleansing
which means simply the removal of these minute vegetable protoplasmic
organisms, which, on being introduced into the suitable fermenting medium
in the bladder (or elsewhere), give rise to the alkaline fermentive, putre-
factive and other processes.

In an interesting chapter on the butyric fermentation of
putrefaction, Schiitzenberger points out that in the process
of putrefaction or putrid fermentation, as it may be called,
there is a formation of butyric acid, one only of a series of
fatty acids formed under similar conditions ; accompanying
this there occurs a transformation of glucose, starch, lactic
acid, albuminoid substances, and the various fruit acids, into
these fatty acids, into carbon dioxide, and into certain
hydrogen compounds. From a very large number of these
substances lactic acid with or without carbon dioxide is
formed, whilst the lactic acid may be further split up into
butyric acid, carbon dioxide, and hydrogen.

From this chapter we may borrow some of the formulz to
show how easy and how rational is the transformation from
these substances into the fatty acids. Other substances
may also be formed, these being further broken down or
separated as the case may be. Glucose, as we have already
seen, is, under the action of the lactic acid ferment, converted
into lactic acid, and this under the action of the butyric.
ferment, a small rod-shaped organism from 1.8 to 18 in
length and 1.8#in breadth is converted into butyric acid,
carbon dioxide, and hydrogen. These micro-organisms

136 BACTERIA.

are straight, somewhat rounded at the extremities, single, or
arranged in short chains of three or four elements ;- if placed
in a nutrient medium similar to that in which yeast grows,
they are reproduced by a process of vegetative division,
especially if this medium be neutral, or slightly alkaline, and
if it be kept at a temperature of 40° C.

The formula of the process of splitting up is given as first
CoH1206 Se 2C,H.O;

1 Molecule Glucose ~ 2 Molecules of Lactic Acid

The two Molecules of Lactic acid are then transformed into
C,H30, + 2CO,
1 Molecule of Butyric Acid 2 Molecules Carbon Dioxide
2M
2 Molecules Hydrogen
In the same way two molecules of malic acid are broken up into two of lactic
acid and two of carbon dioxide, and the lactic acid is again transformed as
above, that is, 2C,H-O; = 2C;HsO; + 2CO, = C,HgO2 +-4CO. + 2H2.
This butyric acid formation is only one of a group, and we find that where
lactic fermentation is going on in an impure condition propioni¢ acid,
C,H.O., acetic acid, C2H,Oz, and valerianic acid, CsH1.O., are produced,
and glycerine fermented for some time with beer yeast, is split up into pro-
pionic acid mixed with formic and acetic acids, giving a kind of compound
of putrefaction products.
A very ingenious formula is quoted by Schiitzenberger to show how

the fatty acids are formed from sugar :— = (CsHi2O6) =

[CnHenO2] (n—2)CH.O,
Fatty Acid Formic Acid
group, being immediately converted into carbon dioxide and hydrogen. The
presence of lime or organic matter appears to modify this process some-
what, and to give rise to the formation of secondary vegetable acids. For
instance, two molecules of malic acid, 2C,H6O,, break up into succinic acid,
C,H6O,, and tartaric acid, 2C,H6O., and from these are formed, from the
tartaric acid the fatty acetic acid, C.H,O, + CO, + H., and from the
succinic acid the fatty valerianic acid, carbon dioxide and hydrogen,
C;Hi.02 + 3CO. + H..

+

Formic acid, the lowest of the fatty acid

These transformations have this in common, that they
are all brought about by the presence of minute organisms,
that the conditions of the development of these organisms
are similar to those met with in other fermentation pro-
cesses, and that theirs are the principal products formed
during the process of putrefactive fermentation, which also
in all cases requires the presence of these minute organisms,
the spores of which, as we have seen, seem to be everywhere

FERMENTATION, 137

present. Here, as in the case of the yeasts, we find both
zrobic and anzrobic organisms. The zrobic forms give
rise to oxidation of certain of the products of decomposi-
tion, one of these, nascent hydrogen, especially taking up
oxygen, as a result of which water is formed; the an-
zrobic organisms, which may be said to complete the
process of putrefactive fermentation, only commence to grow
when the zrobic organisms have used up all the readily
available oxygen such as that dissolved in a liquid, on the
surface of which there has formed a kind of film, by the
intervention of which aeration from the external air is greatly
interfered with.

We have already noted this film growing on yeasts, when it
appears to assist the carbon dioxide developed during the pro-
cess of fermentation in keeping off the oxygen of the atmo-
sphere, and to allow of these anzrobic organisms carrying on
their growth without the further intervention of a free oxidiz-
ing agent. The products of these anzrobic organisms, of
course, differ from the products of the zrobic fermentations
in so far that they contain less oxygen, the sulphuretted
hydrogen, hydrogen, and nitrogen being released in a more
or less free condition and uncombined with oxygen, the
oxygen of the original fluid being used up in the formation
of such substances as leucine, C,H,,(NH,)CO OH ; tyrosine

CH | or (NH,)CO, OH) ; the volatile fatty acids ; some

compound ammonias, and, of course, carbon dioxide ; the
two latter of which, as already seen, may be formed from
urea (CO(HN?),) by simple hydration, the more highly
organized substances, such as leucine and tyrosine, being,
during the process of fermentation, further converted inta
ammonia and the fatty acids.

From the variety of the products of these fermentations
it is evident that we have to do, not with a simple conver-
sion by means of any single organism into the simplest and
ultimate elements of decomposition, but that we have a
series of breakings down or stages of decomposition by which
the highest (and most unstable) organic materials are
gradually transformed into the lowest and most stable. It
will be found, however, that the process is not quite so
straightforward and simple as above stated, as some of the
energy released during the formation of a number of lower

138 BACTERIA.

and more stable compounds is utilized in the presence of
animal and vegetable protoplasm in building up higher and
more stable materials ; though at the later stages of decom-
position these become fewer in number, and eventually
become oxidized into the more stable forms—a free access of
air to the products of anzrobic decomposition always leading
to their oxidation. Hydration almost invariably occurs
during each of these stages.

Schiitzenberger says, ‘‘ Nothing resembles putrid fermentation, with
reference to the derived products, more nearly than the change which takes
place in the constituent parts of yeast, when left to itself without nourish-
ment, deprived of sugar and oxygen.

We see, in fact, the appearance of leucine, tyrosine, sarcine, &c.
This is the first step; the action stops there, and goes no further; the
yeast, or the special soluble ferment which it secretes, is unfit to attack
these bodies again; but if we wait for the development of vibrios, we
shall find the production of ammonia, carbon dioxide, and volatile fatty
acids at the same time that the leucine partly disappears.”

Fermentation by oxidation can only be set up by organisms
in the presence of a free supply of oxygen, and the process in
such cases appears to be due to special organisms which,
attacking the substance to be fermented, remove some of its
constituents, set free others, especially carbon and hydrogen,
which, in their nascent condition, are seized upon by the
oxygen of the air, and so water, or water and carbonic gas,
are formed.

‘In the commonest of these fermentations by oxidation,
the acetic fermentation of alcohol, the free access of air is
as absolutely essential for the growth of the organism as
it is for the special stage of the fermentation of the alcohol
into acetic acid.

If the mycoderma aceti be sowed in wine, freely exposed
to the air at a temperature from 24° to 27° C., chemists hold
that there is hydration of alcohol by the combination of an
atom of free oxygen with two atoms of its hydrogen water, OH,
and aldehyde C,H,O, being formed, the latter of which, by
further oxidation, in turn becoming converted into, C,H,O,,
acetic acid. At a certain stage this process of acidification
stops, owing to the quantity of acid developed, but if this be
removed and fresh alcohol be added, the acidification again
commences, and so on, as long as the mycoderma remains
active, can obtain sufficient oxygen, and sufficient alcohol

FERMENTATION, 139

on which to act. If, however, the supply of either oxygen
or alcohol be cut off, the ordinary mode of life of the
mycoderma is at once interfered with. It uses up part of
its own protoplasm by internal metabolism, continues to act
on the acetic acid, and converts it into simple substances,
water and carbon dioxide, at the same time retaining
molecules of carbon, hydrogen, and oxygen for its own use.
The mycoderma is, then, no longer so much an enzyme-former
or an organism with a thick celled wall, that acts principally
on the suitable nutrient medium by means of its chemical
products, and gives rise to a definite chemical process ; but
is now rather a mass of protoplasm endowed only with
its primary digestive powers, by means of which it can
convert organic matter into its very simplest forms or ele-
ments. There is very complete oxidation, and instead of
alcohol and acetic acid being formed as intermediate products
that can be easily separated, the substances are at once
converted into water and carbon dioxide, the ultimate
products of perfect oxidation.

It is sometimes said that the mycoderma in the true
vinegar fermentation is in a weakly condition; it is cer-
tainly not in its most active condition, but the state appears
to be one rather of quiescence or advanced development, in
which the formation of a thick membrane and the produc-
tion of a separable enzyme are characteristic features, rather
than one that can be spoken of as a condition “ of incomplete
or tardy development.”

To show that the process was not merely a physical one,
as was suggested even by Pasteur, Mayer showed that by
simply heating the acidifying fluid to a temperature at
which the mycoderma is killed all oxidation is arrested, and
he also showed that the temperature at which oxidation of
concentrated alcohol goes on in spongy platinum is at a
temperature above 35° C., at which point it is completely
‘stopped in the presence of the mycoderma aceti, in addition
to which “ physiological acidification” can only take place
in a weak solution of alcohol.

Pasteur’s observations and results led to the adoption
of what is known as the Orleans process of making vine-
gar, which, as given by Schiitzenberger, is as follows :—
“The mycoderma aceti is made up of small, slightly
elongated cells, with a transverse diameter of from 1.5

140 BACTERIA,

to 3H, united in short chains, or curved rods. Con-
striction and division take place between the segments of
the chain as the process of vegetation goes on. A small
quantity of this mycoderma, which occurs as a wrinkled
membrane on the surface of liquids that are undergoing
acetic fermentation, is first sowed on the surface of an
aqueous liquid containing two per cent. of alcohol, one per
cent. of vinegar, and traces of alkaline and alkaline-earthy
phosphates. When the surface is covered with the mem-
brane the alcohol begins to acidify. This action being fully
set up, some alcohol, wine, or beer mixed with alcohol, is
each day added to the liquid, in small quantities ; this is
continued till the oxidation becomes slower ; the acetification
is then allowed to terminate, and the vinegar is drawn off.
The membrane is collected, washed, and employed for a new
operation. It is better always to give the plant sufficient
alcohol, so that its activity may not be exerted on the
acetic acid. Nor ought it to remain too long out of the
liquid, or it would lose its active force ; finally, it is better
to moderate its development, to prevent burning oxidation.”
These last few lines are of special interest, as they show how
markedly favourable conditions of nutrition, and the supply
of special materials on which the organisms may act, modify
the process set up by the acidifying organism ; just as certain
organisms will select glycerine or sugar from a peptone
gelatine medium before they begin to act on the gelatine itself,
so the mycoderma aceti, under certain conditions, confines its
attention entirely to the alcohol, converting it into acetic
acid and then leaving it ; whilst, as soon as the glycerine in
the one case and the alcohol in the other, are completely used
up, or as soon as the activity of the protoplasm becomes so
great that it cannot derive sufficient material from the one,
it immediately attacks the other, and continues the oxidizing
process. Not only so, but the various organisms appear to
be specialized at the different stages of the resolution of
organic matter; each, during the stage at which it can
perform its share of the work, appearing not only to multiply
much more rapidly than the other organisms with which it
is found but actually holding them in check. In this
respect. the vegetable protoplasm of the lower organisnis is
only like the protoplasm of the cells of the higher animal
organisms. In relation to pathogenic processes this is a

FERMENTATION. I4l

point of very considerable interest, for we find that under
certain conditions there comes to be a contest between the
animal cells arid the lower vegetable cells. As Wood well
puts it, “the reaction of the cells upon each other may turn
on the sum of the conditions of existence to which they are
exposed happening to favour one more than the other, thus
when two organisms are sown together in a culture fluid,
which will overgrow the other may depend upon the relative
quantity of the two primarily introduced, the nature and
reaction of the media, and the temperature at which they
are held.” Fermentation, then, is due to the action of highly
specialized cells. That this is so is indicated by the usual
presence of enzymes and also by the fact that they exert this
specific action in their highest degree only under a certain
set of specific conditions which vary in the case of each
organism. Each organism has become adapted to a
certain medium, and is so specialized as to have the power of
splitting it up under certain conditions in a specific way.
This is also indicated by the fact that as the conditions
become unfavourable, less and less of the specific product
(relatively to the bye products) is produced. Although we
shall have more to say in connection with the subject of the
specific infectivity of micro-organisms in disease, it may here
be pointed out that fermentation is due, in great part, to the
action of cells which have the power of developing a special
enzyme function ; that these cells are usually more highly
specialized during the stages when they bring about their
specific action ; that this is associated to a certain extent with
diminished activity of the protoplasm directly on the sub-
stances to be fermented ; that most processes where there is-
the formation of an enzyme, or a special poison, take place
most actively when there is a complete or partial cutting off
of the supply of oxygen ; that the cells in this condition
usually develop a more or less perfect cell membrane which
characterizes the formation of zooglcea masses ; that this in-
complete oxidation also characterizes the formation of
toxines ; that the same rules hold good in the formation of
most of the products of the lower vegetable organisms
and of the individual cells of animal tissues with complete
oxidation and the formation of carbonic acid gas and
water ; that when incomplete oxidation takes place various
specific products make their appearance ; but that even in

142 BACTERIA.

the presence of a full supply of oxygen, protoplasm under
certain conditions cannot bring about complete oxidation,
and as a result some of the intermediary products of fer-
mentation and decomposition may be formed.

lt is evident, then, that all ferments may be classed under
two great heads: firstly, the organized ferments or the active
unstable protoplasmic cells of yeasts, bacteria, of plants and
animals. Secondly, the unorganized ferments or enzymes
which we have spoken of as the separable functions of more
highly developed cells, both of them being, however, as-
sociated with the nutritive and other metabolic changes of
protoplasm, be this protoplasm that of simple vegetable cells
or of highly organized gland cells. Hoppe-Seyler classifies
the whole of the fermentations as follows :—I. ferments which
bring about hydration or cause hydrolysis, these being divided
into (2) those that act like boiling mineral acids, all of which
belong to the enzyme group ; and (4) ferments acting like
caustic alkalies in which we have the process of decomposition
(1) of fats into glycerine and fatty acids as in saponification,
which results from the action both of the organisms directly
and of the unorganized ferments, (2) decomposition of amido
—or ammonia-nitrogen compounds which is also usuaily
associated with hydrolysis. II. In the second group, Hoppe-
Seyler places fermentations in which there is transference
of oxygen from the hydrogen to the carbon atoms. As
examples of this we have (a) lactic acid fermentation in
which there is decomposition of certain carbohydrates into
lactic acid ; this, as we have seen, being always associated with
the presence and activity of a certain group of micro-
organisms, (4) the alcoholic fermentation brought about in a
similar manner by yeasts, (c) putrefactive decomposition
changes, (1) of simple inorganic compounds such as combi-
nations of the fatty acids with lime, or (2) of organic com-
pounds such as fibrin and other proteids. Then we have
also the acetic fermentation already referred to, nitrification
or mineralization, chromogenic fermentation and that series
of changes in which we have the production of ptomaines.

Such a classification is specially important when we come
to consider the relation of the ordinary fermentation pro-
cesses to those that are set up during the course of disease in
animals and in plants.

FERMENTATION. 143

LITERATURE.

Authors already mentioned.. Fligge, Davaine, Roberts,

Tyndall, Fitz. —

Becuamp.—Comptes rendus, t. LVI, pp. 969, 1086, 1231,
1863; t. LXL, pp. 374, 408, 1865; t. LXXxV., p. 1036,
1872; t. XCIIL, p. 78, 1881.

BoutTroux.—Comptes rendus, t. LXXXVI., p. 1, 1878.

Bunee.—Text Book of Phys. and Path. Chem. (Transl. :
Wooldridge). London, 1890.

pg ie pebaaas Zeitung, 1875 ; Die Hefepilze, Leipzig,

_ 1883.

same a AND WipAL.—Gazette Hebdomadaire, p. 146,
1887.

CaGniarp-Latour.—Ann. Chim. Phys., 2nd ser., t. xv., 1820.

Desmazigres.—Ann. des Sc. Naturelles, t. x., p. 4, 1826.

Dusrunrant.—Comptes rendus, t. XLII, p. 945, 1856.

Ence..—Les ferments alcooliques. 1872.

HoprE-SEYLER.—Zeitschr. Physiolog. Chemie, Bd. x., p. 36,
1885.

Hvuepre.—Mitth. a. d. Ges. Amt., Bd. 11, p. 309, 1884.

Huepre anp Woop.—JZancet. Dec. 7, 1889.

Hansen, Cur.—Meddel. fra Carlsberg Lab. Copenhagen,
1879, '81, '83, 86, '88; Allgem. Zeitschr. f. Bierbrauerei
u. Malzfabr. 1883-87 ; Zeitschr. f. d. ges. Brauwesen,
1884, and republished in Munich, 1888.

Hoitm anp Poursen.—Meddel. fra Carlsberg Lab., Bd. 11,
1886.

JORGENSEN.—Micro-Organisms of Fermentation (Trans.),
London, 1889. ,

Kreser.—Schweiggers Journ., 1814, No. x11, p. 229.

Leuse.—Virch. Arch., Bd. c., p. 540, 1885.

Lirsic.—Ann. de Chimie et de Phys., 2nd ser., t. LXXL,
p- 178, 1839. :

Lister.—Pharmac. Journ. and Trans., vol. vut., 3rd series,

Pp. 5555 1878. . :

Maver.—-Die Lehre von den chemischen Fermenten. Heidel-
berg. 1882.

Pasteur.—Ann. de Chimie et Physique, 3me Serie., t. Lvut.,
p- 323, 1860; Etudes sur le vin, 1866 ; Etudes sur le
vinaigre, 1868; Etudes sur la biére, 1876; Comptes
rendus, 1876, t. LXxxulL, p. 173 ; Lecture faite a l’Acad.

144 BACTERIA.

de Méd., 1878, 26 Nov. ; Comptes rendus, 1861, t. LIL ;
1864, t. LVI. |

Reess.—Botanische Untersuchungen itiber die Alkohol-
gahrungs-pilz. 1870.

ScHUTZENBERGER.—On Fermentation. London, 1886.

ScHwann.—Poggendorf Annal., 1837, Bd. 41, p. 184.

Trécut.—Comptes rendus, t. LXI., pp. 432, 553, 1865; t.
LXV., Pp. 513, 927, 1867.

Turpin.—Comptes rendus, t. vit, p. 369, 1838.

van TIEGHEM.—Ann. d. Sc. nat. Bot. Série vi, t. 4, 1878.

WIESNER.—Sitzungsber. der Wiener Akademie, Bd. Lix,,
1869.

WIuis.—De Fermentatione. 1659.

Woon, Cartwricut.—Lab. Reports Roy. Coll. Phys., Edin.,

1890, p. 253.

CHAPTER VII.
PARASITES AND SAPROPHYTES.

Putrefactive Bacteria—Pigment-forming Bacteria—Enzyme-forming Bacteria
—Non-pathogenic, Saprophytic, Parasitic, Pathogenic Bacteria—
Sarcina Ventriculi—Leptothrix Buccalis—Facultative Saprophytes—
Facultative Pathogenic Parasites — Anthrax Bacillus — Pathogenic,
Parasitic, Saprophytic, merely Relative Terms. -

In order that much of what occurs in the following chapters
may be understood, it is necessary that some idea should be
given of the way in which the terms saprophytic, parasitic,
and pathogenic are used. The first of these is not so impor-
tant; the term saprophytic as applied to bacteria is being
gradually supplanted by the term non-pathogenic, especially
in works dealing with the relation of micro-organisms to
disease. Speaking generally, saprophytic bacteria are those
bacteria that have the power of obtaining the nutriment
they require for their building up, and for the carrying on of
their vital functions, from dead vegetable or animal substances
only. They are not able to invade the tissues of living
animals and plants, but they play an important part, as we
have already seen, in bringing about oxidation of various
organic materials and of giving rise to certain definite com-
pounds. To this class belong the putrefactive bacteria, and
many of the pigment-forming bacteria ; the yeasts or fermen-
tation fungi are also to be looked upon as purely saprophytic
in character ; and such organisms as the Bacillus amylo-
bacter, the lactic acid bacillus, and the like, are all probably
to be classified in this group.

The subdivision of the group has been attempted by
many workers, and the saprophytic organisms have been
arranged according to their power of action on the various
carbo-hydrates and on proteid substances. Others have
taken their products as a basis on which to classify
them, and enzyme-forming, pigment-forming, or alcohol-

II

146 BACTERIA.

forming organisms have all been described ; but as their
powers of decomposing different substances and of giving
rise to pigment or to certain enzymes necessarily overlap one
another, or are met with in the same individual under
different conditions, it remains an exceedingly difficult matter
to give any classification founded on the above mixed
characteristics.

Some of the organisms which, at present, are supposed to
be saprophytic in character, may eventually be found to be
parasitic under certain conditions, and it is for this reason
that the term ou-pathogentc is gradually ousting the older
botanical term of saprophytzc.

Parasitic bacteria are those which are able to flourish on
or within the substance of plants or animals. In animals
they live in the various cavities of the body, or in certain
cases in one or other of the nutrient fluids of the body, and
they derive their food from these fluids, or from the nutrient
materials that are taken in by the host. The ordinary terms
of obligate parasites, facultative saprophytes, and facultative
parasites, can scarcely be used in connection with these
minute organisms, as, with very few exceptions, all the
parasitic bacteria have now been cultivated outside the body
and under such conditions that it is evident that some of
those which were at one time looked upon as purely parasitic
have a saprophytic stage of existence ; so that nearly all, if
not all, the parasitic bacteria must be looked upon as facul-
tative parasites, or parasites that can develop almost as well
saprophytically as they can parasitically, or facultative
saprophytes, or saprophytes that can develop almost as
well parasitically as they can saprophytically.

We know of course that all parasitic bacteria are not
pathogenic, and that we have along the alimentary tract,
and even in the respiratory tract, a large number of para-
sitic organisms. The Sarcina ventriculi, for example, first
described by Goodsir, may be taken as a purely parasitic
organism. It is found especially in dilated stomachs. A
drop of fluid taken from the contents of such a stomach
frequently contains a number of organisms which present a
curious appearance under the microscope. They are very
like the typical bale of wool or goods tied with a strong
cord in three directions. Other sarcinz are saprophytic as
well as parasitic, but the Sarcina ventriculi has not yet been

PARASITES AND SAPROPHYTES, 147

satistactorily cultivated outside the body, or if it has, it has
always become somewhat altered in appearance. Sarcine
have also been found in the bladder, in the air passages,
in the intestines, and even in the blood, but most of these
can be cultivated on nutrient gelatine. None of these
organisms are pathogenic so far as at present can be made
out ; they appear to be simply accidentally associated with
certain conditions, as in the case of dilated stomachs, in
which they are not always present, but in which they
frequently occur, apparently because the acid fermentation
that goes on in the accumulated contents, renders them
specially favourable media for the development of these
sarcine, The non-pathogenic parasitic bacteria of the mouth
were amongst the first organisms observed by Leeuwen-
hoek ; they are found on the gums, on the sordes covering
the teeth, and on the mucous membrane of the mouth.
The forms of these we shall mention later. Although most
of these are parasitic and non-pathogenic, one form, the
Leptothrix buccalis (an old term embracing probably several
species, and one not now very generally used), is also patho-
genic in the sense that it invades the teeth, and gives rise
to what is known as‘caries, or rotting of the teeth, but even
this has to obtain help from some of the other non-patho-
genic species found (some of the micrococci), which give rise
to the formation of lactic acid from the sugar of old food,
an acid that, combining with the lime salts of the teeth,
softens them, and so allows of the pensiaiaan of the lepto-
thrix form.

Pasteur has suggested that other facultative saprophytes
are those met with in the alimentary tract, where he thought
they appeared to assist in carrying on the decomposition of
the nutrient materials, assisting, under certain circumstances,
the animal tissues of the body to carry on the process of
digestion without interfering in any marked degree with the
nutrition of the tissues of the host by absorbing any of the
material that could be utilized by these tissues for their own
nutrition. This, however, is now considered to be question-
able. The organism of cholera finding its way into the
alimentary canal, acts as a pathogenic parasite, but here
only from the fact that by its growth and metabolic activity
it gives rise to irritant toxic materials, which exert most
injurious effects both locally and constitutionally.

148 BACTERIA,

As another example of a facultative pathogenic parasite,
the anthrax bacillus may be cited. It may develop actually
in the tissues, as in woolsorters’ disease, where it occurs in
the bronchial mucous membrane; in the lymphatic tissue
spaces, and in the pleural cavities; or in the blood, as in
cases of accidental inoculation. It may also occur in the
blood in woolsorters’ disease, the bacillus readily making its
way from the air vesicles and tubes into the pulmonary
circulation. In this connection it may be mentioned that
anthrax bacillus cannot undergo its whole developmental
cycle within the body ; the vegetative stages only having
been found in parasitic anthrax; whilst growing on dead
tissues at a favourable temperature spore formation is
invariably noticed at some period or other during the life
history of the anthrax rods, so that the bacillus must be
looked upon as a facultative saprophyte. Another point to
which attention may be drawn is that bacteria must be
looked upon as essentially and primarily saprophytic organ-
isms. By a process of long and gradual acclimatization or
adaptation, certain species have become so altered, either
temporarily or permanently, that they are able to exist as
parasites. It will always be found, however, that the
tendency to revert to the saprophytic or non-pathogenic
condition is more marked than their tendency to become
transformed into the parasitic and pathogenic condition. It
has been found, for example, that anthrax bacillus passed
through a series of cultivations in gelatine, or hog-cholera
bacillus similarly treated, loses a great deal of its patho-
genic power. This loss of pathogenic power may take
place in two directions, either by the organism losing its
power of development in living tissues, or by losing its
virulent specific poison. The cholera organism cultivated
outside the body usually loses much of its pathogenic power.
By appropriate treatment, such as cultivating at the body
temperature in specially prepared broth through a number
of generations, this pathogenic activity may, however, be
restored. We must therefore look upon pathogenicity, para-
siticism, and saprophyticism as mere relative terms; the
conditions necessary for the development of the saprophytic
mode of life being more widely met with than are those
in which the parasitic life may become developed, most
organisms become more or less permanently adapted to

PARASITES AND SAPROPHYTES. 149

them, so that it is only under special circumstances that
parasitic and pathogenic organisms are developed. The
importance of this fact will be evident when we consider
the epidemiological importance of the presence and mode
of life of micro-organisms both outside and within the
body.

CHAPTER VIII.
CHOLERA.

Cholera a Parasitic Disease—Early Observations on the Comma Bacillus
—Characters—Methods of Staining—Methods of Isolation—Use of
Plate Method in Practical Public Health Work—Tube Cultures—
Motility of cholera bacilli—Potato, Blood-serum, and Milk Growths
—Behaviour in Water and Sewage—Infection of Man through Agency
of Bacillus — Early Inoculation Experiments — Koch — Nicati and
Rietsch—Macleod—Difficulties—Cholera in Guinea-pigs—Position of
Bacilli in Tissues—Presence of spores doubtful.

On account of its general interest there are few examples
that can more appropriately be taken to illustrate the rela-
tions between a special bacillus and a special disease than
that afforded by Koch’s discovery of the comma bacillus
in cases of Asiatic cholera, and its relations to that disease.
The study of the disease itself, since its appearance in this
country in the great epidemic of 1832, has had a peculiar
fascination for epidemiologists and skilled hygienists. Its
development, behaviour, and whole general history appeared
to be shrouded in mystery, and phenomena, the explana-
tion of which seemed to be utterly beyond the powers of
experts of all kinds to give, were at one period constantly
being observed, recorded, and discussed. Now, however,
through the laborious but brilliant researches initiated by
Pettenkofer at the head of one school, and by Koch in a
very different one, much of this air of mystery has been dis-
pelled, and many of the doubts and difficulties that sur-
rounded the subject have been gradually cleared away as
workers under one or the other of these great leaders have
gained fresh knowledge and elucidated new facts. Take,
for example, the question of the spread of cholera from
its home in Lower Bengal, in the delta of the Ganges,
to surrounding districts and distant countries. At first such
spread was thought to be most erratic and inexplicable.
Now, however, although it appears from time to time to have

CHOLERA, 151

made almost unaccountable leaps and divergencies, it has
been found to follow a very definite line of advance in the
course of the various epidemics, and although there may
have occurred sporadic cases which could be traced to no
definite source of contagion or infection, which have proved
a stumbling-block to many conscientious workers and ob-
servers, it is generally acknowledged that the evidence
accumulated through the researches of Koch and his fol-
lowers, both in Germany and in Great Britain, and of
several enthusiastic workers in France, can leave little doubt
in the minds of most of those who study the subject care-
fully, that cholera is a parasitic disease, that it travels along
the ordinary lines of commerce by railways, caravans and
ships, from the regions in which it is endemic to those
centres of trade and religion which, by their imperfect sanitary
arrangements, by the want of cleanliness of their inhabitants,
by meteorological conditions, and on account of bad water
supply, are ready for its reception and propagation. In the
European epidemics, of which up to 1885 there had been
six exceedingly severe ones during the present century, the
disease has, in every case, followed the lines of trade.

Before the three last epidemics (1865, 1873, 1884) cholera
usually came to Europe by what may be called the Con-
tinental routes—the caravan routes through Persia, Asia
Minor, and Russia; but in the three last it came by the
Mediterranean or maritime route, first by land through
Egypt, brought there by Mecca pilgrims, and thence to
the seaports of France, Italy, and Spain, whence it gradually
made its way northward and inland, spreading over the
whole of Europe.

At the mouth of the Yang-Tsze, as instanced by Macleod,
of Shanghai, cholera breaks out regularly at certain seasons
of the year, but he adduces evidence of great value to show
that although it may be imported’ directly from India,
between which and Shanghai there is at least weekly com-
munication, just as there is between India and the Nile delta
region, itis probably, in the strictest sense, an endemic disease
of that region.

It can be readily understood, after the fearful ravages which
it made in places in which it was not actually endemic,
and after it had decimated the population in certain parts
of India, in Egypt, in the low-lying portions of Persia, and

152 BACTERIA,

Asia Minor and in Europe, that many observers should be
anxious to find out the ultimate cause of the disease, and
as early as 1848 Virchow, and in 1849 Pouchet, Brittan, and
Swaine found numbers of vibriones in the discharges of
choloraic patients, without, however, being able to assign
to them or prove for them any specific ré/e in the causation
of the disease.

Following up these researches Philippe Pacini, setting
himself to look for a causal agent, frequently found in
cholera stools small micro-organisms which were charac-
terized by active and peculiar movements. These observa-
tions, however, as well as those of Klob, who looked upon
the cause of cholera as an accumulation of fungi in the
intestines, and those of Boehm and Hallier (all published
in 1867), who believed that they had found the cause in a
peculiar fungus which was found to grow in certain forms
of grain imported from India, not only did not receive
adequate proof, but were, like all preceding observations,
entirely unreliable. Then followed the experiments by
Hayem and Raynaud, carried on during the epidemic of 1873,
which, however, again were equally futile and without definite
results, and it was not until Koch, going out with the
German Cholera Commission to Egypt and India, was able *
to demonstrate a peculiar species of bacterium as the causal
agent that any definite proof of the micro-organismal nature
of the contagium of the disease could be obtained.

Since that time, however, the amount of work published
on the subject has been so great that cholera has now a
special “ literature” of its own. The report of the German
Commission was speedily followed by that of the French
Government, who sent out MM. Straus, Roux, Nocard, and
Thuillier, the last of whom fell a victim to his assiduity and
zeal in carrying on the work of investigation in Egypt. In
this country Klein and Heneage Gibbes, Cunningham and
Lewis, Roy, Graham Brown and Sherrington, Watson
Cheyne, and Macleod have, with very different voices, some
. supporting Koch’s verdict, others opposing it, reported on
the subject; with the general result that until further and
more convincing opposing evidence is forthcoming, Koch’s
comma bacillus, of which the following is a brief descrip-
tion, must be looked upon as the causa causans of the
disease.

CHOLERA. _ 153

Th: ‘comma’ bacillus, which is now regarded as belong-
ing .» the spirilla, usually occurs as a slightly curved rod,
me: iring from 1 to 2p in length, with an average length
of zbout 1.5m; it is .5 to .6# in thickness, the average
thickness being about one-third to one-fourth of the length.
It is therefore from one-half to one-third the length of
the tubercle bacillus, but somewhat thicker. In place of
occurring as single rods these organisms may be grouped

Photo-micrograph cholera bacilli. x 1000, Long spirilla, comma, S and ‘O shaped
organisms. Some involution forms.

in pairs, or in larger numbers, in which case the curve may
be continuous or reversed, so giving rise to the formation
either of half circles or of S-shaped curves. In cultivations
in meat broth the bacilli may be so grouped that they form
long wavy or spiral threads, each of which may be made up
of 10, 20, or even 30 short turns.

Such are the characters of the organisms which Koch found,

154 BACTERIA.

almost invariably, in the stools of patients during the earlier
stages of the disease, in the contents of the lower bowel and in
the mucous membrane of the lower part of the small intestine.
In fatal cases Drs. Straus and Roux thought that they
could also see certain organisms in the blood of cholera
patients, but they were unable to repeat their observations.
Emmerich also described a bacillus which he said he found
constantly in the blood, organs, and intestinal contents in
cases of cholera. This organism was, however, according to
Fligge, probably a common inhabitant of the intestine, the
Bacterium Coli Communis, and it is now a generally ac-
cepted fact that nowhere, except in the alimentary canal,
have the comma bacilli been found in ordinary cases of
cholera ; as the blood, the liver, the spleen, and other
organs have all been carefully examined, and in no case,
in which no fallacy crept in, could positive results be ob-
tained.

The best method yet described of demonstrating the cholera bacillus in the
discharges is that recommended by Cornil and Babes, who spread out one
of the small white mucous fragments on a microscope slide, and then allow
it to dry partially ; asmall quantity of an exceedingly weak solution of methyl
violet in distilled water is then flowed over it, and it is flattened out by
pressing down on it a cover glass, over which is placed a fragment of filter
paper, which absorbs’ any excess of fluid at the margin of the cover glass.
Comma. bacilli so prepared and examined with an oil-immersion lens
(xX 700 or x 800) may then be seen ; their characters are the more readily
made out because of the slight stain they take up, and because they still
retain their power of vigorous movement, which would be entirely lost if
the specimen were dried, stained, and mounted in the ordinary fashion.

The bacilli retain their curved shape and “rounded”
extremities (which, however, may be either slightly pointed
or thickened), but they seem to be somewhat larger when
thus prepared than when completely dried. During the
very early stages of the disease, and when the period of
reaction is setting in, it is sometimes an exceedingly difficult
matter to demonstrate the presence of the cholera organism
in the dejecta, and several methods have been tried with
greater or less success to obtain such a demonstration. Of
these the plan described by Schottelius is found to be one of
the most useful, especially in the later stages of the disease,
where although the number of cholera bacilli may be com-
paratively small, other bacteria are found, often in con-
siderable numbers, in the intestinal contents.

CHOLERA. 155

A small quantity of the material from the intestine is mixed with double
its quantity of-iaintly alkaline meat broth, which is then allowed to stand
at a temperature of 35° C. for about twelve hours; at the end of that time
the comma bacilli have multiplied to such an extent, especially where they
are in contact with the air, that if a small portion of the pellicle from the
surface is stained and examined, as above, it is found to consist of an
almost pure cultivation of comma bacilli which are, however, some what
shorter than those usually met with. If the cultivation be left undisturbed
for a longer period the bacilli generally grow larger, and eventually spirilla
may be formed, but in the course of a few days the other bacteria grow
so luxuriantly that the comma bacilli no longer predominate, and in many
cases they are almost entirely ‘‘overgrown.” From the earlier ‘‘ maass ”’
cultivation so prepared, in which the relative proportion of cholera bacilli
is enormously increased, ‘‘ plate” cultivations may now readily be made.
The plate method as employed by Koch is the best, and the one least open to
fallacy for obtaining pure cultivations. It is carried out as follows: On a
sterilized platinum needle a drop of the above fluid, of the cholera discharge
or of the contents of the small intestine is taken. This is introduced into
a test tube one-third full of nutrient gelatine, liquefied at as low a tem-
perature as possible by immersing the lower portion of the tube in warm
water. The seeded gelatine is carefully shaken in order that the crganisms
may be disposed widely and equally throughout its substance; three
small drops of this are then taken on a looped platinum wire and added
to a second tube, which is similarly shaken, and from this second tube five
drops are transferred to a third tube. After the contents of each tube have
been thoroughly mixed and the seed material taken for the next tube, the
remainder is poured on toa glass plate previously sterilized ; each of the
three plates is carefully labelled and placed under a bell jar in which the
air has been thoroughly sterilized (see Appendix). The plate from tube
No. 1 is found to contain a large number of organisms, that from tube
No. 2 contains a much smaller number, and that from tube No. 3 a much
smaller number still, so that as the organisms are developed (one colony
from each organism or group of organisms) there is sure to be sufficient
space on one or other of the plates between the different groups to allow
of a careful study of the growths as they extend, and at the same lime there
is a sufficient number of points to ensure the appearance of several growths
of each kind of organism that was present in the original seed material.

If plates made according to Koch’s method be kept in
a chamber in which the temperature is. maintained at
a little over 20° C. small greyish or white points are first
seen; examined with a low power lens, each of these has
a slight yellow tinge and a somewhat wavy margin; as
the “colony” increases in size its colour becomes slightly
deeper, the margins become more and more crenated,
and the surface somewhat granular. Slow liquefaction
is now found to be taking place, and small funnel-shaped
depressions, any one of which seldom measures more
than I in diameter, are seen in the gelatine; at the

156 BACTERIA.

bottom of these depressions yellow pin-like masses are
usually seen. In consequence of this liquefaction the colony
has the appearance of a piece of ground glass with a finely
notched margin surrounded by a clear zone. On the second
or third day small air bubbles are seen, and on the fifth or
sixth day the liquefaction is well marked ; at this stage the
colony has sometimes a delicate pink tinge, a most charac-
teristic feature when present. From these points or colonies
growing, as we have seen, from individual organisms or
small groups of the bacillus, pure cultivations may be made
in test tubes containing gelatine, agar-agar, potatoes, or
broth in or on.which the growth and special characters of
the bacillus may be watched and noted. It is sometimes
made a matter for reproach to bacteriologists that the
methods employed in their work, useful enough though
they may be in conducting scientific experiments, are found
altogether wanting when they are required for actual prac-
tical work. The following story, which went the round of
the medical papers, may. help to remove the idea that bac-
teriologists are mere viSionaries, and to prove that the
science, some of the problems of which they are at-
tempting to solve, is by no means so useless as many would
have us believe. An Italian emigrant steamer, touching
at New York, had on board a child suffering from a sus-
picious form of diarrhoea, though it could not be said that
all the symptoms of Asiatic cholera were present. In order
to determine the true nature of the disease—whether it was
cholera or not—gelatine plate cultivations of the dejecta
were made by a doctor in port, and the vessel was detained
for four days; during that period bacilli identical in ap-
pearance with, and having all the characteristics of, Koch’s
comma bacillus were developed, and it was at once con-
cluded that this was a case of true Asiatic or Indian cholera.
The diagnosis was subsequently confirmed, as a series of
other cases occurred, in all of which unmistakable symp-
toms of Asiatic cholera were developed. An equally in-
teresting and instructive example of the.same thing was
recorded by Pfeiffer and Gaffky when they were working in
Koch’s laboratory. At a time when there was no general
cholera epidemic, Gonsonheim and Finthen in Germany were
suddenly ravaged by a most deadly form of diarrhoea which
in many features resembled true Asiatic cholera. The

CHOLERA. 157

recorders, in order to determine the exact nature of the
disease, made plate cultivations from the dejecta of some
of the patients, and found Koch’s comma bacillus; they
were thus able to put the matter beyond all doubt. How
the organism came to the district, and why the outbreak
remained localized, are questions that still remain un-
answered. During 1890 there was a similar, but less
localized, outbreak of fatal diarrhoea in Spain. Koch's
bacillus was here again separated, and all doubt as to
the nature of the disease at once removed.

In view of these facts the immense importance of bacteriological methods,
as permitting of rapid and definite recognition of the disease, with the possi-
bility of taking precautionary measures as early as possible, and so prevent-
ing a wide dissemination of the disease germs, can scarcely be insisted
upon too strongly.

In a puncture culture in gelatine the growth takes
place along the whole track of the needle, first as a delicate
white cloud, then, as it gradually becomes more and more
marked, it forms a delicate streak, around which there
is usually a clear space, due to the liquefaction of the
gelatine along the tract of the needle. Near the surface the
liquefaction goes on more rapidly than deeper down, and at
the end of forty-eight hours there is a distinct funnel-shaped
area, in which a clear liquid portion usually sinks somewhat
below the level of the gelatine, and it appears as though the
top of the funnel was closed by a small clear glass globe or
air bubble. This appearance is apparently due to the slow-
ness of the liquefaction, time being given for the water to
evaporate from the liquefied gelatine. This is proved by the
fact that if 5 per cent. gelatine instead of 8 or 10 per cent.
be used, the gelatine liquefying more rapidly does not allow
time for evaporation to take place, and the bubble is never
seen, About the fourth day of the growth this “funnel” is
still more marked, but the upper part has become quite clear,
the central thread having fallen to the lower and narrower
part of the funnel, where it may be seen as a comparatively
short spiral, the thread as it sinks being arranged in regular
“ bights ” like those of acable. After a time the whole of the
upper part, say two-thirds, of the gelatine is liquefied and
perfectly clear, then comesa layer of “ white” cholera bacilli,
which rests on the gelatine that has remained solid (this
sediment has usually a yellowish tinge) ; and on the surface

158 BACTERIA,

of the liquefied gelatine a greyish scum is found, in which
“involution forms ” may be frequently observed.
Cultivations made with a platinum needle on the surface
of agar-agar, grow as elevated pale translucent streaks, the
margins of which are well defined, and in the agar imme-
diately below the streak there occurs a slight opalescence
which is very characteristic of the bacillus. The agar, unlike
the gelatine, is not liquefied. As the growth becomes older
it spreads, though not very rapidly, the band increases in
thickness and gradually assumes a brown colour especially
in the opalescent substratum. When the organism grows in
meat infusion it forms a delicate greyish pellicle on the
surface, and in such a medium the movements of the bacillus
are most characteristic. If a single drop taken from the
surface of such a cultivation, or even a very small fraction of
a drop be added to a drop of meat broth faintly tinged with
a 1 in 2,000 solution of methyl violet and examined in the
moist chamber (see Appendix), the bacilli are seen to have
most rapid movements, especially if the temperature be
slightly raised ; Koch says, ‘‘ when they accumulate in num-
bers at the border of the droplet, and are swimming about
actively, it seems quite as if they were a swarm of midges,
between which, diving here and there, are long spiral-shaped
threads, which are also moderately active.’ The shorter
rods become more and more curved, and then stretch them-
selves out, constantly moving, oscillating and twisting.
From numerous experiments carried on by several observers
it appears that this motility is dependent partly on tempera-
ture, but chiefly on the desire for oxygen which these
organisms exhibit. In the “ hanging drop” (see Appendix),
for instance, it will be seen that the cholera bacillus separates
itself from most other micro-organisms by. its movements in
search of a supply, of oxygen, which always bring it to the
margin of the drop ; once there, however, it loses its motility
and remains in its acquired position, where its wants
are supplied without any effort on its part. The cholera
bacillus also grows on potatoes, but here it should be noted
the ‘growth does not go on at the ordinary temperature
of the room, but only when the temperature is raised
to 30° or 35° C. A fragment of blue litmus paper
placed on the surface of a slightly moistened boiled potato
indicates, by turning red, a slight acid reaction, which is due

CHOLERA. 159

(according to Koch) to the presence of an organic (pyro-
malic) acid. Here the acid reaction of the potato is the
factor that interferes with the growth of the bacillus, and it
is only when the temperature conditions become more
favourable than usual that any growth can take place.

The growth when started appears as a thin, moist “ light
greyish brown” mass, very similar in character, as Koch
points out, to the glanders bacillus as grown on potato,
except that it has not the deep or chocolate brown colour so
characteristic of that organism.

A most interesting fact has been observed in this connection. Potato
cultivations of bacilli from the various cholera epidemics vary somewhat in
colour; there are also other slight modifications in the appearance of
the different masses, by means of which a skilled observer, who has
cultivated the various organisms from the different localities and epidemics,
is enabled to distinguish the bacilli of each epidemic; and it is stated
that in some of the German laboratories the directors can take up half a
dozen potato cultivations and say, This is from the Naples epidemic, that
from Egypt, and so on through the whole series. There are undoubtedly
differences, and to the most inexperienced eye the colour of the Shanghai
cholera growth may be seen to differ slightly from that of the Egyptian and
that of the Naples growths.

On blood serum and in milk these organisms grow most
luxuriantly. They may cause slight liquefaction of blood
serum, but in milk, which forms for them an exceedingly
good nutrient medium, they give rise to no noticeable altera-
tions ; it may therefore be readily understood how deadly the
-cholera organism may become if it once finds a resting place
in milk. Its presence cannot be recognized by any peculiar
or characteristic appearance, by taste, or by smell, as it only
gives rise to a faintly aromatic and sweetish smell, which
can scarcely be distinguished, except by the most practised
nose, from the slightly aromatic smell of the milk itself.
Dr. Simpson in Zhe Indian Medical Gazette records a
naturally prepared experiment, which shows at a glance
what an important part these milk cultivations may play in
the spread of cholera. On board the ship Ardenclutha, in
port in Calcutta, ten men partook of the milk supplied by a
native milk-seller who came to the ship daily ; of these ten
men, four died of cholera, and five suffered from exceedingly
severe diarrhoea, the tenth, who escaped, had taken only a
small quantity of the milk. After careful investigation it
was found that the milk had been watered with 25 per cent.

160 BACTERIA.

of water, from a district in which it was known that several
people were suffering from cholera. A case had been im-
ported into this district on the second day of a certain
month, the dejecta from this patient drained into a tank,
near which the milkman’s house stood ; on the seventh day
of the same month the first new case occurred among the
milkman’s neighbours, and on the same day the first case of
diarrhoea occurred on board the Ardenclutha, and two days
later a case of undoubted cholera occurred in the ship.
These facts are in themselves interesting and instructive,
but they are rendered doubly so by the fact that of the other
members of the crew, fourteen in number, who had taken
none of the watered milk, not a single one was attacked by
diarrhoea or cholera.

Cholera bacilli are so little fastidious in their diet, that,
within certain limits, they are satisfied with anything, but
there are certain things at which even they draw the line—
soup mazgre, for instance, they abhor, as also sour things ;
acids are to them a deadly poison ; on the other hand, how-
ever, they are somewhat exclusive in their habits, and the
presence of other and more vulgar bacteria they cannot
brook for long. For example, if intestinal contents or
dejecta from a case of cholera are sprinkled on moist soil or
damp linen kept at blood heat the comma bacilli increase
at an enormous rate for the first twenty-four or thirty-six
hours, and under these conditions, as already seen, there
may be obtained almost pure cultivations of the specific
organism, but on the third or fourth day it begins to die
out, and other bacteria are found to be asserting themselves
most strongly.

Babes has found that at a temperature of 30° C. the bacillus will grow upon
various kinds of meat, on eggs, on various vegetables, and on moistened
bread. It also multiplies in the dejecta from healthy patients, on cheese,
in coffee, chocolate, caw sucrée, and other kinds of fluid sugars ; but it can-
not exist for twenty-four hours on acid fluids or vegetables, on mustard or
onions, or in wine, beer, or distilled water. Distilled water and soup
maigre are never sufficient for its nutriment, and if the strength of any of
the usual cultivation media be reduced to about a fortieth of the original
strength, there is gradual diminution in the number of organisms; even
in water which contains an ordinary amount of organic and inorganic
material in solution, multiplication of the comma bacillus does not take
place. Where, however, there is a very large accumulation of organic
matters, as at the margin of stagnant water, where there are large
quantities of nutrient materials “from the presence of a variety of solid

CHOLERA, 161

particles in suspension and of pieces of mud,” development occurs, “ especi-
ally on the floating sohd fragments.” Koch’s experiments, however, have
shown that cholera bacilli mixed with spring water were still alive after the
lapse of a period of thirty days. In Berlin sewer water they lived only six
to seven days, mixed with excrement only twenty-seven hours, and in cess-
pool water they were not alive at the end of twenty-four hours. Babes’
experiments, as already stated, did not bear out these statistics in their
entirety.

Infection through the agency of milk has already been
mentioned, and water has been referred to as a vehicle by
which the bacillus may be carried. Macnamara gives an
experience of infection through water, for the absolute
accuracy of which, in the case of such a skilled observer,
Koch contends there can be little doubt. In a region in
which no cholera prevailed, the dejecta from a sporadic
case of cholera became accidentally mixed with some water,
which, after remaining exposed to the heat of the sun for a
whole day, was partaken of by nineteen persons, of whom
five were attacked with unmistakable cholera within thirty-
six hours ; the evidence here given was so conclusive that
little doubt can be entertained as to the relation of the
dejecta to the water, and to the subsequent cases of cholera.
It should here be mentioned, however, that Klein, who does
not believe in the specific nature of Koch's cholera organism,
in order to prove his position, drank a quantity of fluid
which was said to contain cholera material without feel-
ing any ill. effects; and that Bochfontaine swallowed
cholera dejecta in pills without suffering any incovenience ;
but in both cases there is ample evidence that the persons
experimenting upon themselves were suffering from no gas-
tric or intestinal derangement of any kind, and that the
gastric juice was sufficiently active to prevent the multipli-
cation of the cholera organisms, and it is probable that the
fourteen men of the nineteen not attacked in Macnamara’s
case were in a similar impregnable condition. As positive
evidence to set against the above is a most striking case that
occurred in connection with Koch’s cholera courses, carried
on in the Hygienic Institute in Berlin. A doctor who had
been for eight days engaged on work in the cholera course
became affected with a slight disturbance of digestion, which
was accompanied by diarrhoea ; he continued to get worse,
and was at length so ill that he started for home, where he
was almost immediately attacked with symptoms of true

12

162 BACTERIA.

cholera, rice-water stools, great weakness, unquenchable
thirst, diminished excretion of water, except by the bowel, and
spasmodic contraction of the feet and toes. He was very
anxious to have this rice-water material examined for the
comma bacillus, and a quantity was despatched to Koch,
who was able to demonstrate in and cultivate from it, true
comma bacilli; the patient recovered. There wereno other
cases of cholera in Germany at the time, but the doctor had
been examining and making cultivations of the cholera
bacillus shortly before he was attacked, and there can be
little doubt that he had, through inattention to the rules of
the laboratory, in some way or other, ingested some of the
bacilli with which he had been working. °

The description of these cases of infection or non-infection
of individuals by cholera material introduced into the
alimentary canal, naturally opens up the way for an account
of the results obtained experimentally.

Thiersch was the first to make experiments on animals
with cholera dejecta. He fed white mice with scraps of
filter paper, impregnated with decomposing cholera dejecta,
but he found that plain filter paper was equally efficacious
in causing the illness of the white mice. He was quickly
followed by Burdon Sanderson, who carried on a similar
series of experiments, and obtained much the same results.
Dr. Richards, of Goolando, also attempted to produce cholera
in pigs by feeding them with large quantities of cholera
material, but he found that the animals died in from fifteen
minutes to two and a half hours after the administration of
the poisonous material, and the contents of the intestine of
the first pig that died, when given to another, produced
absolutely no symptoms. It was argued from this that the
poison had not multiplied, but had been destroyed or ab-
sorbed, or rendered inactive in the stomach of the first host,
so that none was left to act on the second pig ; death in the
first case being produced by rapid intoxication through the
introduction of a large quantity of some active poison, and
not by a poison developed in the intestinal canal as in the
case of true cholera. This poison, whatever its nature,
had no effect on dogs.

Following on these came a series of experiments by Koch,
who attempted to produce symptoms of cholera by feed-
ing and inoculating in various ways, mice, monkeys, cats,

CHOLERA. 163

dogs, poultry and other animals with cholera dejecta and
with comma bacilli; in‘'no case could he produce cholera,
and on search being made for the bacilli in the stomach and
intestine, they were invariably absent, apparently having
been destroyed in the stomach, as in only a few instances did
they reach the intestine at all, and then in very small
numbers, To demonstrate the difference between these and
certain other bacteria, a mouse was fed with a red micro-
organism that had been isolated in a pure condition ; and
after a time its intestinal contents were disseminated on
potatoes, on which small red colonies of the same organism
shortly made their appearance, showing that their vitality
was not appreciably affected by their passage through the
stomach. Having found that the comma bacillus was so
destroyed, Koch thought that if the organism could be intro-
duced directly into the large intestine, so as to escape the action
of the gastric juice, it might be able to retain its vitality
and multiply; the results obtained, however, were in all
cases negative, even when purgatives were previously given
to induce an altered condition of the intestine. The only
cases in which partially successful results were obtained were
those of a series of rabbits into which pure cultivations of
the bacillus were injected directly into the circulation, and
of a number of mice, in which the cultivations were injected
into the abdominal cavity ; the rabbits recovered in one or
two days, after suffering from marked symptoms of intoxi-
cation ; but the mice died at the end of the first or second
day, bacilli in these experiments being found in the blood.
Koch argued from these observations (1) that the gastric
juice of the healthy stomach killed the micro-organism ;
- (2) that even when it found its way into the intestine, it was
passed along it so rapidly that it could not take any effect on
the healthy or even slightly irritated mucous membrane, or
that it was actually very rapidly destroyed in the intestine
itself. In guinea pigs, in which he attempted to produce
cholera, he found that there was great acidity of the gastric
juice, and that the peristaltic movements of the intestine
were very strong and rapid.

It will have been concluded from what has been stated
that both naturally and in artificial cultivations there is
some poison developed during the growth of the bacillus,
which, when introduced in considerable quantities, produces

164 BACTERIA.

a condition of intoxication, which would account for the
death of those animals in which partially successful results
were first obtained. Further, it is evidently quite possible
that this intoxication may account for the very rapid deaths
which occur during certain epidemics, and also for the pre-
liminary diarrhceas by which the intéstine, in some cases at
any rate, is prepared for the reception and multiplication
of the cholera organism itself. It must, indeed, be looked
upon as one of the common causes of this diarrhcea, If
these facts be borne in mind it is possible, even without
analyzing the further experiments on animals, to understand
the immunity against cholera experienced by Bochfontaine and
Klein, and the susceptibility of the doctor who was attending
the cholera course, whilst he was suffering from indigestion
and diarrhoea. It may also be understood how infection
occurs by milk and through water after what has been
described, and in accordance with these facts is the experience
of all those who have had to deal with cholera epidemics.

Utilizing the experience gained by Koch in his experiments, Nicati
and Rietsch performed a series of operations, by means of which they
claimed they were able in a certain proportion of cases to produce
typical cholera symptoms. They introduced pure cultures of the comma
bacillus into the upper part of the intestinal canal, having previously
tied the bile duct; but as Koch pointed out later they were successful in
producing true cholera, probably, only in those cases in which the intestine
was somewhat injured during the manipulation to which it was necessarily
subjected, and its peristaltic action interfered with, and it is very naturally
objected that death might be due not to the action of the cholera bacillus at
all but to this rough manipulation. The observers themselves believed that
it was the presence or absence of bile in the small intestine which determined
the success or failure of their experiments.

In order to avoid the operation of opening into the
duodenum, Koch thought that the two great factors in
bringing about the destruction of the bacilli might be
thrown out of court, first by neutralizing the acid reaction
of the stomach by means of 5 c.c. of a 5 per cent. solution
of carbonate of soda ; and, secondly, by interfering with the
peristaltic action of the bowel. He found in test experi-
ments that the intestinal contents remained distinctly alka-
line for six hours after the introduction of such a solution.
But he also found that the organism still passed through the
stomach alive, and that it failed to produce any changes in
the small intestine, the food and the bacilli passing from the

CHOLERA. 165

stomach to the cecum in a few minutes; the peristaltic
action of the intestine causing such rapid passage of the
bacillus that it was completely powerless to produce any
characteristic symptoms of cholera, a fact the importance of
which was accentuated when it was found that the only
guinea pig in which any choleraic symptoms were observed,
and in which there was any increase in the number of bacilli
in the small intestine, was probably suffering from an attack
of peritonitis, due to the animal having aborted just previous
to the experiment—a condition in which, as has long been
recognized, there is invariably interference with the peristaltic
action of the intestine. It was possible, then, that this second
factor might be neutralized by the introduction into the
peritoneal cavity of some reagent, which would by its
action cause partial or complete paralysis of the small
intestine. For this purpose he first used opium, but he
afterwards found that alcohol was equally efficacious. After
administering the soda solution, and injecting into the
stomach of the guinea pig 10 c.c. of broth, to which one or
several drops of a pure cultivation of bacilli had been added,
he injected into the abdominal cavity tincture of opium, in
the proportion of 1 c.c. to every 200 grammes of the animal’s
weight ; the results he obtained were indeed startling.

Of course objections were raised to the method, and it was argued that some
of the animals died from an overdose of opium, some from blood poisoning,
and so on. Subsequent observers found, indeed, that the dose of opium
was somewhat too large, as some animals experimented on never awoke
from the opium sleep. Macleod, of Shanghai, found that he obtained the
best results by giving ‘‘ repeated doses of 1 c.c. or less of the tincture till
the animal was stupefied sufficiently to lie on the side or back for ten
minutes when placed in that position.” ‘‘ Several times,” he says, ‘‘the full
dose recommended by Koch had to be given, but usually a smaller one
sufficed.” . Control-animals treated, according t-Macleod’s method, and in
all respects in the same way, with the exception that they received sterilized
broth instead of cholera material, always recovered.

The contents of the bowel or dejecta from cholera patients
passed into the stomachs of guinea pigs so prepared, pro-
duced cholera symptoms and death. Giving the results of
of his experiments, Macleod states that from one of the
animals that died after a dose of cholera material, the small
gut contents were collected into a sterilized vessel, and
injected (by means of a fine indiarubber flexible catheter)
in doses of 2 cc. into the stomach of two other animals.

166 BACTERIA,

These two animals died, and the contents of their small
intestine were used in the same way, and so on through ten
generations. Of twenty-one animals thus treated, two
recovered, nineteen died. The doses varied from 0.5 to
2.5 C.C.

It is sometimes argued that in consequence of the difference of thé
symptoms in animals (especially guinea pigs), and in man, undet these
conditions of infection by the stomach, the disease cannot be the same, and
it is pointed out that vomiting and profuse diarrhoea are entirely absent in
cholera, experimentally produced. Against this objection may be put the
fact that there is usually in the guinea pig a large accumulation of fluid
transudation in the small intestine and even in the stomach; whilst in the
very acute forms of cholera the pathological appearances presented in the
intestines are almost identical with those found in man. In exceedingly
acute cases the peritoneum presents a dark colour and a peculiar glistening
mucous appearance, which is very characteristic, and seems to be associated
with the tarry condition of the blood. The mucous surface of the intestine
is usually congested, and in the intestinal canal is a considerable quantity
of serous fluid, in which are floating the small white rice-like bodies which are
merely portions of desquamated epithelium and mucus. There is usually, at
this stage, marked injection of the vessels of the solitary glands, and at”
the periphery of the Peyers patches, and the longer the patient remains alive
the more accentuated become these appearances. It is only in the later
stages of the disease, or during the period of reaction, that the swelling
of the follicles is very marked, and that more or less extensive ulceration
of the mucous membrane may be observed. The dejecta, of course, are
similar in character to the contents of the bowel, are watery owing to the
great amount of serous effusion, and contain the rice-like bodies in which
are found the comma bacilli. Bacilli are also found lying free in the
watery fluid. The dejecta have little or no odour, and when they are
allowed to stand they separate into two layers—an upper slightly grumous
layer, and a lower grey deposit. ‘‘ Neutral, or slightly alkaline, they con-
tain a very small proportion of organic or inorganic salts—one to two per
cent.—which consists of chloride of calcium, carbonate of ammonia, potash,
salts, and a small quantity of urea. They contain little or no albumen, and
during the first day or two no bile pigments.”

If microscopic sections of the lower part of the small
intestine be made, and stained according to Léffler's
method,' the bacilli may be seen with the aid of a high

« First stain in a solution of Leonhardi’s Dresden methyl violet ink, and
then in an alkaline solution of fuchsin, which is made up as follows :

100 c.c. aniline water.

1 c.c. of a solution of one per cent. of caustic soda.

2 grammes of solid fuchsin.

The whole is well shaken, after which the slo gag may be left in it for
twenty-four hours ; the sections are then washed in distilled water acidu-
lated with a drop of acetic acid, dehydrated in absolute alcohol, cleared up
in cedar oil, and mounted in Xylol balsam.

CHOLERA. 164

fiagnifying power (x 800) beneath the loosened epithelium
of the villi, in the follicles, and in the surrounding connec-
tive tissue. They are especially numerous near the ileo-
cecal valve, and are distributed, not in masses, but usually
in ones, twos, and threes, scattered thickly through these
tissues.?

On post-mortem examination of the guinea pigs, in which cholera had
been experimentally produced, ‘“‘the blood was fluid, thicker and darker
than natural, the tissues of the thoracic and abdominal walls were re-
markably dry, the small intestine was throughout distended, congested, and
paralyzed-looking, and occupied a much larger proportion of the abdominal
cavity than usual. The czecum was distended with fluid or semi-fluid con-
tents. If the animal died early the fluid was not quite clear in the small
gut, there being present traces of food; still the watery character was very
manifest, and mucous flakes were abundant. If the animal died on the
second or third day no food remains were to be seen, and the fluid in the
small gut was the counterpart of the typical cholera stool of man. In
either case the comma bacilli were demonstrated microscopically, and by
cultivation as in man.”

While the organisms in the broth injected could be frequently counted in
a microscope field, in a drop of the small bowel contents from an animal
having received such broth, the bacilli might be so numerous that counting
them, without dilution of the fluid examined, was an impossibility. On
floating the bowel in water, the stripping of the epithelium could be well
demonstrated ‘‘immediately after death,” a fact which appears to dispose of
Macnamara’s assertion that this separation of the membrane is always a
post-mortem appearance. In animals which died about the eighth day
the appearances were very similar to those met with in cases of Asiatic
cholera, when the patients have succumbed during the stage of reaction,
and no bacilli can be found in the intestinal canal or in the surrounding
tissues. In‘ the earlier stages the bacilli can be demonstrated in sections
lying under the partially-detached epithelium, or in the lumina of Lieber.
kuhn’s follicles.

In fluid cultivations, as has already been mentioned, the
comma bacilli, or little groups of them, assume various forms,
all of which, however, may be looked upon as made up of
more or less perfect spirals. At the point of division, just
before division take places, there is a small clear space which,
by some authorities, has been looked upon as a spore. In some
of Macleod's preparations this appearance was so marked that
it was difficult to convince oneself that it was not due to
actual spore formation, but as drying for twenty-four hours
completely destroyed the activity of the bacillus in which
these clear spaces were most marked, and as they could not

* Itisnotmy intention to give the complete pathological anatomy of cholera,
except so far as it is associated with the presence of the cholera bacilli.

168 BACTERIA,

be stained by any of the ordinary spore-staining methods he
was convinced that he was not dealing with true endospores.

On three occasions Hueppe-was able to demonstrate in moist
chamber cholera cultivations on agar-agar small brilliant
spore-like bodies which he called “ Arthrospores.” These
were placed sometimes at the extremities, sometimes in the
middle of the comma bacillus. They appear to develop
where the conditions of nutriment, temperature, or moisture,
are somewhat unfavourable to the development of or
to the very existence of the bacillus. This is proved by
the fact that the protoplasm in the bacilli, in which these
arthrospores are present, is stained very imperfectly with
methyl blue; the clear bodies themselves taking on a brownish-
red tinge. Hueppe maintains that he has observed these
bodies germinate out into comma bacilli, and he holds that
they are much more resistant to various germicidal reagents
and unfavourable conditions than the vegetative form of
bacillus. Several observers have tried to repeat these ex-
periments, but, as any apparent spores that have been
formed invariably failed to resist the action of drying, such
“‘ Arthrospore ” containing bacilli, must, for the present, be
“sail ‘upon as involution forms, similar to those described
below.

When these are formed the ordinary dimensions and forms of the comma
bacillus as previously given are sometimes departed from, and in old agar-
agar cultivations, and in old gelatine cultivations in which the surface has
become somewhat dried, spirals are frequently found of which the con-
stituent bacilli are considerably thicker than ordinary comma bacilli, the
large curved forms remaining attached by delicate terminal filaments. In
most cultivations there are slight modifications in both form and size of
the cholera bacillus, which appear to be associated with the difficulty or
facility with which they obtain food, water, and oxygen, and also with the
temperature at which they are grown. For example, if ten per cent. of
alcohol be mixed with the gelatine, or if the gelatine medium contains
but little beef extract or peptone, and if the temperature at which the
media are kept be very high or very low, say 45° C. on the one hand
or 20° C. at the opposite extreme, a large proportion of spiral fila-
ments is formed; whilst if the medium be well adapted to the nutri-
tion of the organism, and the cultivation be kept at a temperature
of 36° C., short comma bacilli are almost exclusively developed. It is
a curious fact, however, that, once developed, these various forms may
persist cr pass on their characters unchanged for one or two generations,
even though they are now inoculated into ordinary fresh peptonized gelatine
medium, and Cornil and Babes say that if there are inoculated simul-
taneously, on peptonized gelatine, a fresh culture of filaments, another of
ordinary comma bacilli, and a third with the short and only slightly curved

CHOLERA: 169

bacteria which are formed under various conditions, the same typical naked-
eye appearances of the cholera bacillus are always obtained in each case,
but on microscopical examination it is found that the first still develops in
the form of spiral filaments, ‘the second as comma bacilli, and the third as
short bacteria, and that these characteristics are carried on to the fourth
generation. :

With all these modifications in form they still produce typical cholera
symptoms when injected into the alimentary canal of animals. In addition
to these changes there frequently appear at one extremity of the bacillus
small cystic dilatations which are due, according to Virchow, to a kind of
cedematous degeneration. As these dilatations make their appearance in
certain bacilli, others of them shrivel up, and tadpole and spindle-shaped
organisms are formed which may break down into small granules and frag-
ments. In the later stages of degeneration these various involution forms
are completely sterile. In the earlier stages, so long as they take on the
aniline colouring matter pretty freely, successful inoculations may usually
be made, but if the unfavourable conditions are continued in the new
cultivations, the organisms soon lose both their power of taking up the
staining reagents and of reproduction. :

It has been stated that the comma bacillus is entirely an
zrobic organism, and there can be little doubt that its vege-
tative activity is much more marked when the organism is
grown in contact with the oxygen of the air. It is certainly
more resistant to the action of germicidal agents, and exhibits
movements in a much more marked degree. It had been
observed, however, that comma bacilli do not cease to multi-
ply even when their supply of free oxygen is entirely cut off,
but under these conditions, as Wood has pointed out, the
bacteria are much more sensitive to external influences, and
are very readily destroyed by acids and other germicidal
agents. He has found, however, that where free oxygen is
cut off, as Pasteur had observed in the case of other ferments,
the bacilli produce a relatively much larger quantity of the
specific toxine, or poison, than when oxygen is present,
and by a number of ingenious experiments he showed that
when they were grown on albuminous substances with com-
plete exclusion of oxygen, and of substances from which
oxygen could be easily derived, the cholera poison was pro-
duced more energetically and more rapidly than under the
ordinary conditions of zrobic cultivation ; probably because,
under these conditions, much larger quantities of albumen
must be split up to meet the energy requirements of the
organism.

Wood and Hueppe from this argue that the bacillus gives
rise to such important changes in the intestine because there

176 BACTERIA.

we have quantities of albuminoid material which can bé
rapidly broken up by the action of the bacilli even in the
complete, or almost complete, absence of oxygen, which is
supposed to rule in the intestinal canal, as a result of which we
have the formation of large quantities of toxine and rapid in-
toxication. But as they are voided in the dejecta after growth
under these conditions, the cholera organisms are more easily
destroyed than at any other time or under any other condi-
tions.

It is very significant that in cholera, yellow fever, and typhoid fever,
three diseases in which the manifestations are chiefly in the intestinal canal,
and in which the evacuations apparently contain the living poison, dzvect
infection from the stools appears to be the exception rather than the rule,
and Wood explains this as due, at any rate in part, to the state in which the *
specific organisms associated with these diseases are present in the stools,
and are dependent upon the conditions present in the intestinal canal.
Should they find their way during this stage into the stomach of the living
person they, being more susceptible, would be much more liable to be
destroyed by the acid gastric juice, or to undergo attenuation ; but if they
are allowed to live outside the body, even for a short time, they become
more resistant in character, they multiply more readily, they are less par-
ticular about the nature of their food, and consequently they are much more
dangerous.

CHAPTER IX.
CHOLERA (continued).

Pettenkofer’s researches—Saprophytic and Parasitic Stages of Cholera
Bacillus—Temperature Conditions—Relation to Epidemics—Moisture
—Ground Water—Flushing—Cholera in Shanghai Endemic but
Intermittent—Cholera Endemic at the Mouth of the Ganges— Vitality
of Cholera in Old Cultures—Relation of this to Quiescent Periods
during Parts of the Year— Gastro-intestinal Irritations prepare for
Cholera—Chinese Vegetables—Cholera Poison Formed in the Intestine
absorbed into Body—Inoculation against Cholera—Gamaleia’s Experi-
ments—Germicides useful in attacking the Cholera Bacilli—Water
Supply, Pilgrimages, Feasts predisposing to Cholera-—Quarantine
except in Harbours Useless—Time and Place Dispositions.

PETTENKOFER’s indefatigable researches on the relation of
ground water and the drying zone to cholera epidemics have
thrown much light on many obscure points, and have opened
up the way for further work. He holds that the increase of
cholera is due entirely to the increase of the “drying zone”
near the surface of the soil—z.e., the lowering of the level of
the “ ground water,” and that the rise and fall in the level of
this ground water is the principal factor in the production of
conditions necessary for the outbreak of epidemics of various
kinds. On these grounds he has itaken up a very strong
position against the spread of cholera directly from patient
to patient. There can be little doubt that many of Petten-
kofer’s observations are entirely in accordance with this view,
but equally can there be little doubt that all his interpreta-.
tions of the facts he has collected are not entirely accurate,
although, as Hueppe points out; Wood's observations offer
solutions of questions that have hitherto been unanswerable.
The instances are almost innumerable in which there has been
a most remarkable immunity against the passage of cholera
from individual to individual ; where the dead bodies have
been buried immediately the disease has afterwards occurred,
not in those who have had to carry out the actual burying of

172 BACTERIA.

the dead cholera patient, but in those to whom has fallen the
duty, a day or two later, of washing the soiled linen used by the
patient during life. In the first case the attendant is exposed
to the action of the bacillus as it comes from the patient, but
at a time, it is argued, during which the organisms are more
readily destroyed, either in the dejecta, or by drying ; but the
attendant who has to wash the soiled linen may be attacked
by the bacillus after it has had ‘time and opportunity to
develop on the damp sheets, in the presence of air, and when
it has acquired a greater power of resistance, and has become
much more dangerous. It has now, in fact, lost its anzerobic
habit, and has become adapted to its new surroundings, with
the result that it is much more resistant and is better qualified
to live outside the body and to resist the action of ordinary
germicidal reagents, or of the acid gastric juice, should it find
its way,.into the stomach of a fresh host.

In similar fashion may be explained the fact that when
the drying zone becomes limited, the cholera bacillus appears
to die out more readily. It appears that the micro-organism
passing directly from the feces into very damp soil contain-
ing insufficient oxygen to satisfy its saprophytic requirements
is, on accountof its feeble resisting powers, “suffocated” at once,
or within a very short period. Where, however, the depth of
the drying zone increases, there is more air (oxygen) in the soil,
the organisms are more able to multiply in its presence, and,
taking the field against other putrefactive organisms, gradually
become more and more hardy, acquire the zerobic and sapro-
phytic habit, and thus become more dangerous to the inhabi-
tants of the locality in which all this occurs. It must be re-
membered, however, that the term “drying zone” is entirely a
relative one, and that it may still contain, as it usually does,
sufficient moisture for the wants of the cholera organism. That
the organism takes some little time to pass from the zrobic to
an anzrobic condition is evident from Koch’s early experi-
ments, in which he made plate cultivations of cholera bacillus,
and then, before the gelatine was perfectly set, covered about
one-third of the surface with exceedingly thin glass—cover
glass thickness—or split mica. He then found that colonies
grew as usual, and became visible to the unassisted eye in the
uncovered portion of the gelatine and for a very short distance
under the covering plate (2 mm.). He observed, however,
that where the air (oxygen) was cut off, the colonies did’ not

CHOLERA, 173

increase in size sufficiently to allow of their being distin-
guished without the aid of a lens; the power of growth to
the size of a colony visible to the naked eye being attributed
by Koch to the presence of the small quantity of available
oxygen that remained in the nutrient gelatine—growth
ceasing as soon as this oxygen is exhausted,—the colony
remains of small size.

A second experiment was made by inoculating nutrient
jelly contained in a small glass with comma bacilli ; this “was
placed under the receiver of an air pump, another glass pre-
pared in a similar way being placed outside the air pump as
a control experiment. It then appeared that those bacilli
under the air pump did not grow, while those outside the

; receiver did well. But if those which had been under the
receiver were afterwards exposed to the action of the air, they
then began to grow. They therefore had not been destroyed ;
they only wanted the necessary oxygen to be able to continue
their growth. A similar result is obtained when cultivations
are placed in an atmosphere of carbonic acid ; whilst those
cultivations placed for control purposes outside the carbonic
atmosphere grow in the usual manner, those subjected to the
action of a stream of carbonic acid remain inactive and undergo
no development. Here also, however, they do not die; for
after they have been exposed to the carbonic acid for a con-
siderable time, they again begin to grow as soon as they are
taken out of it.” In place of carbonic acid, hydrogen may
be used, and the result is much the same.

It cannot be assumed from these experiments that the organisms require
to obtain free oxygen from the air in order that they. may continue their
growth, for, as has been pointed out by Hueppe and Wood, if they are
grown on a suitable medium, such as unchanged normal albumen, they are
able not only to exist, but to produce unusually large quantities of their
specific poison, as they are able by the dissociation of such a medium to
obtain all that they require for their growth and development.

All that can be argued from Koch’s experiments is that the bacillus has
a saprophytic stage (although this was apparently not at first recognized by
Koch), during which it grows and multiplies, but only under ordinary con-
ditions where free oxygen can be obtained from the atmosphere. During
its parasitic existence it becomes so far modified, that, still growing with
great vigour, it produces its toxine more easily or in greater quantities in
the absence of oxygen, other conditions being favourable. During this
stage, however, it is much more readily affected injuriously by acids and
other germicidal reagents, and is therefore much more easily destroyed
than when it has had time to acquire its full saprophytic faculties,

174 BACTERIA.

Mention has been made of the conditions as regards
temperature that are necessary for the growth of the cholera
bacillus when other conditions are slightly adverse. As the
result of an extensive series of experiments, first carried on by
Koch, and afterwards repeated by other observers, it has been
found that the comma bacillus flourishes most luxuriantly,
and is most productive, at a temperature ranging between
30° and 4o° C., although it can grow at a much lower tem-
perature, and even at several degrees above the higher point
mentioned. At 20° C. it flourishes luxuriantly upon a
peptonized gelatine medium.

As early as 1884 Koch described the growth of the
cholera bacillus at a temperature of 17° C., but as might
be expected the growth is not nearly so prolific, and it is
certainly considerably less rapid than at a slightly higher
temperature. A temperature below 17° C. seems to be
inimical to the growth of the bacillus, as at 16° C. develop-
ment may be said to have almost ceased. It is, however,
remarkable that an intense degree of cold does not deprive
the organism of its power of growing, for, if after being ex-
posed to great cold for some time, it is again placed under
favourable conditions, it appears to regain its powers of
rapid multiplication. Koch, to test this, submitted a culti-
vation of the bacillus for one hour to a temperature of 10°
C. (10° C. below freezing point), with the result that the
nutrient medium was completely frozen. This was now
thawed, and a cultivation made at a higher temperature and
under favourable conditions; the bacillus began to grow
again almost immediately, and appeared, indeed, to have lost
none of its vitality. These experiments accord well with,
and verify the observations that have been made on, the
appearance and spread of cholera epidemics in the hot and
cold seasons. Most of the cholera epidemics seem to have
attained their maximum virulence during or at the end
of the hot season of the year. In those regions that are
periodically visited it usually breaks out in the autumn, when
the external temperature is most favourable to the growth of
the bacillus as a saprophyte, and when in consequence the
micro-organism is enabled to live for a longer period outside
the body, to give rise to numerous progeny and thus to
multiply the possible sources of infection. On the other
hand epidemics are of frequent occurrence, even in the

CHOLERA, 175

coldest seasons of the year ; nay, in some cases the appear-
ance of the cold damp season is the signal for the outbreak
of a cholera epidemic, because not until the return of the
cold weather, after the hot dry summerand autumn months, is
there that dampness of soil and atmosphere which 1s essential
for the existence of the organism, in addition to which the soil
remains at a comparatively high temperature for some time
after that of the atmosphere has fallen, Again, the external
cold, though it may paralyse the organism for a time, does
not destroy its vitality, but, keeping the organism in a
passive condition, actually enables it to retain this vitality
for a considerable period, so that, when it finds its way into
dwellings, in which the atmosphere, owing to bad ventilation,
is not only raised to a high temperature, but also contains a
considerable quantity of moisture and organic matter, it is at
once introduced to conditions that are essentially favourable
to its saprophytic existence; it again begins to thrive
luxuriantly, and becomes a possible source. of infection.

The fact must not be ignored, also, that seasonal temperature plays not
only a most important part in the determination of the amount of moisture
in the air and in the soil, but also in the production of currents of air by
which the organisms may be carried from point to point (though these
currents, when the air is dry, or in hot, clear weather, play but a small part
in the dissemination of the disease), and by its effects on certain kinds of
vegetation, which indirectly have bee proved to play an important part in
the distribution of the cholera organism.

On the effect of heat on the amount of moisture in the
atmosphere, and consequently on the depth of the ground
water, it is scarcely necessary to speak at any great length,
but there can be little doubt, from Pettenkofer's observations,
that such factors do play a most important part in condition-
ing the growth and multiplication of the cholera organism.
The rapid removal of stagnant and upper ground water (if
complete) is inimical to the saprophytic growth of the
cholera organism, as even organic matter in a state of dust is
absolutely useless as a nutrient material for the cholera
bacillus. Where, on the other hand, there 1s a considerable
quantity of moisture in the atmosphere, even though the
temperature be high, stagnant water is often found.

Koch says: ‘* On the surface or in the ground, in marshes, in docks, which

have no outlet,in places where the ground is formed like a trough, in sluggish
rivers and the like . . . there a constant nutrient solution can be formed and

176 BACTERIA.

may accumulate in the neighbourhood of animal and vegetable decaying
matters most readily and give the micro-organisms opportunities for growth ;”
and he further says, ‘* that whenever the water has a swilt current, or is in
a constant state of change, both on the surface and in the ground, these con-
ditions occur less easily, or sometimes not at all, for the continuous current
prevents a localized concentration of nourishment in the fluid sufficient for
the pathogenic bacteria. The connection between the sinking of the
ground water, and the increase of many infective diseases, we might explain
thus : that with the sinking of the ground water, the current which exists in
it becomes very much lessened, Besides, the mass of water lying superfici-
ally at disposal will be considerably diminished, and therefore such a
concentration as I have described as necessary for the growth of the
bacteria will be produced much sooner.”

These conditions are necessarily closely associated with
temperature, with the nature of the sub-soil, and the pre-
vailing winds and air currents, and the consequent affection
of the quantity of moisture in the atmosphere, and the pro-
duction of rain must also be taken into account, as even the
transmission of the cholera organism is affected or interfered
with by great dryness of the atmosphere. Where there is
great dryness of the atmosphere the small quantities of
cholera dejecta, which are likely to be left unnoticed, and
therefore left disinfected by the attendants of the patient are
so rapidly dried that they are speedily rendered inert, and
cannot convey the infection further ; whilst the materials on
which, in ordinary circumstances, they would thrive, are so
dried on the surface that although the organisms. may find
their way on to them in a living condition, there is not
sufficient moisture left to allow of the development of the
probably somewhat weakened organism.

As Fliigge points out, such conditions “ only occur where there is a very
great deficiency in the saturation of the air with moisture—for example, in a
desert . . . It is conceivable that in.a desert climate such as is present in
Mooltan and Lahore during the greater part of the year, and where every-
thing dries up, as it were, under one’s eyes, the conditions favourable for
the spread of the cholera may only be present at most during the some-
what moister or so-called ‘rainy’ season (July to October).”

Extreme wet may, however, exert an influence in interfer-
ing with the saprophytic growth of the organism, especially
if the rains be heavy and continuous. The conditions men-
tioned by Koch, as inimical to the collection of organic
material on which the organism may grow, are brought into
play, infective dejecta are removed from the surface soil,
from the surface drains and from the sewers, rapid and con-

CHOLERA. 177

tinuous currents are created, organic matter of all kinds is
washed away, air is driven out from the subsoil owing to the
rising of the ground water, and all the conditions become
unfavourable for the growth of the organism. The fact that
vegetation has been mentioned, as associated with climate in
the production of cholera, requires some explanation, which
will be given later. Dr. Macleod, writing on the subject of
climate itself, says :

‘* Cholera makes its appearance in Shanghai every summer with start-
ling regularity, Before the end of July it is hardly met with, by the end of
August it is well marked, in September it is in full swing, not quite so
virulent in October, and in the beginning of November an occasional case
may be heard of, after which time it disappears entirely till the following
late summer. For twenty years this has gone on with unfailing regularity
under the observation of medical men now resident, and for how long
previously no one can estimate.”” He then goes on to describe the weather
in June as damp and hot, in September as hot, damp, and muggy, in
October as cool and wet during the first part, and as frosty towards the end,
“at Christmas there is usually ice, there may be snow.”’ He then says, ‘* so
far as temperature of the air is concerned, we enjoy tropical heat for nearly
a couple of months before the disease breaks out ; it is most virulent in the
hot, damp September, and does not disappear until after the hoar-frosty
mornings are experienced ” (the end of October).

Dealing with the same subject Koch has stated that in
no part of the world, of which the climate, the geology, and
the epidemiology are known, does cholera occur all the year
round except in the province of Bengal, and he says :

‘* All authors are agreed, that the delta of the Ganges is the true home
of cholera, and I have come to the conclusion that this is the case, and that
there are no other places of origin of cholera in India. For the only district
in India, where cholera prevails continually year after year in a uniform
manner, is the delta of the Ganges. In all other places it shows marked
variations, or it may even disappear altogether for a shorter or a longer time.
In certain places, for example in Bombay, it never entirely disappears, but
it is highly probable, that on account of the unusually active trade with the
rest of India it is constantly being imported there afresh.” He then
describes this tract as a perfectly flat country only slightly raised above the
sea level, which, during rainy seasons, is almost entirely under water in the
lower part of the delta, where the “Ganges and the Brahmapootra break up
into a network of water courses, in which the sea water, mixing itself with
the river water, flows hither and thither with the tide, and at flood time
places large tracts of the Sunderbunds under water. A luxuriant vegetation
and an abundant animal life have developed in this uninhabited region,
which is inaccessible to man not only on account of the floods and the
numerous tigers, but is avoided principally on account of the pernicious
fever which attacks everybody who remains there even for quite a short
time. One can easily imagine how dense the vegetable and animal matter

13

178 BACTERIA.

ny

is which is given up to decomposition in the marshy districts of the
Sunderbunds, and that here an opportunity is afforded for the development
of micro-organisms, such as exists in scarcely any other place on the globe.
Peculiarly favourable for this are the regions between the inhabited and
the uninhabited parts of the delta, where the excrements of an unusally
thickly populated country are washed away by the current, and, flowing
here and there, are mixed with the brackish water of the Sunderbunds,
already teeming with decaying matter. Under these peculiar conditions
quite a distinct fauna and flora of micro-organisms must be developed
there, to. which in all probability the cholera bacillus belongs. For every-
thing points to the cholera having its origin in this district. All the greater
epidemics have begun with an increase of cholera in the southern portion
of Bengal. Jessore, from which the first intimation of the epidemic of
1817 came, lies on the borders of the Sunderbunds ; and Calcutta, which is
now the fixed home of the cholera, is connected with the neighbouring
Sunderbunds by a marshy and sparsely inhabited tract of land. Now the
comma bacillus finds in this district, contiguous to its presumptive home,
the most favourable conditions imaginable to implant itself and to spread
from one individual to another.”

In the large towns in this cholera region there appears to
have been no diminution in the mortality from the disease
during the British occupation ; and until a new water supply
was obtained and it was no longer necessary for the natives
and others to take their water from tanks, from the Ganges,
or from the Hoogley, even a better sanitary and drainage
system appeared to have little effect. Once this supply was
obtained, the mortality fell to about one third, and even a
considerable portion of that third is to be accounted for by
the fact that it was, and is, very difficult to impress on the
natives the necessity of avoiding the old contaminated
sources of water supply. Although Koch brought out these
remarkable statistics he is still, as he says, “ not a supporter of
the exclusive drinking-water theory,” and he considers “ that
the ways in which cholera can spread itself are extremely
varied, and that almost every place has its own peculiarities
which have to be thoroughly investigated, and the regula-
tions which are to serve for the prevention of infection in the
place in question must be drawn up accordingly.” It is a
remarkable fact that Koch never speaks or appears to think
of the possibility of the existence of the cholera bacillus as a
saprophyte. If it enters food or water it is the result of an
accident and the organism can remain there, capable of
development only for a short time and under exceptionally
favourable conditions. Thus he contends that: cholera is
endemic in the Ganges only because there is a complete

CHOLERA. 179

chain of cases from one year’s end to the other by which the
infection is handed on. Hueppe, on the other hand, main-
tains, as above stated, that the bacillus can have a distinct
saprophytic existence, and. points to the fact that in places
analogous to the delta of the Ganges it has a marked
tendency to become endemic.

As an example of this we may summarize the results of
Macleod’s observations on cholera in Shanghai. He says that
“the remarkable regularity of the time of outbreak, period
of duration, and time of cessation of the disease, so far as
I am aware, has no parallel on record.” The country round
Shanghai is strikingly like that of the deltas of the Ganges
and the Nile ; “in each there is the alluvial deposit, rich in
organic matter, and the high ground-water level, yet in
the Ganges delta the disease is prevalent all the year round ;
at the mouth of the Yang-Tssze it occurs with the regularity
of a crop at the same season yearly ; in the delta of the
Nile it occurs only occasionally but at the same season as at
the mouth of the Yang-Tsze. The two latter regions have
at least weekly communication with India. Some causes or
combination of causes are prevalent all the year round at the
mouth of the Ganges; at one season every year, viz, late
summer and autumn at the mouth of the Yang-Tsze ; and
at the same season, but not every year at the mouth of the
Nile.”

There must then in these three regions be perfectly distinct but local
conditions which must determine the difference in the behaviour of cholera
in these various regions, The general characters of the soil, the ground
water, and position are much the same, but there are well-marked differ-
ences as regards temperature, population and methods of cultivation. In
the delta of the Ganges cholera is absolutely endemic, it is never absent ; the
poison, whatever may be its nature, is therefore always present. ‘“ From
the position and climate, the temperature of the soil varies little throughout
the year, and is never so low that some vegetable growth is not active.
At the mouth of the Yang-Tsze (where cholera is prevalent only during
certain parts of the year), the poison is endemic or is introduced shortly
before the time that cholera breaks out each year, in which case it
is curious that it should always be at the same time, there being no means
of communication with a cholera infected country opened up specially at
that time, communication wlth India being weekly. Here the climate
admits of a greater range in the soil temperature than at the mouth of the
Ganges, there being great extremes of both heat and cold. The soil heats
more slowly than the air, hence perhaps it is the determining cause for the
spread of the disease in the late summer and autumn, as already described,
and of its absence in winter and spring.” It is evident from this statement

180 BACTERIA.

that cholera does not show itself during the summer when the air tempera-
ture is at its maximum, but only when sufficient time has elapsed to allow
of the rising of the ground temperature or that of the earth, and as a matter
of fact the disease occurs in Europe during the autumn months often when
the general or atmospheric temperature has actually begun to fall, but when
the earth temperature has attained its most equable maximum, when of
course we have the optimum temperature conditions for the growth of
the cholera organism in the soil. ‘‘ At the mouth of the Nile the con-
ditions are much the same as near Shanghai, except that the extremes of
heat and cold are greater at the latter. Both have at least weekly communi-
cation with India, both communicate through the tropics ; whilst Shanghai
is at a distance of at least three weeks in time, Egypt is but two. The
regularity of the yearly outbreak at Shanghai points to an endemic pvison,
the irregularity at the mouth of the Nile to the occasional introduction of
the poison. Late summer is also the period for the Egyptian epidemics of
any extent.” :

Bearing on this, we have the fact that a cholera
epidemic may remain dormant for months or a whole
winter ; especially as the organisms retain their capacity
of reproduction if they -are kept moist and are supplied
with oxygen. According to Nicati and Reitsch, cholera
bacilli were found alive eighty-one days after they had
been placed in the harbour water of Marseilles. Koch
found cholera bacilli capable of reproduction after they
had grown one hundred and forty-four days on agar, but
in one hundred and seventy-five days these cultivations
were found to be dead. Macleod found that pure culti-
vations renewed but once a month retained their virulence
for a year. Taking all these facts into consideration, it
is easy to understand how in Shanghai the organisms
may remain dormant for a certain period, and then under
favourable conditions begin to grow again with increased
activity. As Koch puts it—‘‘ One can easily imagine that in
superficial layers of earth, in marshes, and so forth, the cholera
bacilli may find conditions in which they can exist preserved
from death for five months or even longer, just as well or
even better than on our moist agar jelly.” Here then are
three places which, although they haye certain features in
common, are characterized by certain differences, all of which
appear to depend on climatic conditions. In the one case
the cholera is prevalent during the whole year ; it is in fact
endemic and continuous. In the second case cholera appears
with the utmost regularity at certain seasons of the year, and
then as regularly disappears. In this case the disease appears

CHOLERA. 181

to be endemic, but on account of some local circumstances
connected with temperature and vegetation it is developed
only in crops--at harvest time, as it were. In the third case,
in the Nile delta, the disease is extremely intermittent in its
outbreak, and occurs only in those years in which the condi-
tions as regards temperature, moisture, &c., are specially
favourable for its development. In connection with local
peculiarities as favouring the growth of the bacillus and the
spread of the cholera, Macleod makes several very interesting
observations, some of which bear out in a most remarkable
manner Koch’s statement as to the general laws that
govern the spread of the disease, and also as to the minor
local factors that determine individual outbreaks. - The
population in Shanghai he divides into three classes : resident
foreigners, amongst whom deaths from cholera average less
than 2 per 3000 per annum ; amongst the seafaring foreign
population the average is very much higher, as of the sailors
who come to port there die from cholera from 15 to 30 per
1000 yearly ; whilst among the Chinese, even in the settlement,
there are rarely fewer than from 200 to 300 deaths in a single
season. What part then do local peculiarities and customs play
amongst these three sets of people? It is a fact, generally
recognized, that any disturbance of the digestive function is
the principal predisposing cause of the disease in a cholera
outbreak—a fact that is explained by the absence of the
ordinary amount of acid from the gastric juice in . these
cases ; such gastric derangement is specially met with after a
bout of drinking. Macleod says, “ Among the sailors a night
ashore is the usual precursor of the disease, and commonly
that indicates a large consumption of liquor.” This, how-
ever, does not account for all the cases that occur, and he
says, “Occasionally men who have not been ashore are
attacked. A sailor belonging to an American ship was
attacked two or three days after arrival ; he had not been
ashore, the ship had touched at no port for weeks before, and
the water supplied on board had been in use during the
voyage. The captain reported that he had specially en-
couraged the men to eat fresh vegetables largely, and that
he had seen the man referred to sitting on deck munching
lettuce the day before he became ill (three of this ship’s
crew died of cholera). These vegetables had been supplied
by Chinese bumboats.”

182 BACTERIA.

The custom amongst the Chinese “‘is to carefully collect human excreta
and add these to water ; this mixture is then used for watering vegetables,
&c., so that the leaves come in for a fair share. All European and Chinese
night soil is collected daily and carried away to be used for agricultural pur-
poses. No night soil finds its way into drains, and there are no water closets,”
and all refuse not used directly for watering vegetables is carried into the
country and used for agricultural purposes, so that if the poison be in the
excreta, in China at least, its deposition in the ground is secured, and it has
certainly been discovered empirically by European residents that vegetables
cannot be taken with impunity. ‘‘ Chinese vegetables are regarded with
suspicion during the cholera season, more especially such as are uncooked.”

When the relation of the comma bacillus to the dejecta
from cholera patients is borne in: mind, there can be little
wonder that the consumption of, Chinese lettuces should be
followed by an attack of cholera. In connection with the
conveyance of cholera by means of drinking water, it may be
objected that the Chinese do not drink water as a rule, but
indulge in weak tea ; but here again it is stated that water
for household use is stored in an earthenware or wooden
vessel placed in or near the kitchen, and into this vessel:
others are dipped from time to time. The water is obtained
from creeks or wells. Vegetables may be seen hanging over
these vessels or lying on tables, so that though the vegetables
are cooked, tables, dishes, cloths, and all that come in con-
tact with uncooked vegetables furnish abundant opportunity
for contamination of food after it has been cooked, where
such filthy habits prevail as amongst the Chinese. They
do not use milk as do foreigners, but they supply it, and from
what has been said of vegetables and Chinese habits, unless
the supply is known to be well cared for, it cannot be regarded
as beyond suspicion of forming a vehicle for the distribution
of cholera, typhoid, and other poisons. It is evident, then,
that although no one set of factors alone can be looked upon
as determining or explaining all outbreaks of cholera, the
bacillus, so far as our present knowledge goes, must be
looked upon as the essential factor in the causation of the
disease ; this bacillus, like every other organism that we
know throughout the whole animal and plant kingdoms,
being, to a great extent, dependent on its environments for
its very existence. In considering this question let it be
understood most distinctly that though the bacillus is proved
to be the cause of the disease, it is not necessary to assume,
as some people seem to suppose, that the careful, and in

CHOLERA. 183

themselves complete, observations of the great epidemio-
logists of this and other countries are valueless ; and as in
time a more perfect knowledge of the conditions favourable
or unfavourable to the growth of the organism is obtained,
it may be expécted that all observations made: with care by
those whose only desire is to get at the truth, will fit in and
take their place in a large and comprehensive scheme
through which there will ultimately be a possibility not only
of understanding the disease more thoroughly, but of re-
meving or combating its causes.

It has already been pointed out how Pettenkofer’s obser-
vations may be made to agree with Koch’s ; it has been seen
that the apparently contradictory statements on the subject
of the spread of cholera along the lines of pilgrimage and trade
have been more or less satisfactorily explained and reconciled
on the assumption that the bacillus is the cause of the disease.
Then, too, the well-known facts of the association between
cholera and gastric and intestinal disturbances of various
kinds ; between it and consumption of various articles of food
and of water from sources which are probably contaminated,
or in which the presence of the cholera organism has been de-
monstrated, and the facts connected with climatic conditions,
ground water and subsoil drainage, temperature and special
local conditions, may all be satisfactorily explained on Koch’s
comma bacillus theory. These explanations, taken with the
fact that the comma bacillus is invariably found during cer-
tain stages of the disease, that it has never yet been found in
any but typical cases of cholera, that with it the disease has
been produced in animals, experimentally, and one might
also say in man, though accidentally (at the cholera course
in the Hygienic Institute in Berlin), make it impossible for
us to shut our eyes to the fact that in the cholera bacillus we
have the only suggested causal agent that will allow of a
satisfactory explanation of the mass of observations made up
to the present. It had already been demonstrated that the
bacillus was found only in the intestinal canal, when it
naturally suggested itself to Koch that the symptoms of
cholera were to be explained by the theory that in this
disease there was absorption from the intestine of some
soluble poison produced by the bacillus 2% sz ; that there
was, in fact, a local formation of the poison, but a general
absorption into the system (the “ Intoxication” theory). This

184 BACTERIA.

theory was at once accepted as being from every point of
view more far-reaching and more satisfactory than the older
one which explained all the symptoms by referring them to
loss of water through the excessive intestinal discharges ; a
symptom or effect was, in fact, looked upon as the cause of
other symptoms. The earlier observations on ptomaines and
sepsines, and Pasteur’s and Hansen’s later observations on
ferments, naturally led to a search being made with the
object of finding out any specific products of the cholera
organism. Koch himself records the fact that he succeeded
in preparing cultures of the comma bacillus which were so
intensely poisonous, that when injected into animals, either
subcutaneously or into a peritoneal cavity, there were set up
in a few minutes all the symptoms which occurred in
animals suffering from cholera a day or two after infection—
“paralytic weakness of the hinder extremities, coldness of
the head and legs, and prolonged respiration, a condition
which usually leads after some hours to death.” Buchner,
who demonstrated, as he believed, the formation of butyric
acid during the growth of pure cultivations, was unable to
corroborate by any experiments of his own this view of
Koch’s, but Pouchet and Villiers were both able to extract
from the dejecta or from the organs of cholera patients cer-
tain products which they deemed to be characteristic.

The former, with the aid of chloroform, extracted from cholera dejecta an
extremely toxic oily liquid which, as it becomes oxydized in the presence of
air and light, takes on, first a rose, and then a brown colour. It readily
combines with hydrochloric acid to form a chloride, but again breaks down,
im vacuo, or when the temperature is raised, or on the addition of an alkali.
It gives the reactions characteristic of the alkaloids, and gives the blue re-
duction coloration with ferro-cyanide of iron and perchloride of iron.
Villiers succeeded in separating from the organs of a single cholera patient
a couple of centigrammes of a peculiar alkaloid. On treating this with
hydrochloric acid, there separated out a number of acicular crystals: these
crystals have since been described by various observers, and it is stated that
in this form the basic substance, whatever it may be, exerts comparatively
little poisonous action, but that as soon as it is again set free from the acid
combination, by the addition of an alkali, such as soda or potash, it exerts
not only an extremely caustic local action, but also when injected into a
guinea pig produces muscular tremblings and very great irregularity of the
heart’s action, the animal-dying at the end of about four days. It is im-
possible, however, to be quite certain that these reactions were obtained with
Villiers’ pure acicular crystals, ang not with one or other of the sepsines or
toxines, as there is some little doubt as to the exact nature of the poison
obtained in several of the series of experiments, Brieger, going beyond

CHOLERA. 185

these researches, was able to isolate, especially from old cultures, substances
analogous to, or identical with, cadavarine, putrescine, and choline. Carrying
his experiments further, and cultivating the comma bacillus in media con-
taining creatine, he produced a toxine or specific poisonous product which
produced, when injected into the animals, dyspnoea, muscular tremors,
cramp, and death. To this he gave the somewhat formidable name of
Methylguanidin. Carrying his observation still further, he succeeded in
separating from a precipitate obtained by the addition of a mercurial salt,
two other toxines, both of which appeared to be more or less characteristic
of the cholera growth.

It will be noted that in all the experiments made, in which
the toxines were obtained even from pure cultures of
Koch's comma bacillus, the time required for their production
and the quantities separated are out of all proportion to what
occurs in actual cases of cholera, where, from the rapidity of
the course of the disease and the severity of the symptoms,
a large quantity of the poison must be developed in a very
short time. Most of the artificial experiments, however,
have béen made on different media (usually houillon contain-
ing peptone) and under very different conditions from those
which obtain in an animal. In a series of investigations
carried out by Wood in Hueppe’s laboratory, an attempt
was made to grow cultures under precisely those conditions
that are met with in the human intestine, with the result
that a very rapid toxine production, and an additional proof
of the bacillary origin of the cholera virus were obtained.
He took for his nutrient medium normal albumen, and thus
obtained the earlier products such as the albumoses that
occur in the breaking down of albuminoids. The presence
of these would of course account for the greater toxicity of
his cultures under such conditions.

One of the earliest observations made by epidemiologists was
that one attack of cholera protects for a certain time and to a
certain degree against a second, and from this it was now
argued that it should be possible to obtain a system of pre-
ventive inoculation ; and Gamaleia, basing his work on what
was already known of preventive inoculation in other
diseases, commenced a series of experiments by means of
which he hoped to construct such a method of inoculation
against cholera. In this he was ultimately successful. He
found that if, from a culture of the vzbrz0 Metschnikovz, an
organism almost identical with the comma bacillus, both
morphologically and physiologically, in beef broth, he took

186 BACTERIA.

the pellicle which formed on the surface at the end of five
days, sterilized it in an autoclave at 120°C., and then injected
from two to six c.c. of the fluid expressed from the sterilized
mass into the muscles of a guinea pig, the animal was rapidly
and completely protected against the action of Koch’s comma
bacillus, introduced in the ordinary fashion. By further
experiments he found that by simply keeping for several days
the sterilized mass in which the dead bacilli still remained
a much larger quantity of the poison could be obtained—
sufficient, in fact, to kill the animal when given in the same
dose as before, but still protecting the animal when exhibited
in smaller doses. (Here the organisms could no longer be
producing the poison, but it appears as though there was
stored up in their body a considerable quantity which could
only diffuse out into the fluid after a certain lapse of time.)

To Gamaleia also we. owe the knowledge that this
organism may become very much modified in various ways,
and some of the experiments he carried out provide us with
an explanation of some.of the most important facts connected
with the increase and decrease in virulence of type, in cases
that occur during the “ rise and fall” of an epidemic.

He was able to increase the virulence of the special comma bacillus in a most
remarkable manner. After obtaining a growth of the organism in broth he
introduced a smadl quantity of the culture into the lung of a white rat ; this
was followed by an-acute form of croupus pneumonia accompanied by
marked pleurisy, the animal rapidly succumbing. With the fluid that ac-
cumulated in the chest a second animal was inoculated in a similar fashion,
with the result that this animal died more rapidly than the first. This
inoculation was continued through « whole series of animals, until finally
the rats succumbed very rapidly indeed, and an organism was found not in
the intestine, but in very large numbers in the blood."

In addition to drying, acid, and the other destructive
reagents already mentioned, it. has been found that a very
considerable number of chemical reagents arrest or prevent
the growth of the cholera bacillus.

Koch’s list contains alcohol, 10 per cent., sulphate of iron, 2 per cent.
(this latter acts first as an acid, and secondly as a precipitant of the

* That this may have a bearing on Wood's observations as to the viru-
lence of the organism in the zrobic and anzerobic conditions is most evident,
and is well worth further investigation,

CHOLERA. 187

albuminoid materials on which the organism lives), alum, i per cent.,
camphor, .33 per cent., carbolic acid, .25 per cent., essence of peppermint,
+5 per mille, sulphate of copper, 4 per mille, quinine, .2 per mille, corrosive
sublimate, 1 per cent. " ;

Babes has found that the organisms will not develop in a gelatine
nutrient medium which contains .5 per mille of corrosive sublimate, .1 per
mille of carbolic acid, .2 per mille of sulphate of copper, .8 per mille of
salycilic acid, .1 per mille of thymol, 2 per mille of iodine, 2.2 per mille of
bromine, 7 per cent. of alcohol, .8 per mille of sul phate of quinine, .5 per mille
of acetic acid (the action of which, however, depends toa certain extent upon
its interference with the degree of alkalinity of the original gelatine, as the
bacillus cannot grow in a medium in which there is more than a merely
appreciable trace of acidity). He also tried a very interesting experiment

_in which he inverted a gelatine plate on which was placed a cholera
culture over a watch glass or shallow vessel containing a drop of essential
oil of mustard; no development of the organism took place, and if the
plate was left for twenty-four hours the organisms.were all killed, as there
was no subsequent development ; so that oil of mustard is an excellent
disinfectant. Other active volatile principles act in very much the same
manner.

The experiments with gelatine cultures are of special value
because they allow demonstration of the effect that disinfec-
tants have on the bacillus as it grows on specially favour-
able nutrient media. Reference has already been made to the
fact that putrefactive organisms and their products exert a
prejudicial effect on the cholera organism, but it should be
remembered that when the conditions are favourable there is
usually such rapid proliferation of the cholera organism for
the first day or two, that the others are left behind in the
race for existence. It is, however, a case of the hare and
the tortoise, for after a time the cholera organisms die out,
and never again obtain a permanent footing unless a fresh
supply of nutrient material becomes accessible. It is im-
portant to remember this in connection with the spread
of cholera by water supply, as it explains the immunity
enjoyed, after a time, even in the most cholera-stricken
regions. In India, in the regions in which the cholera is
endemic, the wells, as a rule, are merely surface tanks into
which sewage and surface water may be drained, and
which are frequently on the same level as, and connected
with, the cesspools, so that even the water supply contains a
considerable quantity of organic matter in which organisms
of all kinds can flourish most luxuriantly; whilst these same
wells, being merely -dug-out pits beneath the slightly raised
houses, are open for the reception of sewage and excreta

188 BACTERIA.

of all kinds, especially in times of illness, when neither
patients nor nurses have strength or time to see that these
are properly removed. ‘This source of danger is so evident,
and is so in accordance with what one would expect, that
efforts have been made to remove, as far as possible, all
organic material which might serve as nutrient material for
infective organisms from the soil and ground water, and
also to remove as rapidly and as completely as possible not
only dejecta, but also the water employed for cleansing linen,
clothing, and utensils “ without allowing them to come in
contact with the surface of the soil, with wells,” with
vegetables, and the like.

Most ‘hygienists are agreed that it is necessary not only
to have a pure water supply—z.e.,a supply free from all pos-
sible contamination, derived from wells so deep, or from
reservoirs so far from human habitations, that there is
no possibility of contamination by sewage, dejecta, or surface
drainage, and so carefully conveyed by conduits and pipes
that no such contamination can take place during distribu-
tion—but that a water supply should be so ample that
dirtiness is heavily discounted. With all the improvements
that have been made in the drainage system and water
supply of Lower Bengal, cholera has only diminished about
60 per cent., so that there still remain certain factors that
favour the spread of cholera, and every now and again such
a spread or outbreak may take place with extreme rapidity,
and may involve a very wide area. Cleanliness, however,
both general and personal, may be said to be the most
important factor in the prophylaxis of cholera. Fligge,
for example, states most emphatically and explicitly that
“the average cleanliness of the population has the greatest
influence in this respect. The more cleanly the method
of handling the sick and the infected clothes, the more care-
fully contamination of the soil, of the water, and of various
other objects with the dejecta is avoided, the fewer will
be the sources of infection. The more carefully the hands
are cleansed and the articles of food prepared, the more will
the paths of spread from existing sources of infection be
diminished. It is evident that in this respect marked dif-
ferences must exist between more and less civilized countries ;
between new and well-built, and old and cramped cities ;
between poor and wealthy neighbourhoods; between the

CHOLERA. 185

portion of a city inhabited by the poor and that in which
the better classes dwell.’’ The habits of people, then, es-
pecially as regards their food, play a most important part
in the propagation or restriction of cholera. -The organism
is almost invariably introduced by the mouth, so that in
addition to passing into the alimentary canal by con-
taminated water directly, it may also be introduced by
utensils, or food washed or sprinkled with such water, and
Koch gives an excellent example of this when he describes
the market women of Marseilles as being in the habit of
sprinkling the vegetables exposed for sale with water from.
the street gutter, into which he proved that comma bacilli
were constantly making their way, so that any one partaking
of these vegetables uncooked, just as in the case of the
American sailor at Shanghai, was taking in an unknown
quantity, but a very appreciable and deadly dose, of the
cholera organism and its poisonous products. Not only do
these uncooked vegetables offer a nidus and an excellent sub-
stratum for the growth of cholera organisms, but they often
produce that condition of slight indigestion which along
with an overloaded stomach. is one of the most favourable
for the development of the comma bacillus in the human
alimentary canal.

For the same reason feasts, fasts, Saturday night carousals
and Sunday dyspepsias, pilgrim festivals, arrivals in port,
and similar events are all predisposing causes of cholera,
as in all cases there is a disturbance of the digestive function
and a lowering of the system, due either to excess in
drinking or in eating, or to gastric disturbance and lowered
vitality resulting from abstinence from the use of food for
too long a period. It is found, too, that wherever people
assemble in large numbers in excess of the ordinary popula-
tion, the strain on the sanitary arrangements is always
excessive, and further, is necessarily accompanied by care-
lessness in the selection and preparation of food. Cholera
depends for its existence, outside those places in which it is
endemic, on these fairs and pilgrimages, and only by
controlling them and by attending most thoroughly to the
sanitary conditions at the points where people are massed
together can there be any hope of preventing the outbreak
and spread of this insidious and deadly disease.

It is not necessary here to do more than mention the pre-

190 BACTERIA,

disposing effect of autumnal gastric disturbances and slight
diarrhcea, which have, in many epidemics, been the in-
variable precursors of the true Asiatic or Indian cholera.
It is known that during an epidemic one attack of cholera,
especially a severe one, exerts a great protective influence
on those who survive, and as these consist almost ex-
clusively of the people who under ordinary circumstances
are again most exposed to infection, there may be natural
breaks in the line of extension of an advancing epidemic ;
breaks which have been used, by some authorities, as evi-
dence of the sporadic outbreak of the disease.

So much has the study of Koch’s comma bacillus tinged
and affected all our ways of looking at cholera epidemics
that we now consider the conditions under which the
bacillus can multiply and be carried from point to point,
and the conditions that favour its development and mul-
tiplication, instead of dealing with cholera itself as the
entity with which we have to contend. The epidemiologist
has now assumed the ré/e of biologist in the widest sense
of the term. The bacteriological hygienist agrees with the
“localist” that it is necessary to get rid of all conditions
in which the cholera poison can be propagated and dis-
tributed, and that to this end the ground: water should
be kept as free as possible from sewage, that all areas should
be properly drained, and that everything should be done
to render the “drying zone” as little congenial to the
bacillus as possible ; but he goes further, and insists that
the poison should not be allowed to be introduced into
localities in which it does not already exist, for he believes
that however favourable the conditions may be, there will
be no outbreak of cholera until the bacillus is introduced
and gains a foothold. He therefore insists on careful in-
spection of all ships coming from India or from other cholera-
stricken regions, though he is firmly convinced that it is
only in seaport towns that inspection or quarantine can
be of any value ; that if cholera makes its way beyond the
seaport no quarantine or sanitary cordon can stop its spread ;
that once beyond port the most careful isolation of all
patients and disinfection of every article of clothing, feeding
utensils, &c., should be rigidly carried out and insisted upon;
that the dejecta should be mixed with large quantities of
carbolic acid, concentrated hydrochloric acid, or strong cor-

CHOLERA. 191

rosive sublimate solution; that the comma bacilli in
rooms should be thoroughly dried and aired by throwing
open the windows for several days, and that instructions
should be issued as to the necessity for thoroughly personal,
culinary, and household cleanliness, as to the avoidance
of all water except that known to be pure, as to careful
boiling and cooking of drinking water and food, as to the
necessity of paying attention to the slightest gastric derange-
ment, and as to the avoidance of all excesses in both eating
and drinking. He pays attention then, first, to all relating
to the power of the individual to resist any attack of the
organisms. All place dispositions favouring the production.
of conditions of gastro-intestinal disturbance are to be care-
fully neutralized, and in the same way all time dispositions,
such as autumn diarrhoea, determined by the unripe or
overripe fruits so abundant in the latter half of the year are
to be met and counteracted; and second, to everything
relating to the rendering of all the environments unfit for
the development of the comma bacillus, so that the number
of centres from which infection may spread may be kept
down as much as possible.

LITERATURE.

Authors already referred to. Cornil and Babes.
Bazses.—Virch. Arch., Bd. xcrx., p. 148, 1885.
BocuFonTAINE.—Comptes rendus, t. Xcix., p. 845, 1884;

t.c., p. 1148, 1885.

Borum.—Die kranke Darmschleimhaut in der Asiatischen
Cholera mikroskopisch Untersucht. Berlin, 1838.
BrieGer.— Untersuchungen iiber Ptomaine. Berlin, 1885,

1886.
Brittan anpD Bupp.—London Med. Gazette. Sep., 1849.
Bucuner.—Arch. f. Hygiene, p. 361, 1885.
Bujywip.—Centralbl. f. Bakt., Bd. rv., p. 494, 1888.
CantTani.—Deutsch. Med. Woch. 1886.
Cueyne, Watson.— Bret. Med. Journ. April 25, May 2,
g, 16, and 23, 1885.
CunnINGHAM.—Scientific Memoirs Med. Off. Indian Army,
Pt. 11., 1886, “on Cholera,” Calcutta, 1885.

192 BACTERIA.

Davaine.—Comptes rendus de la Soc. de ‘Biol., 2me Serie.,
t. 1, p. 129, 1859.

Denexe.—Deutsch. Med. Woch., No. 3, 1885.

EMMERICH.—Deutsch. Med. Woch., No. 50, 1884.

ERMENGEM (VAN).—Recherches sur le Microbe du Choléra
Asiatique. Paris, Bruxelles, 1885.

FINKLER UND Prior.—Deutsch. Med. Woch., No. 36,
1884; Centralbl. f. Allg. Gesundheitspflege, Bd. 1,
Heft 5, u 6.

*FLUEGGE.—Deutsch. Med. Woch. Jan., 1885.

GarrKy.—Arbeiten a. d. k. Gesundh, Bd. ., 1 2 Heft, p. 39,
1884.

GaMALeIA.—Annales de |’ Institut Pasteur, t. 1, 1888, t. m1,
1889.

Ha.iier.—Das Cholera Contagium. Bot. Unters., Aerzten u.
Naturforschern. Mitgetheilt. Leipzig, 1867.

HuerPe.—Berlin Klin. Woch., Nos. 46-47, 1889.

Kvess.—Ueber Cholera Asiatica. Basel, 1885.

KLEIN AND H. Gispes.—Rep. English Cholera Commission.
1885. ‘
KLEIN.—The Bacteria in Asiatic Cholera. London and New

York, 1889.

K1ios.—Path-anat. Studien u. d. Wesen des Cholera Pro-
cesses. Leipzig, 1867.

Kocu.—Deutsch. Med. Woch., No. 45, 1884; Berlin Klin.
Woch., No. 374, 1885.

Lewis.— Med. Times and Gazette. Sept. 20, 1884.

Mac Leop AND Mities.—Lab. Reports, R.C.P., Edin., vol.1.,
p. 161; Proc. Roy. Soc., vol. xv1., p. 18.

Macnamara.—2&rit. Med. Journ., vol. 1., p. 502, 1884.

Mitter.—Deutsch Med. Woch., No. 9, 1885.

NicaTI uND RietscH.—Comptes rendus, t. xcix., pp. 928-
929, 1884.

Pacini.—Arch. de Med. Militaire de Bruxelles, 1855.
PETTENKOFER.—Zum Gegenwartigen Stand der Cholera-
frage. Munich, see Lancet, July 3 and 1oth, 1886.

PreirFerR.—Deutsch. Med. Woch. 1885, 1886.

PoucHEeT.—Comptes rendus., t. xxvul, p. 555. April 23,
1849.

Roy, Grades BROWN AND SHERRINGTON.—Proc. Roy. Soc.

No. 247, p. 173, 1887.
ScHOTTELIUS—Deutsch. Med. Woch., No. 14, 1888.

CHOLERA. 193

Straus, Roux, Nocarp anp THUILLIER.—Comptes rendus
de la Soc. de Biologie. Paris, t. 1v., 1883.
SwainE.— Lancet, pp. 368, 398, 1849.
VILLIERS.—Comptes rendus, t. c., p. 91, 1885.
WEISSERZUG, FRANK.—Zeitschr f. Hygiene. 1886.
WoopHEApD AND Woop.—Edin. Med. Journ. April, 1890.
* Full lists of references given.

14

CHAPTER X.
TYPHOID FEVER.

Typhoid Fever a Bacterial Disease — Recklinghausen’s Observations—
Klein—Eberth—Klebs—Coats—The Bacillus—Method of Staining—
Position in Tissues—Gaffky’s Observations—Pure Cultures—Excretory
Products—Experiments on Animals—Mixed Infections--Action of
Light and Heat on Typhoid Bacilli—Pseudo-typhoid Bacilli.

From the curious nature of the symptoms of typhoid fever,
and from the fact that after complete recovery from an
attack there appears to be a certain immunity (for a certain
period at any rate) against a second, although relapses are
of comparatively frequent occurrence, it was early supposed
to be the result of the presence of some specific micro-organ-
ism within the body, probably in the deeper tissues of the
wall of certain parts of the intestinal canal, and in those
organs, such as the spleen and lymphatic glands, that are
specially connected or associated with that canal. Although,
as early as 1871, Recklinghausen described in abscesses that
were formed during the course of an attack of typhoid fever,
microbes which he considered to be specific, and although
Klein in this country found several varieties of micro-
organisms in typhoid lesions, the specific organism was first
accurately described, and distinguished from others, by
Eberth and Klebs abroad and by Coats in this country, all
of whom give very exact descriptions of the typhoid bacillus,
These bacilli are short, somewhat thick rods, about 2 to 3# in
length and .3 to .5# in breadth ; they are usually distinctly
rounded at the ends, where the protoplasm is always rather
more deeply coloured by aniline dyes than the central
portion, which was at one time supposed to be a spore,
though more recently this lighter coloured portion has been
looked upon as evidence of a process of degeneration.
These bacilli are said to be stained with difficulty, but I have
found that if the sections in which they are present are first

TYPHOID FEVER. 195

allowed to remain for about ten minutes in a 1-5th per cent.
solution of corrosive sublimate and then stained by Gram’s
method, the bacilli are most deeply stained, although Fraenkel
and others state that the colour is invariably discharged if
Gram’s method be used. They may also be prepared by
Kihne’s method of first allowing them to remain in a con-
centrated watery solution of oxalic acid, washing them
carefully and afterwards staining with methyl blue dissolved
in a 1 per cent. solution of ammonium carbonate. Sections
may also be stained for twenty-four hours in L6ffler’s alkaline
methylene blue, after which they are rinsed in water, which
removes sufficient of the colour ; the water is driven out with
aniline oil, the sections are allowed to dry on the slide and
mounted in Xylol Balsam.

The bacilli are found in the adenoid follicles, or lymphatic
tissue of the intestine, in the mesenteric glands, in the spleen,
and in the liver, and more rarely in the kidneys. They are
usually collected in little clumps, and single bacilli are seldom
if ever met with. These clumps, although readily enough
recognized when seen, are as a rule so sparsely scattered
through the tissues that it is often a difficult matter to find
them, even in characteristic cases, and as Fliigge says, “ It is
only after the examination of a large number of sections that
one or several of these deposits can be found.” Gaffky,
working in the Hygienic Institute in Berlin, was first able to
make pure cultivations of this bacillus, and in 1884 he gave
a very complete description of the bacilli that he was able to
examine or to cultivate in twenty out of twenty-two cases of
typhoid fever of which the examination was committed to his
charge. The bacilli, when obtained pure, and cultivated in
fluid, grew out into very long.threads, both threads and short
bacilli apparently being motile, having a peculiar wavy motion ;
quite recently this motion has been found to be due to the
presence of groups of lateral flagella which, waving back-
wards and forwards, impart to the organism its peculiar
snake-like movement. The bacillus can grow perfectly well
both in the presence of free oxygen and also when oxygen is
cutoff, but, as in the case of the cholera organism, it appears
to-have somewhat different functions and different powers
unde¢r the two sets of conditions ; outside’ the body in the
presence of oxygen it. appears, to. develop. great “ resistant”
power and a saprophytic habit, whilst in the anzrobic con-

196 BACTERIA.

dition, especially in the intestine, although its power of
breaking up the albuminoid substances presented to it and
of developing its specific toxines is greatly increased, its
capability of resisting antiseptic substances is considerably
diminished. In plate cultivations made from the organs of
typhoid patients, Gaffky found that the bacilli developed in
the deeper part of the gelatine as small white points, whilst

Photo-micrograph of Typhoid bacilli in Lymph follicle of Intestine of a child in
which the Typhoid lesions were very characteristic. x 500.
on the surface they grow as moist-looking greyish colonies
with irregular margins. Under a low magnification the small
rounded points are seen to be slightly granular ; they have
a sharply-defined margin and are of a dirty yellow colour ;
the superficial growths, although spreading somewhat
rapidly, are thin, sometimes almost transparent, and have a
yellowish tinge when seen in the sunlight ; the margin is

TYPHOID FEVER. 197

irregular and is marked by large and small indentations.
There is no liquefaction of the gelatine around the growth.
In a test tube cultivation the growth appears all along the
line of the puncture and also on the surface. The surface
culture has a peculiar mother-of-pearl look, it gradually
spreads over the whole of the gelatine, forming a kind of
bluish-grey film, whilst down each side of the needle track
there is a delicate zone of the same bluish-grey colour,
surrounded in turn by a peculiar opalescent milkiness. The
most characteristic growth, however, occurs on_ sterilized
potatoes. It is characteristic in that, even when there is a
most luxuriant growth of the typhoid bacillus, it cannot be
recognized by the naked eye, even at the end of three or
four days, except by a peculiar moist appearance of the
potato, which, taken along with the appearances in milk
and on gelatine so far as is at present known, distinguishes
the growth of this organism from all others. It will be
remembered, however, that the potato is slightly acid, and
it appears that this acidity is necessary for this typical growth,
for on potatoes rendered slightly alkaline there appears a
yellowish or dirty grey growth with sharply defined margins
—a growth quite different from that above described.
Chantemesse and Widal utilized the power of the micro-
organism to grow in acid to help them to obtain pure
cultivations. They prepared special tubes of gelatine by
adding to each 10 cc. of the nutrient medium, 4 or 5
drops of 1 to 20 per cent. solution of carbolic or phenic
acid ; they did this in order to prevent the development of
those microbes that bring about liquefaction of gelatine.
They were perfectly successful in their attempts, although, as
we have seen elsewhere, it does not follow that their expla-
nation was absolutely correct ; at any rate they were able to
separate pure typhoid bacilli which had all the characteristic
appearances both on the gelatine plates and under the
microscope.

The organism itself, unlike many other bacteria, seems to
form an acid and not an alkaline excretory product. It
grows in a variety of media, and as I have already said, it
appears under certain conditions to become quite saprophytic
in habit, this being evidenced by the ease with which it may
be cultivated outside the body, and quite recently Wolffhigel
found that it may develop readily in both milk and water to

198 BACTERIA,

which it has gained access. This bacillus, then, has been
found in the body ; it has also been found by Pfeiffer in the
dejecta of typhoid patients, and now it has been found in the
water supply, so that the chain of evidence is, so far, pretty
conclusive. It is a somewhat important fact that the typhoid
bacilli may remain active for a considerable length of time
in the stools, for the bacilli in faeces kept in a sterilized
tube for fifteen days remain alive at the end of that period,
and vigorous cultivations could be made from such material.
Experimenters were now.confronted with the difhculty that
typhoid fever is seldom met with except in the human subject.
Experiments were made on animals by injecting cultures of
the typhoid bacillus into the aural vein of rabbits, with the
result that about 50 per cent. of the animals died, and on
post-mortem examination it was found that the spleen and
those glands usually affected in typhoid were somewhat
swollen. A. Fraenkel was able to kill monkeys with in-
jections of the bacillus, and Chantemesse and Widal point
out that they can produce a septicemia by injecting a con-
siderable quantity of a culture of the bacillus into the
peritoneal cavity of a mouse. They also found, repeating
Fraenkel and Simmonds’ experiments of injecting cultiva-
tions into the vein of the ear, that this was followed by
diarrhoea and rapid emaciation at the end of several days,
although the animals frequently recovered. When they
were killed at the height of the disease, lesions corresponding
to those met with in typhoid fever were found in the intes-
tine, and bacilli were also found in the organs.

The method that was used in the inoculation of cholera
was afterwards resorted to, the contents of the stomach were
rendered alkaline, the peristaltic movement of the intestine
was paralyzed by means of full doses of opium, and the typhoid
bacilli were injected into the alimentary canal ; most of the
animals died, and numerous bacilli were found in the intestines
and even in the glands, but none could be demonstrated in
the blood. It was observed, however, that it was not neces-
sary to have an active bacillus present in order to cause very
serious symptoms, and even death, with typhoid cultivations ;
and it soon came to be recognized that these symptoms were
due to regular poisoning or intoxication by toxines and tox-
albumens, both of which were described by Brieger as being
present in typhoid discharges and in pure cultures of the

TYPHOID FEVER. 199

typhoid bacillus. It was in typhoid fever, in fact, that this
investigator carried on some of his earliest experiments on
the poisonous metabolic products of pathogenic organisms,
growing in albuminoid substances. As regards the relation of |
this special bacillus to the disease, it stands on exactly the same
footing as that between the cholera organism and cholera, and
it follows that most of the points that have been accentuated
when we were considering cholera may also be accentuated
in this instance. Klein pointed out that in typhoid lesions,
especially in the intestinal canal, several organisms were
usually associated, and other observers have agreed that in
typhoid there is, very frequently, what is known as a
“ mixed infection "—z\e., in addition to typhoid bacillus other
organisms appear to be present and to play an important
part. Streptococci, and septic organisms, are frequently
found in the tissue of the spleen, liver, and wall of the intes-
tine. It is supposed that some of these organisms play their
part in preparing the intestine for the reception of the
typhoid bacillus, and it is maintained that a condition of
irritation and a removal of the epithelium, brought about by
the action of other micro-organisms on the wall of the
intestine, may be necessary to prepare the way for the
entrance of the typhoid bacillus. The intestine is, in fact, pre-
pared just as a field is prepared by the farmer by ploughing
and manuring for the reception of the seed that he intends to
sow. The fact that the bacilli can grow on potatoes without
becoming evident to the naked eye indicates the possibility of
a similar growth occurring on other articles of diet, which,
taken into an alimentary canal that has been previously
prepared by gastro-intestinal disturbances—diarrhcea and
similar conditions—may set up the disease.

It may be appropriate here to consider the action of light
on typhoid bacilli, as although the first observations on the
germicidal action of light were made on other organisms, in
this country by Downes and Blunt, and these experiments
were continued by Tyndall and a number of other workers
both in this country and abroad, the more recent experiments
on the action of light on bacteria have been carried out on
typhoid bacilli. That bacteria are influenced by the action
of light either to their advantage or their harm is very
evident. In the one case it will be found that certain of
the colour-producing organisms cannot exert this function

200 BACTERIA.

unless they are very well supplied with both air and light,
whilst on the other hand such organisms as usually grow in
the body appear to become markedly weaker as regards their
power of growing and of giving rise to their special dele-
terious products if they are freely exposed to the light.
Recently Dr. Janowski has made a number of experiments
by exposing growths of the typhoid bacillus to the action of
light, and has found that it exerts a distinctly depressing
action on the typhoid organism, an action entirely inde-
pendent of any oxidation of the food material that might
occur under the action of the chemical rays, these chemical
rays acting directly upon the protoplasm and rendering it
incapable not only of further development but of continuing
alive. In order to prove his thesis he took a gelatine tube
in which typhoid bacilli had been sown and exposed it to the
action of the light on a cold winter day; a similar tube
inoculated with the same bacillus was wrapped up in a layer
of black paper and then in one of white paper, this also
was exposed in the same position. The light in this case
delayed the development and the multiplication of the
organism in a somewhat marked manner, as in the two pro-
tected from the light, growth took place in three days, whilst
in that exposed to the light, it did not commence for five
days. Of course the growth here referred to is measured by
the size of the colony that can be seen with the naked eye
and although both were probably growing during the whole
time, the rate of multiplication in the one was very consider-
ably greater than in the other. In order that there might be
no doubt as to the identity of the organism and the quantity
sown in each tube, a U-shaped tube (a double Pasteur tube)
was taken and the inoculation was made; the fluid was
thoroughly mixed by passing from one limb of the tube to
the other, then one limb was protected as above and the
other was exposed to the light ; similar results were obtained,
the bacilli in the limb that was exposed to the light being
considerably delayed in their development. He found that
direct sunlight acting on fluid cultures of the typhoid bacillus
kills the organisms in the short space of from four to seven
‘hours, but diffused light requires a considerably longer period
to entirely arrest the development and multiplication of the
organisms. Instead of analysing the rays of light by means
of a prism, as Englemann and others had done, qeaowikl

‘

TYPHOID FEVER. 201

made use of solutions of alum, bichromate of potash,
Bismarck brown, fuchsine, methyl blue, gentian violet, &c.
He found that the yellow and brown solutions filtered out
the chemical rays and prevented the action of light upon
the organisms almost as efficaciously as the black and white
papers, but found that the other fluids—fuchsine, methyl
blue, gentian violet, &c.—had little more effect in preventing
the injurious action of light on the bacilli than distilled
water or alum solution. He therefore comes to the con-
clusion that the hurtful action of both diffused light and
direct sunlight is due in very great measure to the chemical
rays of the solar ‘spectrum—the rays at the other end of
the spectrum exerting comparatively little influence on the
organism. This entirely accords with Englemann’s observa-
tions ; he found that certain chromogenic bacteria when
examined in a drop of water and illuminated by the rays
from a micro-spectral objective, invariably made their way to
that part of the spectrum furthest away from the violet end,
thus indicating that they were attempting to evade the
chemical rays which appeared to be hurtful tothem. This
question of the action of light, especially on pathogenic
bacteria, is one of very great importance, and Duclaux’s
dictum that fresh air and sunlight are two of the most
powerful agents that we have with which to combat the
onslaught of the bacteria of disease cannot be too strongly
insisted upon. Bacteria, especially those of disease, seek out
the dark places for their habitation, and as the exclusion of
light to a very great extent necessitates the exclusion of fresh
air, they find in these holes and corners places of rest whence
they may go out to do all the harm of which they are
capable.

Janowski also made an elaborate series of experiments on
the effect of high temperature on the typhoid bacillus. He
found that a temperature of 55° C. continued for ten minutes
was quite sufficient to render sterile cultivations of this
bacillus, but if this same temperature were continued for
only five minutes he could not rely upon obtaining complete
destruction of the organism. He also came to the conclusion
that an extreme degree of cold, especially when continued
for some time or where frequently repeated, had a moderately
injurious effect upon the vitality of the typhoid bacillus ; a
temperature of 14° C. being sufficient to kill the bacilli when

202 BACTERIA.

growing in a fluid medium. When they were allowed to
dry, however, this did not appear to hold good to nearly the
same degree.

These experiments are interesting in their bearing on the
outbreaks of typhoid fever at certain parts of the year,
especially in countries where the cold appears to be exceed-
ingly intense, and where one would naturally expect the
development of the bacilli to be interfered with, but where
as a matter of fact such is found not to be the case.

Quite recently Cassedebat, examining the drinking water
supplied to Marseilles, which is a very hotbed of typhoid fever,
was not able to find the characteristic bacillus in any one of
250 cultivations made of seventy specimens of water, but
curiously enough he found three other bacilli which in many
respects resembled the true typhoid bacillus most remark-
ably, although they differed in certain essential character-
‘istics. He points out that they all grow in the phenic acid
gelatine, and he further states that several other organisms
offer quite as great resistance to this acid as the typhoid
bacillus itself. They all present clear spaces or deeply stained
masses of protoplasm which may easily be mistaken for spores,
but these, like those in the true typhoid bacillus in which as
we have seen similar bodies occur, are all killed at a tempera-
ture of a little over 45°C. The pseudo-bacilli are very im-
‘perfectly stained by Gram’s method. They exhibit a lateral
and oscillatory motion as well as a forward motion. The
plate cultivations are so much alike, that unless all four can
be examined simultaneously, it is a very difficult matter to
distinguish one from the other ; their growths on potatoes,
in broth and in milk resemble one another in a most re-
markable manner, except that they develop with different
degrees of rapidity, and vary somewhat as regards the alkali-
nity and acidity of their products at the end of about thirty
days, and also as to the degree and time of appearance of
turbidity that is produced when these organisms grow in
broth. In consequence of these slight differences the use of
the various aniline staining reagents, added to such culture
media as broth or milk in which the colours undergo
changes under the different reactions, has been resorted to
and described by Cassedebat, who was able by their use
to distinguish one organism from the other. The ordinary
cultivation methods are quite sufficient to distinguish these

TYPHOID FEVER. 203

four forms as a group from most others, for which the
typhoid bacillus itself has at different times been mistaken,
whilst in addition to the differences above mentioned none
of the pseudo forms are quite so toxic to white mice as the
Eberth-Gaffky bacillus, and one of them is quite innocuous.
Although Cassedebat was not able to find the true form in
water taken from a supply that was open to contamina-
tion, he found that this was not because the bacilli could not
live in water, as in distilled water to which a cultivation was
purposely added he could easily distinguish its presence at
the end of forty-four days, and when added along with half
a-dozen other forms he still found it living and active at the
end of seventeen days. As the result of his observations he
comes to the conclusion that the true typhoid bacillus does
not occur in water so frequently as is sometimes represented,
and that one or other of the pseudo-typhoid bacilli has in
certain cases been mistaken for it.

LITERATURE.

Authors already referred to. Brieger, Fligge, Fraenkel.
Atmguist.—Typhoid feberns bakterie. Stockholm, 1882.
Bazes.—Journ. de l’Anatomie, p. 39, Jan., 1884.

BEUMER UND PEIPeR.—Zeitschr f. Hygiene, p. 489, 1886.

Birco-HirscHFELD.—Zeitschrift f. Epidemiologie, Bd. 1,
1874.

Hanes GAD ExrutcH.—Berlin Klin. Woch., No. 44, p. 661,
1882.

CassEDEBAT.—Annal. de l'Institut Pasteur. Oct. 25, 1890.

CHANTEMESSE ET WipaL.—Arch. de Physiol, p. 217, 1887 ;
Annal. de l'Institut Pasteur, t. 1, p. 54, 1888

Coats.— Brit. Med. Journ., vol. L, p. 421, 1882.

Crooxe.— Brit. Med. Journ., vol. i, p. 15, 1882.

Downes anp BLunt.—Proc. Roy. Soc., vol. xxvr., No. 184,
Dec. 6, 1877.

Downgs, BLUNT, AND TYNDALL.—Proc. Roy. Soc., vol. XXVIII.
No. 191, Dec, 19, 1878.

Exsertu.—Virch. Arch., Bd. Lxxxt., p. 58, 1880; and Bd.
LXXXIIL, p. 486, 1881 ; Volkmann, Klin. Vortrage, 1883.

FRAENKEL UND Srimmonps.—Centralbl. f. Klin. Med. p.
737, Oct. 31, 1885; Der aetiologische Bedeutung des
Typhus Bacillus, 1886.

204 BACTERIA.

GAFFKY.—Mitth. a. d. Gesundheitsamt, Bd. 11, p. 372, 1884.

JAMIESON.—Vature, vol. xxtv., No. 620, Sept. 15, 1881.

Janowski.—Centralbl. f. Bakt. u. Parasitenk., Bd. vut.,
Nos. 6-9, 1890.

Kuess.—Arch. f. Exp: Path., Bd. xmm., p. 381, 1880.

KL EIn.—Reports of the Med. Officer of the Local Gov.
Board, 1874.

Kiune.—Zeitschr. f. Hygiene, Bd. 1, p. 553, 1886.

Lérrier.—Centralbl. f. Bakt. u. Parasitenk, Bd. v1, Nos,
8-9, and Bd. vu., No. 20, 1890.

PFEIFFER.— Deutsch. Med. Woch., No. 29, 1885.

RECKLINGHAUSEN.—Wurtzburger Zeit., June 10, 1871.

SIROTININ.—Zeitschr f. Hygiene. 1886,

SocoLorr.—Virch. Arch., Bd. Lxvi.

CHAPTER XI.
TUBERCULOSIS.

Tuberculosis a widespread Disease—The Tubercle Bacillus—Koch—Baum-
garten—Spores seen by Watson Cheyne—Relation of Organisms to
Tissues—Bacillus in Tuberculosis of Animals—Tubercle Bacilli as
Saprophytes—Bacilli cultivated outside the Body by Koch—Methods
—Temperature Relations—Cultivation on Different Media—Channels
of Infection—Ransome— Williams—Cornet—Conditions of Infection—
Methods of Disinfection—Tuberculosis at Different Ages—Tubercle
in Milk—Diagnosis of Tuberculosis in Cattleh—Tuberculous Meat—
Koch's Method of Treatment—Nature of Virus and Mode of Action
Is Immunity conferred?—Koch’s Method a New Departure—Sterile
Products cause Marasmus Maffucci—Indications for Treatment.

TUBERCULOSIS, one of the most widespread and deadly diseases
with which we have to deal, not in this country only, but in
the whole of Northern Europe, has now certainly been proved
to demonstration to be due to the presence of a specific
micro-organism. Almost innumerable researches have been
carried on with the object of finding out and piecing to-
gether the various facts in the life-history of this organism,
the products to which it gives rise, the conditions under which
it can multiply in the human body and in the animal body,
the nature of the very grave changes produced in the tissues,
the mode of transmission directly and indirectly from one
body to another, and, last, but not least, the possibility of
combating the ravages made on the body by this organism,
by interfering with its growth or retarding its development,
either outside the body or after its introduction into the
tissues.

Tuberculosis and Phthisis or Consumption account for
such an enormous percentage of deaths in our colder north-
ern latitudes, that the subject has come to be one of intense
interest, not only to physicians and surgeons, but to all well-
educated people, and the subject of the treatment of tuber-
culosis is—unfortunately perhaps for patients—taken up

206 BACTERIA,

almost as exhaustively in the daily papers as it is in special
treatises and in the medical journals. It has long been known
that tuberculosis was an inoculable disease, but it was only
quite recently (1883) that Koch and his pupils were able to
demonstrate that a specific organism could be separated from
tuberculous tissue and cultivated outside the body—the
cultivated organism having all the characters of the organism
found in the tissues—and that when introduced into certain
animals, this organism was capable of producing tubercular
disease, the organism in turn being again demonstrable in
the new tubercular growth.

It was for long found to be an exceedingly difficult matter to demonstrate
any specific micro-organisms in tubercular tissues by means of aniline or
other nuclear stains and Baumgarten’s method * was introduced after he
had failed to attain his object by any of the ordinary methods. The
difficulty he had, however, was that, although the tubercle bacilli un-
doubtedly resisted the potash solution, other organisms were also more
resistant than were the animal tissues, so that there was no great differen-
tiation except in size and form between the tubercle bacillus and other
bacteria. While Baumgarten was working out his method, Koch had
completed a series of investigations, the outcome of which was that he
proved that by tlie addition of a small quantity of an alkali to the aniline
stain the dye was rendered capable of penetrating the resistant outer
membrane of the tubercle bacillus. It was afterwards found that aniline,
thymol,. turpentine, or carbolic acid, added to the stain, bring about
the same results; these substances, acting, apparently, as mordants on
the tissues. The next step in the process of demonstration of the tubercle
bacillus was taken when it was found that this organism differed from
others in the fact that it retained the staining reagent most tenaciously,
even the strong mineral acids, which readily discharge the colouring-matter
from nuclei and other bacteria having little effect, if acting for a short time
only, in taking out the stain from tubercle bacilli; in sections stained in an
aniline colour mixed with one of these substances and then treated with
an acid, a most beautiful differential staining was obtained, the stained
bacilli standing out most prominently frdm the unstained tissues.

Tubercle bacilli when stained are seen as delicate rods or
threads 1.5 to 3.5#in length and about .2 in thickness, though
these dimensions are by no means constant even in the same
preparation, and under different conditions the variations in
size are sometimes very marked. As in the case of anthrax
and cholera bacilli, the methods of staining and preparation
exert a marked influence in determining the apparent size

* The sputum, after being dried on a cover glass and then heated to
coagulate the albumen, was simply soaked in a solution of caustic potash.
Sections were treated in the same manner.

TUBERCULOSIS. 207

of the organism. The length is sometimes given at 2.6
and the breadth as from .2 to .S#. They are usually
described as from a quarter to a half the diameter of a red
blood corpuscle and longer than the bacilli of mouse septi-
cemia, Or 0.0015 to 0.0035 m.m.in length. These bacilli
always appear to have the larger dimensions when treated
by Baumgarten’s liquor potassze method. The bacilli are

Photo-micrograph of Sputum, from a phthisical patient, containing large
nucleated epithelial cells and characteristic tubercle bacilli. x 1000.

usually slightly curved, or two are arranged end to end
so as to contain an angle. At first they were described as
not containing spores, but it was demonstrated by Watson
Cheyne that from two to six spores may frequently be seen
in these rods; the spores occurring as small ovoid or rounded
clear spaces placed at intervals in the stained thread. In
some cases they are so prominent that they appear to

208 BACTERIA.

project beyond the straight outline of the bacillus ; sometimes
this is so much the case and the spores are packed so closely
together, that when examined under a sufficiently high power
the spore-bearing thread has been described as a chain of
cocci; by some it is maintained that this appearance is
present only if the specimen is imperfectly stained, too much
heated, or too long treated with a strong acid ; whilst, on the
other hand, certain observers assert that the tubercle bacilli
also occur in the form of regular chains of cocci. The bacillus
is non-motile.

Fliigge holds, however, that it is ‘always possible, in carefully
prepared specimens and with the aid of good lenses, to convince one’s self
that the if va chain of cocci does not exist, but that the delicate
contour of the bacillus can be for the most part traced through its whole
length, and that it is only within this contour that the alternation of stained
and unstained zones gives the deceptive appearance of stained cocci
separated by narrow intermittent spaces.”

The association of this organism with tubercular disease is
undoubted ; it is found in the lungs and sputum in various
forms of consumption, it is found also in tubercular ulcers
of the intestine, around the vessels in tubercular inflamma-
tion of the membranes of the brain, a condition which
occurs frequently in children, in tubercle of the liver and
of all other organs of the body, and in tubercular eruptions
of the skin such as lupus. In all these cases the bacilli are
found most abundantly at those points where the disease
appears to be spreading into the surrounding tissues, and
especially where there is the formation of large multi-
nucleated epithelioid cells. If these bacilli are present
in considerable numbers in such an area, there will
usually be found in the immediate neighbourhood a small
portion of tissue that has undergone marked degenerative
changes, the cell protoplasm is somewhat hyaline or glassy
looking, and takes on any staining reagent, except perhaps
picric acid, very badly; the nucleus is also considerably
altered, especially in that it no longer .stains, or is stained
very imperfectly, with carmine or the aniline dyes. At such
a stage there can frequently be demonstrated, in these cells,
imperfectly stained tubercle bacilli, though in some cases
the bacilli stand out sharply and very brilliantly from
the glassy or homogeneous looking cell. After a time
the cells lose their outlines; they become more and more

TUBERCULOSIS. 209

indistinct, until eventually nothing but the ghost of a cell is
left. This occurs where we have what is known as the
cheesy or caseous degeneration, in which condition nothing
but broken-down nuclei and very rarely a tubercle bacillus
can be distinguished. It would at first sight appear as
though the tubercle bacilli had disappeared, root and
branch, from this caseous centre, but if a small portion of
the cheesy material be inoculated into an animal susceptible
to tubercle, flourishing groups.of tubercle nodules all con-
taining tubercle bacilli are produced, first near the seat of
inoculation, and then at points situated at some distance from
the point of primary infection. The results of such experi-
ments naturally led the opponents of the bacillary theory
of tubercle to assume that the poison of tubercular virus
was not associated with the bacillus, which they contended
was merely of accidental or sequential and not of causal
occurrence, as it could not be found in material which
undoubtedly produced the disease ; but after the demon-
stration of spores in the bacilli, and bearing in mind what was
known of spore formation in other organisms, most observers
were very naturally led to the conclusion that, although the
bacilli are not to be demonstrated in these infective caseous
masses, spores in enormous numbers are probably present,
and that from these, bacilli are developed as soon as the sur-
rounding conditions of nutrition, moisture, and heat are
again sufficiently favourable. The fact that it is so difficult
to stain spores made it no easy task to demonstrate the
accuracy of this theory, but it may now be held, as the out-
come of inoculation and other experiments, that there can be
no reasonable doubt on the subject. It is a curious fact that
whilst in the human subject tubercle bacilli appear in many
cases to be actually contained within the giant or other large
cells, in some herbivora, and in fowls, the bacilli sometimes
occur single or in small masses, apparently outside the cells.
There are several explanations given for this, but the most
rational appears to be that where the bacilli are few in
number, and where they are being rapidly destroyed by the
tissue cells, by far the larger proportion are taken up by, and
are seen in, these cells; this being specially observable in
cases of chronic tuberculosis, whilst in those cases where
the tubercle bacilli are relatively numerous, as in cattle, and
even more markedly in the fowl and in the horse, the

15

210 BACTERIA.

destruction of the tissue cells takes place so rapidly, in con-
sequence of the invasion of each cell by a large number
of bacilli, that nucleus and protoplasm break down com-
pletely and the little group of bacilli is set free, or at any
rate is not embedded in a mass of protoplasm, but is merely
mixed up with the granular débrzs of the cell, from which,

Micro-photograph of Tubercle bacilli, found in the scraping from the lung
a cow suffering from Perlsucht. x x000.
or along with which, it may be carried to other parts of the
body. ,

Having found the tubercle bacillus almost invariably accom-
panying tuberculous disease, Koch, to complete his proof,
wished to separate the organism in the form of what is
called a pure cultivation, in order that he might study
its life-history, and that he might determine whether
the organism when introduced alone into the animal body,

TUBERCULOSIS. 2it

could give rise to tuberculosis. With all the ordinary
nutrient media then at his command he entirely failed to
obtain any growth of the organism outside the animals in
which it led its parasitic life. It depended so much upon
these conditions of parasitic life that when removed from
them it was no longer able to grow and multiply : it might
still remain alive; in fact, Cornil was able to demonstrate
that at the ordinary temperature of the room the tubercle
bacillus, kept in ordinary Seine water, continued to exist, but
not to multiply, for seventy days. At length, however, Koch
overcame the difficulties with which he had to contend, in a
most ingenious manner, and he succeeded in growing as a
saprophyte what had hitherto been demonstrated only as
a parasitic organism. He argued that as the tubercular
process developed but slowly, he would have to obtain a
medium which would remain unaltered for a considerable
length of time when placed in a temperature at which the
organism could grow and multiply. He was satisfied too,
from his early experiments, that only special substances
would serve as nutrient material for this fastidious organism,
and he ultimately found that solidified blood serum was by
far the best medium on which to cultivate it, as it alone of
the many substances which he had then tried supplied all the
requirements of the organism. ‘This blood serum contains
all the elements necessary for the nourishment of the
organism ; it remains solid at the normal temperature of
the body, at which temperature it may be kept for a long
enough time to allow of the development of the slowly
growing bacillus, whilst a small amount of water might be
left in the test tube along with the serum without dis-
solving it, thus serving to supply the moisture requisite
for the perfect growth of the bacillus. In consequence of
- the slowness of the growth above referred to, it is an ex-
ceedingly difficult matter to obtain pure cultivations of the
tubercle bacillus should it once become mixed with putre-
factive bacteria, and it was for long deemed almost an
impossibility to separate it from these other forms: this
difficulty has now, however, been overcome. Most of Koch’s
earlier pure cultivations were obtained by taking as seed
material, tubercular lymphatic glands from freshly-killed
guinea pigs, which had been inoculated, some three or four
weeks before, with tuberculous material.

212 BACTERIA.

As this method may be looked upon as almost classical it will be well to
give the description in Koch’s own words. ‘‘ A number of knives, scissors
and forceps are heated in a flame sufficiently to free them from any adherent
bacteria. They are then laid ready in such a manner that no further con-
tamination of the instruments can take place. Meanwhile the animal
immediately after being killed is fastened to a dissecting board. In order
to avoid the flying off of particles of dirt, hairs, &c., when the skin is
incised, the fur of the animal is freely moistened with a 1 to 1000th solution
of corrosive sublimate. With a pair of scissors and forceps, both still hot,
the skin is now divided and turned back on each side sufficiently to free the
lymphatic glands of the axilla and groin ; but the glands, if they are to be
used to start cultivations, must not be touched with the instruments em-
ployed for cutting through the skin. With another pair of scissors, also
heated, a piece I to 2 c.c. cube is cut out of the side wall of the thorax,
and the surface of the lung laid bare. A number of tubercular nodules
are thus rendered accessible, and a few are removed as quickly as possible
with fresh instruments, which must, however, be cooled for this purpose.
In order to set free the bacilli contained in the nodules, the latter are cut in
pieces or crushed with the scissors, or, better still, between two scalpels that
have just been heated and allowed to cool. The substance thus subdivided
and crushed is removed by means of a platinum wire fused into a glass rod
(which, immediately before use, has been heated and allowed to cool),
introduced into the test tube, spread out on the surface and well rubbed
about. During this operation the test tube must be held obliquely or
almost horizontally between the thumb and forefinger, and the cotton wool
plug held meantime between the other fingers of the same hand in such a
way that no contamination of it by other objects can take place. The
transference of the substance into the solidified serum, which may for
brevity be designated inoculation, must take place as quickly as possible in
order that no germs of extraneous organisms from the air may alight on the
inoculation material or enter the test tube. It is desirable also to conduct
the experiment in a room where no dust is flying about, and in the same
way all unnecessary movements by which dust from the clothing, &c., is
mingled with the air are to be avoided, as experience has shown that it is to
particles of dust that the germs of micro-organisms suspended in the air
adhere.

“ In spite of all these precautions we cannot be perfectly sure of preventing
the entrance of a few solitary germs, and it is necessary in each case to
inoculate several (five to ten) test tubes, so that if we fail to obtain a pure
cultivation in one or two tubes, we shall yet have others that are free from
impurity. ~

**The process is the same as that above described for obtaining seed
from a pulmonary tubercle, when lymphatic glands, tubercles from the
spleen, &c., are to be used to start a culture. The process must always be
carried out with heated instruments, which must be changed every time a
fresh stratum is laid bare. All preparatory incisions which do not come in
contact with the inoculation substance itself are to be made with hot
instruments, but the inoculation material is to be cut out with a cool pair
of scissors and forceps. It is necessary to change the instruments constantly
in order that impurities adhering to them after the division of the skin and
superficial layers, may not be carried into the cultures. ;

‘* When the organs of a recently killed or dead animal could be obtained,

TUBERCULOSIS. 213

and the inoculation with substances containing tubercle bacilli was done in
the way just given, I invarjably succeeded in obtaining pure cultures. The
result was uncertain, on the contrary, when material from human corpses or
from cattle with ser/sucht was used, as it was always impure on the surface,
and, moreover, was not always quite fresh when it reached me. In these
cases I first rinsed the surface of the object repeatedly with a solution of
corrosive sublimate (1 to 1000) and then cut away the upper parts in layers
with red-hot instruments, which were changed repeatedly ; finally, I took
the material for inoculation from a depth which justified me in concluding
that it would be free from the bacteria which had entered the tissue after
the death of the animal. In this way I generally succeeded in obtaining
pure cultures even from this kind of material, particularly from small
superficial pulmonary cavities, the outer wall of which, after treatment with
solution of corrosive sublimate, was removed with hot instruments.

“After the inoculation of the solidified serum with material containing
bacilli has been accomplished, the vessels are placed in the incubator and
kept constantly at a temperature of about 37°C. Every incubator is not
suitable for the culture of tubercle bacilli. Growth takes place but very
slowly, and the vessels must therefore remain in the incubator for weeks.
So that if the incubator is so constructed as to favour rapid evaporation of
liquids from the culture vessels, the serum gets dry before visible colonies
of tubercle bacilli have developed. For example, an apparatus cannot be
used in which the heat is unequally distributed, so that the vapour constantly
present condenses in the cooler parts, ¢.g., on the glass cover, and has to be
continually replaced by moisture given off from the culture glasses. D’Ar-
sonval’s thermostat is very convenient ; the warmth is equally distributed in
it, and the blood serum remains almost unchanged.

‘‘For the first few days no alteration is to be observed in the cultures in the
incubator. If, however, there is a change, and drops or spots of white or
other colour form on the surface of the serum, increase more or less rapidly
in size, render the fluid at the bottom of the glass turbid or cause the serum
to liquefy, it is a sign that the culture is not pure, and that foreign bacteria
have entered and choked the growth of the tubercle bacilli. If these drops
or spots are examined they are found to consist of bacilli or micrococci
which, by Ehrlich’s method of staining, assume a different colour from the
tubercle bacilli, and are distinct from them also in size and shape. In the
tubes free from these impurities, the first signs of the growing colonies of
tubercle bacilli are not visible to the naked eye for ten to fifteen days.
They then appear as whitish points and small spots lying on the surface of
the serum ; they have no lustre, and consequently stand out clearly from
their moist surroundings. They are best compared to tiny dry scales
adhering loosely to the surface of the serum. The number of the scales
and the extent of surface covered by them vary with the richness of the
infecting material in bacilli, and with the extent of the surface over which
it was rubbed or spread out.

“The individual scales attain only a limited size, so that if few are present
they remain distinct ; but when numerous and closely packed, they coalesce
finally and form a very thin, greyish-white, lustreless covering on the serum.
After a fragment of the tubercular lung of a guinea-pig has been rubbed on
serum, small whitish colonies of tubercle bacilli appear close to the greyish-
red bit of lung, and also in its neighbourhood wherever it has been pushed
over or pressed on to the surface of the serum by means of the platinum

214 BACTERIA.

wire used to distribute the bacilli as widely as possible. The colonies in
some cases are relatively few in number, because only a few bacilli were
present in the pulmonary tubercles, as the examination of sections shows.
In other cases the little colonies are much more numerous; in many, as
especially after inoculation with the contents of cavities very rich in bacilli,
they soon coalesce and form a coherent membranous mass.’

From the cultivations so made other tubes were inoculated
by lifting off the small scales with a needle bent at right
angles, breaking the scale up somewhat so as to cover the
point of the needle with the bacilli, and then drawing it
along the surface of the solidified blood serum. At the end
of ten or eleven days there became visible to the naked
eye, and very much earlier if examined with a lens, a
regular thin superficial layer spreading along each side of
the track of the needle. This organism does not bring about
the slightest liquefaction of the blood serum ; the scales are
somewhat dry ; they are exceedingly thin, and spread only
over the surface, no growth making its appearance in the deeper
layers of the nutrient medium. Tubercle bacilli cultivated in
this manner through seventy generations still continue to
act on animals in the same way as the original or first
culture. Similarly the bacillus grows only on the surface of
a fluid, forming a very delicate, thin film, the organism being
essentially zrobic in its habit. The film growing on solidified
serum is loosely attached to the surface, and any fluid intro-
duced, however gently, floats off a considerable portion in
larger or smaller flakes, which do not break up, but gradually
sink tothe bottom. Koch observed, from the behaviour of the
films when growing on the surface of fluids, or broken down
and introduced into fluid media, that the tubercle bacilli were
non-motile. He described the appearances of these colonies
under the microscope, and came to the conclusion that the
growth was perfectly characteristic, as compared with any
other organisms known up to that time. They have the
appearance of lines or short threads thrown into curves like
worms, or snakes, or flourishes of a pen, thinner or thicker
according to the age of the colony, each thread being com-
posed of a large number of individual bacilli, arranged with
their axes in the long axis of the thread, running parallel to
one another, but with a small space between each, these
being apparently occupied by some zooglceea medium. This
arrangement can be best brought out by pressing down a

TUBERCULOSIS. 215

cover glass on to the surface of a colony as it grows on
the serum, and then removing it without sliding it in any
way and staining by any of the usually recognized methods.

As regards the conditions under which this organism
exists, we have already seen that a certain amount of
moisture is absolutely necessary for its growth, and Paw-
lowsky was able to cultivate it, even on potato, when he
took the precaution to keep a considerable quantity of
moisture in contact with the growing organism, and watched
the potato for a considerable length of time, the growth not
being visible to the naked eye for three weeks or a month.
Koch’s great difficulty in his earlier experiments was, as we
have already seen, to obtain a substance which, in addition to
containing all the other elements necessary for the nutrition
of the bacillus, would remain sufficiently moist for the
requirements of the bacillus when exposed to a pretty high
temperature for a considerable length of time. As regards
temperature, it was found that, although there was an actual
cessation of growth and development below 28° or 29° C.,
on blood serum the organism might be exposed to very low
temperatures for a considerable length of time without
losing its power of again becoming active when returned to
favourable environments. It grows best at about 37° C.,
but as soon as the temperature rises beyond 38° C. the
development of the bacillus in the cultivation tubes begins
to diminish in activity, and at 40° C. (Koch originally gave
this limit as 42° C.) it ceases entirely to grow and multiply.
It is a remarkable fact that the temperature at which the
bacillus develops best is exactly that of the human body
(37.8° C.). In the cow and the horse, where the other con-
ditions must also be favourable, the temperature is about
38.3° C., in the calf a little over 39° C., whilst in the hen we
have the maximum temperature of 40° C.

Klein, in a series of experiments reported in 1886, finds
that it is possible to inoculate successfully with tubercle
taken from the human subject, also that it is possible to
inoculate from a guinea-pig to a cow, but when inoculation
is made from a cow to a fowl the experiment breaks down,
and there is no tubercle produced, so that, not only can one
modify the activity, and the power of growth of these
bacilli outside the body by altering the temperature at which
they grow, but it is also within the range of possibility

216 BACTERIA.

to modify the bacilli by introducing them into different
animals whose normal temperatures and other general
metabolic conditions are different. This modification has
more than nominal value, for it has been proved experi-
mentally that, although the organisms in human and in
bovine tuberculosis are morphologically identical, they are.
not absolutely the same in all their vital and pathogenic
characteristics. For instance, tubercle bacilli taken from
a phthisical patient and introduced into the tissues of a cow
will soon set up an acute general tuberculosis, whilst bacilli
taken from a case of per/sucht, or ordinary bovine tuber-
culosis, almost invariably give rise to the per/sucht form of
tuberculous disease, and rarely, or never, to the acute
generalized form. It would appear that in these cases the
microbe becomes adapted to the special conditions present
in each host, and consequently becomes less suited to the con-
ditions in others. It might be objected that in the case of the
fowl the bacilli are present in enormous numbers, and that,
therefore, their action should be more virulent; but, in
answer to this, it may be pointed out that increased activity
of growth is not necessarily always associated with increased
virulence. In illustration of this fact, it may be pointed out
that, after many experiments, Nocard and ‘Roux were able
to obtain most luxuriant cultivations of the tubercle bacillus
on agar-agar or on blood serum, to which 6-8 per cent. of
glycerine had been added. The organisms on these media
grow so rapidly that they are quite visible at the end of four
days, and at the end of twenty days, and not four weeks, as
on ordinary blood serum, the growth seems to have reached
its maximum, when it appears as a pale grey, thick, mamil-
lated or reticulated, mass. In the same way luxuriant
growths may be obtained in bouillon to which a similar
proportion of glycerine has been added, small opaque flakes
or flocculi first making their appearance at the surface, and
then sinking to the bottom, where they remain. This
growth in glycerine broth may take place at a comparatively
low temperature, 18°-20° C., though it then goes on very
slowly. Earlier generations of such cultivations produce
typical tubercle nodules that grow with extreme and charac-
teristic rapidity when inoculated, but after several generations
of such pure cultivations have been made in these glycerine
media, the virulence may become distinctly diminished,

TUBERCULOSIS, 217

although the growths are as luxuriant as, or are actually
more luxuriant than ever. We have in fact a kind of
reversion to the saprophytic condition of the culture, a
condition accompanied by diminished parasitic virulence.
It is possible, therefore, that the higher temperature that is
met with in cattle along with other, conditions there present
may have a distinct effect in diminishing the virulence of
the organism, whilst at the same time it may play an im-
portant réle in causing its parasitic and vegetative activity
to be increased within the body of these animals, though
this is not necessarily accompanied by increased vegetative
activity outside the body. As Koch pointed out at the
International Medical Congress, of 1890, the tubercle cultures
from fowls were quite distinct and could not be passed
on as such from animals to animals of different species
or by growth at different temperatures, and he con-
cludes that although nearly related to the ordinary tubercle
bacillus they are specifically distinct. It should be noted,
too, that tubercle bacilli grown on glycerine agar are,
according to Nocard and Roux, somewhat shorter than
the bacilli met with in tubercular sputum, that they
also contain numerous ovoid spores, but that otherwise
they are exactly like those described by other observers in
various animals and on other media. As has been already
mentioned, there was necessarily a doubt whether any disease
could be tubercular if it was not possible by special methods to
demonstrate in histological preparations the presence of the
bacillus. This histological demonstration is, however, after
all, a clumsy method, and in many cases it has been found
possible to obtain demonstrations of the tuberculous nature
of a disease by inoculation experiments when the organisms
have been so few that they have escaped the notice of the
most careful observers. It may therefore be confidently
anticipated that more and more proof of the tuberculous
nature of lupus, scrofula, cold abscesses, and bone disease will
be gradually accumulated.

As early as 1843 it had been demonstrated that tubercular
material from dead subjects when inoculated into rabbits
produced tuberculosis; in 1865 these experiments were
repeated and extended by Villemin, and other observers have
from time to time confirmed the results that were then
obtained. Koch’s observations and experiments have now,

218 BACTERIA

however, placed the matter on a much surer footing ; he has,
as we have seen, succeeded in separating a specific bacillus
from tuberculous material, with which he has been able to
produce tuberculosis with the utmost certainty, so that,
instead of dealing with comparatively large fragments of
diseased tissue, he has worked with particles so small that
they can only be distinguished with the aid of the best
microscope and the use of special’ methods of preparation,
This, of course, has cleared up many points which hitherto
have been very obscure, and, most important of all, it has
enabled us to determine the channels by which human beings
may become affected with the disease, especially in connec-
tion with the respiratory passages and by way of the
alimentary canal, and through wounds or damaged tissues.

It has been demonstrated that in the sputum of patients
. suffering from phthisis, in those cases where the softened
lung tissue is breaking down and is being expectorated, an
enormous number of tubercle bacilli may often be met with,
though they may be present in comparatively small num-
bers ; so frequently is this the case, that the presence (in
different numbers from day to day) of tubercle bacilli in
the sputum, or their absence, is relied upon as a diagnostic
feature by attention to which the physician is enabled not
only to determine the rapidity with which breaking down is
going on, but even to obtain very considerable assistance
in arriving at a decision as to whether a disease is tubercular
or not.*

Until quite recently not the slightest attempt has been made either to
alae! the diffusion of such sputum, or to disinfect it in any way, and it

as been said, with some considerable degree of truth, that a very large
quantity of virulent tuberculous material has been allowed to be freely dis-
seminated, with the result that it must have been the lot of many individuals
to contract tuberculosis by means of the inhalation of particles of dried
tuberculous sputum in which active tubercle bacilli or their spores were
necessarily entangled. That the sputum contained the elements which

* In making an examination of sputa for tubercle bacilli, the fact that
the bacilli are most numerous in the small yellow caseous points should
always be borne in mind. Such points should be carefully searched for,
crushed between two cover glasses, dried, stained, and examined. Single
bacilli may be found elsewhere, but masses can, as a rule, be found in these
disintegrating points only. It is only where disintegration has commenced
that any reliance can be placed on the numbers of tubercle bacilli as
affording any assistance in forming a diagnosis and prognosis,

TUBERCULOSIS, 219

were the exciting causal agents of the disease had been experimentally
proved, even before the actual discovery of the bacillus was made, and
dogs, which had been in the habit of taking up the sputum of tuberculous
_persons, had been known to contract the disease, an observation that was
fully corroborated by further experiment. Similarly it had been related
how barn door fowls in a country district, which for a long time were
perfectly healthy, were suddenly attacked by an outbreak of tuberculosis
after a phthisical patient had come to liveat the farm. The expectorations
of this patient were voraciously devoured by the fowls, with the result that
tuberculosis of a most virulent nature broke out in a most extraordinary
fashion amongst the brood. Feeding experiments were also made to
corroborate this accidental experiment ; and more recently similar acci-
dental experiments have been recorded both in France and in this country.

In the case of certain micro-organisms the products of
putrefaction exert such a deleterious influence on them that
they are destroyed very readily and rapidly. Again, in the
case of the cholera bacillus, desiccation at once proves fatal,
not only to its growth, but also to its actual virulence and
power of infection. In the case of the tubercle bacillus,
however, observers, both in France and in Germany, very
early pointed out that putrefaction and drying could exert
but little influence on the number of the bacilli, whilst dry-
ing alone interfered only slightly with their virulence, as it
was quite easy to inoculate rabbits with sputum that had
been dried at a temperature of 30° C. Later, Galtier found
that maceration and putrefaction for a period of five days,
and even intermittent freezing and melting, did not interfere
with the transmission of the disease by means of the bacillus.
Other observers have demonstrated that the bacillus remains
virulent after it has been exposed,in sputum, for forty days, and
even after 186 days if it is carefully protected from the action
of the air. It is, of course, concluded from these experiments
that, difficult as it is to cultivate the specific micro-organism
of tuberculosis outside the body as a saprophyte, the parasitic
form (or its spores) still retains its vitality and power of
development for a considerable length of time—and under
what would appear to be very unfavourable circumstances—
even when removed from its host. The way was thus
being thoroughly prepared for Dr. Georg Cornet’s researches
on infection in hospitals and rooms where phthisical
patients were treated. Dr. Ransome had, early in the con-
troversy, demonstrated the presence of tubercle bacilli in the
air respired by tuberculous patients; and Dr. Williams, in
1883, suspended glass plates smeared with glycerine for a

220 BACTERIA.

period of five days in one of the ventilating shafts of the
Brompton Hospital for Consumption. Washing off the
glycerine “with distilled water, the fluid was mixed with a little
mucilage and evaporated down to one-half, and then examined
for the bacilli,” and in this he was able to demonstrate bacilli
in fair numbers. In a thoroughly purified ward a number
of non-phthisical patients were placed, with the result that
no bacilli were found in the air carried off by the extraction
shaft ; whilst in another ward, filled with consumptive
patients, the washings from a plate exposed for fourteen
days in the extraction shaft contained numerous bacilli.
Similar experiments have been made by other observers, but it
was left for Cornet to make a systematic examination of the
dust in rooms were phthisical and non-phthisical patients
were treated. By numerous careful inoculation experiments,
he demonstrated the fact that the expectorations from
phthisical patients are a source of a very real and appreci-
able danger. The bacilli are not only exhaled, in small
numbers no doubt, but he finds that they are also con-
tained in very considerable numbers in the dried sputum
obtained from handkerchiefs, bed linen used by phthisical
patients, and in the sputum that has made its way on to the
floor and walls through the dirty habits of many of the
patients. His experiments extended over a very considerable
period, and to the rooms of private patients in hospitals, in
lunatic asylums, &c. ; he even found bacilli in the streets
and open spaces in a certain proportion of cases where
tuberculous patients were collected together. These results
have the greater value from the fact that in no case did he
consider his experiments complete unless the dust with
which he was experimenting, when inoculated into animals,
produced the disease. It follows from all this that infection,
as the result of inhalation of the dried virus, is one of the
most common forms with which we have to contend, this
being especially the case in older people, amongst whom
pulmonary tuberculosis is most commonly met with, but in
whom, as in children, pulmonary catarrh seems invariably to
precede the tubercular disease. In children, the catarrhal
inflammation of the lungs that so frequently accompanies
such conditions as measles, scarlatina, diphtheria, whooping-
cough, and other similar conditions, may very frequently
become tubercular in character.

TUBERCULOSIS. 221

‘There seems, in these cases, to be, first, a weakened con-
dition and impaired power of resistance of the epithelial
cells lining the small bronchi, the smaller air passages and
the air vesicles making up the spongy tissue of the
lung. The bacilli in the air and dust then finding their way
to a surface already weakened and specially prepared as it
were for their reception, recommence their parasitic life,
multiply, and make their way further into the tissues, where
they set up the changes associated with tubercular disease.
It is evident from all this that much work still lies ready
to our hand in connection with the spread of tubercle,
and ‘that if we could only persuade people to look upon
tubercle as an infectious disease similar in character to
scarlet fever, though not so rapidly developed, much
would have been done to prevent its spread, and a great
advance in preventive medicine would have been made.
Through the work of Koch, Cornet, and others, the Ger-
mans have come to look upon perfect cleanliness in the
treatment of phthisical patients as absolutely essential.
Pocket handkerchiefs and bed linen used by phthisical
patients are most carefully sterilized by means of bichloride
of mercury, hot air, steam, or other germicidal agents ;
patients are strongly enjoined not to expectorate except into
receptacles specially made for the purpose, receptacles that
can be carried about, can be most readily cleaned, and in
which expectorations can be easily disinfected. Of course,
the results of all this are not yet manifest, but it may be
confidently anticipated that within a comparatively short
time a considerable diminution in the number of phthisical
patients in Germany will have to be recorded ; not to be
compared, perhaps, with the diminution of cases of other
diseases, but still a very appreciable one.

As a single example we may take the Grand Duchy of Baden, where
there was a diminution of deaths from tuberculosis from 3.08 per 1,000
inhabitants in 1882 to 2.80 per 1,000 in 1887, or no less than .28 per
1,000. Were this to be equalled in the British Isles, and the patients were
not carried off by other diseases, the saving to our community would be
nearly 10,000 lives per annum.

We may here mention the various disinfectants used. By far the best is
heat, especially moist heat. Sunlight, or even ordinary daylight, will, ac-
cording to Koch, kill tubercle bacilli in from a few minutes to five or six days.
Koch has also proved that a number of ethereal oils, some of the so-called
aniline or tar dyes, mercury in the form of vapour and silver and gold com-

222 BACTERIA.

=

pounds, all exert an inhibitory effect on the growth of tubercle bacillus ; the
compounds of cyanogen and gold, especially, even in a solution diluted to one
part of cyanide of gold to two millions of thesolvent substance, checking the
growth of tubercle bacilli. Carbolic acid also kills the bacilli if it is allowed
to act in considerable strength and for a lengthened period ; but none of
these can, for a moment, be compared as regards not only efficiency and
cheapness, but for facility of application, with steam or boiling water. All
disinfection should be just as completely carried out when a phthisical patient
leaves a room or ward as if a case of scarlet fever had been treated there ;
the walls, floor, and even the roof should be thoroughly washed and disin-
fected by means of hot water or lime. During treatment in such apart-
ments no patient should be allowed to expectorate on the floor; and glass
or porcelain spitoons, which can easily and safely be boiled in water, should
be placed for the reception of all sputa. During the time that corridors,
steps, and rooms are being cleaned and disinfected, they should be kept
quite moist, in order that as little dust as possible may arise from the clean-
ing operations. No room that has been occupied by a phthisical patient
should be used until it has been thoroughly disinfected ; the bedding and
curtains should be well boiled, the blankets steamed, the mattresses dis-
infected, all the furniture washed with soap and water, the carpets and
upholstering thoroughly beaten in the open air, the dust of which should
be caught in straw (such straw and the paper taken from under the carpets
should always be burned), the floor thoroughly washed with soap and hot
water, and the wall paper rubbed down with freshly baked bread. In
San Remo all these methods of procedure have been brought under the
notice of the hotel-keepers, and they are advised to carry them out in
connection with the whole of their rooms at the end of each season, not
only the sleeping rooms, but also the public rooms.

The alimentary canal is probably the next most important
channel of infection. Evidence of infection by this channel
was first obtained by the feeding experiments already referred
to, but further evidence of it has been noted in the ulcera-
tion of the intestine that is so frequently met with in
phthisical patients, and which appears to be due to the action
of bacilli contained in the sputum passing down the gullet
through the stomach in cases where the gastric juice is not
very active, and so to the intestine in which ulceration occurs
in the course of the tubercular process as the result of the
pathogenic activity of the bacilli. In children this mode of
infection is comparatively common, and tubercular ulceration
of the intestine, or tubercle of the glands connected with the
intestinal tract, is of frequent occurrence.

In 127 cases of tuberculosis in children that I examined this tubercular
ulceration was found in 43; whilst in 100 cases, or nearly 79 per cent. of
the whole, the glands were in some stage or other of tubercular degenera-
tion. It would thus appear that tuberculosis connected with the intestine
is of frequent occurrence in children, and we should therefore argue that

TUBERCULOSIS. 223

the infection, in these cases at any rate, frequently takes place by the ali-
mentary canal. The age at which these tubercular glands were found is
very significant ; during the first year of life there were 4 cases ; from 1 to
2% years, 33; from 3 to 54 years, 29; from 6 to 74 years, 12; from 8 to 10
years, 133; and from 11 to 15 years 9 cases. In 14 cases these glands only
were affected.

Bolitz (Inaugural Dissertation, Kiel) gives similar figures, but on a more
extensive scale.

Out of 2,576 children whose bodies were submitted to a post mortem ex-
amination in Kiel during the years 1873-1889, there were 424 cases of
tuberculosis, or 16.4 per cent. of the whole mortality.

The following shows the percentages of the whole of the deaths from
tuberculosis at each of the diferent ages :—

Still-born children . 0.0 per cent. { Up to2-3 years old 33. per cent.
6

Upto4weeksold 00 ,, » 3-4 yy 29. ”
” 5-10 ” 0.9 ” » 45 ” 31.8 ”
»» 3-5 months old 8.6 ,, »» 5-10 55 55 34:3
» G12 ,, » 18.3 ” »» 10-15 5, 2 30.1 ”

» I-2years ,, 268 ,,

Here, again, as where the lung is attacked, we must look
upon the bacillus as the exciting cause, but tissue weakness
as the predisposing cause. These conditions may be summed
up as follows :—

_(a@) The presence of the bacillus tuberculosis in such a
position and for such a length of time that it obtains a coign
of vantage, so to speak, from which to attack the tissues of
the body.

(6) Some weak point in the epithelial surface “made by
disease, or due to irritation or bad food,” by which the organ-
isms may attack the deeper tissues in sufficient numbers to
ensure their being able to hold their own in the struggle for
supremacy that ensues.

(c) The comparatively low vitality of these deeper tissues
brought about by imperfect nutrition or irritation ; the cells
of which they are composed being no longer able to deal
successfully with any large number of bacilli that can under
ordinary circumstances find their way thus far.

As regards the possibility of the bacillus tuberculosis being
present in the intestinal canal, it should be remembered that
the class of patients amongst whom abdominal tuberculosis is
most rife, consists of infants, which during the first year of
their life, and sometimes for a longer period, are suckled at
the breast ; after this, however, the diet is extremely mixed,
and as a rule it is extremely unsuitable ; but it is in by far the

224 BACTERIA.

larger proportion of cases, even amongst the poorer classes,
partially, at any rate, composed of cows’ milk. During this
first year of their life, children with tuberculosis of the
mesenteric glands, or of those glands connected with the
intestine, form a very small proportion of the cases of infantile
tuberculosis. Whilst the child issuckled by its mother there
is little tubercle, but after this first year there is a very rapid
rise in the mortality from tubercle. It isa somewhat singular
fact that although tuberculosis is frequently met with in
young married women, tubercular disease of the breast is
extremely rare, so rare, indeed, that one observer, Dr. Huber-
maas, who took great interest in this subject, was able to
collect the records of only some eight cases. In cattle, on
the other hand, where the mammary gland carries on its
functions when the animals are placed under conditions
which are far from healthy, or at any rate far from normal,
this tubercular disease of the milk gland is not by any means
of infrequent occurrence.

Some of the earliest experiments from which actual proof
of the infective nature of tuberculous material was obtained
were those made by Gerlach and Chauveau, who used the milk
of tuberculous cows to feed young animals ; though tubercu-
losis was not produced in every case, the former was successful
in a sufficient number to justify his conclusion that there was
some specific virus in the milk of tuberculous cows which
could, when ingested, produce tuberculosis of the alimentary
tract, or of the glands associated with it. Numerous experi-
ments on young pigs, some of them accidental, others de-
signed, and others on calves and hens, have been recorded,
in which tuberculosis has evidently followed their being
fed with tuberculous milk. At the International Medical
Congress held in Copenhagen in 1884, Professor Bang, of the
Royal Veterinary School in that city, gave the results of a care-
ful examination of twenty-seven cases of tubercular disease
of the udder in cattle, in the milk of which he was able to
demonstrate the presence of tubercle bacilli, both directly
under the microscope and in the sediment obtained by means
of the use of acentrifugal separator. With this milk, or with
the separated sediment, he was able to produce tuberculosis
both by inoculation and: by ingestion. Another observer,
Nocard, was able to demonstrate the presence of the specific
bacillus in milk in eleven cases, and Professor M’Fadyean and

TUBERCULOSIS. 225

I also found tubercle bacilli in the milk from six cows out of
six hundred examined. So certainly are the bacilli found in
cases of tubercular disease of the udder that certain authori-
ties maintain that it is possible to differentiate between the
simple and the tubercular inflammation of the udder in the
cow merely by means of a microscopic examination of the
milk. (See Appendix).

This question of tubercle in milk has now assumed such importance that
much attention has been given to it abroad, and in Denmark a most
complete system of inspection has been instituted in connection with one
of the largest milk supply associations in the world ; and private enterprise,
guided by Prof. Panum and Mr. Busck of Copenhagen, has indicated
a way in which the State might tread with very great advantage. In
connection with this association, six special veterinary surgeons, in addition
to local practitioners, are constantly employed in keeping watch over the
cattle that supply the milk to it. One of these veterinary surgeons alone,
specially retained, examines eight hundred cattle fortnightly, and makes
most careful notes (notes that I had an opportunity of examining) of the con-
dition of every animal. Bang, to whose energy and observations the opening
up of the tuberculosis question is very greatly due, contends that a diagnosis
of tubercular disease of the udder can generally be made without difficulty
during life and in the very early stages of the disease. With this conclusion
it is somewhat difficult to agree, but much more may be done in the way of
careful and systematic examination than is done at present, and in Denmark
they have certainly systematized the examination of cattle to a far greater
extent than we have succeeded in doing. As it may be of interest to
mention the points on which the veterinary surgeon should depend in
making his diagnosis, they may, so far as I was able to follow the routine
of a number of inspectors, all of whom however differed slightly in their
methods, be briefly summed up in the following :—

(a) First of all the sub-maxillary glands are examined ; these are easily felt,
and any change is readily made out.

(4) The glands at the root of the neck and those in front of the haunch
bones are always carefully examined. The glands in the flank should be
equal in size, about the size of the middle finger, and not hard. Mere
enlargement, even when considerable, is, however, not looked upon as of
great importance if it is perfectly equal on the two sides.

(c) The animal is made to cough by means of pressure on the trachea,
and the lungs are carefully examined during and after the coughing.

The condition of the skin over the flanks is carefully observed ; it should,
in a healthy animal, be “ loose,” like that of a dog, soft and pliable; any
adhesion, hardness, or harshness, should be carefully noted.

(d) The udder is carefully examined for inequality of size and for any
induration. It is a somewhat curious fact that tuberculous disease usually
affects the hind quarters of the udder, which become hard and knotty, but
not painful ; whilst in acute inflammation of the udder, the anterior quarters
are quite as much affected as the posterior ; the pain is usually very acute,
and the process is accompanied by much more marked febrile symptoms.

(e) Then the glands above the bar high up between the quarters, are

I

226 BACTERIA.

most carefully examined. In cases of tubercular disease of the udder these
glands are invariably affected, are unequal in size, and the larger one,
corresponding to the affected quarter, is usually considerably indurated.

) Careful auscultation is carried out at least once a month. The fore-
foot of the side that is being examined being always well advanced. The
normal expiration sound lasts half as long as the normal inspiration, and
if this rhythm is deviated from in any way, a further and thorough examina-
tion of the lungs should always be made.

) The examination is continued still further if the slightest suspicion of
tubercular disease is aroused by the above investigation, and an examination
per rectum is made, with the object of determining whether there is any
tubercle of the peritoneum or not. As the onset of the disease in the udder
is so rapid, and as, as yet, it is held by most observers that the bacilli may
make their appearance in the milk, even where the udder is not directly
affected, it follows that if there is the slightest suspicion of the existence of
tubercular disease in a cow, the milk from that animal should not be put
into the milk supply, and as a matter of fact, on the Danish farms above re-
ferréd to, it is not sent to town, but it is either thrown out, or after being
most thoroughly disinfected by prolonged boiling, is given to the pigs.

(4) The farmer keeps a record of the quantity of milk given by each cow,
and a note of what is done with it; and any milk that is put out of the
supply by the veterinary surgeon, or by the farmer himself, on account of
suspected disease, is paid for by the company, or the difference between the
full value and the value as pig food.

other inflammatory condition of the udder is carefully noted, and
even then the milk is withdrawn from the regular supply.

(4) A small quantity of milk is always drawn off by the veterinary sur-
geon, who carefully notes its colour. If it is too thin and watery looking he
immediately condemns it; whilst if it loses the peculiar blue tinge that
freshly-drawn milk from a healthy cow almost invariably has, and takes on
even a slight yellow tinge instead, the milk from the infected quarter is not
used for any purposes, although the milk from the other quarters may be
used, after being thoroughly boiled, for the feeding of pigs.

The authorities of the association insist rigidly on the fortnightly in-
spection, because it has been observed that very great swelling may appear
as a sign of udder tuberculosis in from ten to fourteen days, as in this posi-
tion the onset of the tuberculous disease is usually much more rapid than in
the lungs, in which the process in a very large majority of cases appears to be
far more chronic in character. In all cases, the condition of the glands
must be systematically and carefully observed.

In the light of recent events, too, it is to be hoped that Koch’s tuberculin
may be utilized in the diagnosis of tuberculosis in cattle, and that satis-
factory results may thus be obtained.

In consequence of the rapid onset of the disease diag-
nosis without regular inspection is almost impossible,
and it is probable that the swelling of the udder is only
one of the later manifestations of the disease, the glands
above the udder apparently being a far more reliable
index. Professor M’Fadyean and I were so much struck

TUBERCULOSIS. 227

with this difficulty of diagnosis, that in a paper read at a
meeting of the Pathological Section of the British Medical
Association, held in Dublin, August, 1887, we stated that it
appeared to us “that where, as is very frequently the case
with cows kept in towns, a complete history of the diseased
condition of the'udder is not obtainable, a differential diag-
nosis of mammitis (inflammation of the milk glands) is by
nO means easy, except by microscopic demonstration of the

Tutercle bacilli arranged around a closed up milk-duct in a case of
Tuberculous disease of the udder of acow. x 1000.
bacilli in the milk ; which may also fail if a most careful
search is not made” ; and Principal Walley, dealing with
the same subject, says, “that he could not undertake to
diagnose, with accuracy, tubercular mammitis in every case,
nor even in a majority of cases”; and he states that in
specimens of the udders of tuberculous cows he has ex-
amined, after death, he has found good examples of tubercu-
losis in the mucous membrane of the milk sinuses without

228 BACTERIA.

the occurrence of any induration of the milk gland, “ indeed,”
he adds, ‘“ I may say that no veterinary surgeon could, during
life, have diagnosed the existence of tubercular mastitis
(tubercular inflammation .of the udder) without the aid of
a microscope.” That tubercle bacilli make their way into
the milk, when there is tubercular ulceration in the milk-
ducts, can be readily understood, and has frequently been
shown ; but it has also been demonstrated by Professor
M’Fadyean that in cases of tuberculosis, bacilli may be
found lying free in what are otherwise apparently healthy
milk ducts. Fresh evidence is being accumulated every
day, but these facts alone, when considered along with
the occurrence of bacilli in milk, with the feeding experi-
ments already recorded by so many observers, and taken in
connection with the great prevalence of tubercle‘in certain
classes of animals, afford strong presumptive evidence that
milk is a source of tubercular infection, especially in children,
and in those in whom, on account of imperfect nutrition
and impaired digestion, the walls of the alimentary canal are
less able to resist the invasion of the organism known as
Koch's Bacillus Tuberculosis.

The danger of contracting tuberculosis from taking meat
from cattle affected with the disease is perhaps not so great
as that associated with the drinking of tuberculous milk ;
it is nevertheless one with which the sanitary authorities
will have to deal, and one to guard against which it is
necessary to take very considerable precautions. There can
be little doubt that in those cases where the disease is
localized to any one of the viscera at the time of the
death of the animal, there is little danger to be anticipated
from eating the well-cooked flesh from other parts of that
animal; and if we could be absolutely certain that the
localization was complete, all would be well. Even in cases
where the flesh is taken from animals in advanced stages of
tuberculous disease there would be a certain proportion of
cases in which no evil results would follow, and one observer
who made sixty-two experiments with such flesh boiled for
ten or fifteen minutes, found that only 35.5 per cent. of the
inoculated animals became tuberculous. Even in cases of
generalized tuberculosis Nocard failed in thirty-nine out of
forty cases to transmit the disease by means of raw muscle
juice injected into the peritoneal cavity of guinea pigs, but

TUBERCULOSIS. 229

he succeeded in the fortieth. Other observers, however,
have been more successful, and in two rabbits I was able
to produce tuberculosis by injection into the peritoneal
cavity of the raw juice expressed from the intercostal
muscles of a tuberculous cow after all tuberculous pleura
had been carefully “stripped”; whilst the juice taken
from the muscle of the thigh injected into two other rabbits
was perfectly innocuous. The danger of infection by the
consumption of meat from tuberculous cows may have been
much exaggerated, but that there is a very appreciable
danger must most certainly not be lost sight of by our
Medical Officers of Health and the Veterinary Inspectors of
the Board of Agriculture.

Koch, as we have seen, had discovered a specific or-
ganism which he had been able to cultivate outside the
body ; he had inoculated and produced tubercle ; and he
had found certain germicidal agents which were capable of
destroying the organism outside the body. He, and the
many interested workers who had investigated the subject,
had found that it was more difficult to inoculate certain
animals successfully than others. It had been observed even
that certain individuals of the same species were much more
refractory to the action of the virus than others, and it
very naturally suggested itself to those workers, that there
were two conditions which would have to be determined
before any systematic and organized attack could be made
on the tubercle bacillus within the body. The tubercle
bacillus itself might be developed under such conditions that
its virulence might be more or less modified. For example,
Koch found that all his cultivations made on blood serum
retained their power of growing in animal tissues in a most
extraordinary degree, and for long this substance was the
only nutrient medium used for the cultivation of the tubercle
bacillus. It was found, indeed, that the other combinations
used: for the cultivation of other organisms up to that time
were valueless as nutrient substrata for the tubercle bacillus.
Nocard and Roux, however, found, as we have seen, that on
the addition of a certain percentage—6-8 per cent.—of
glycerine to peptonized broth, solidified by the addition of
agar, they could obtain a nutrient medium on which the
tubercle bacillus would grow most luxuriantly. After it had
passed through several generations of cultivations on this

230 BACTERIA.

medium, the virulence of the organism, or its power of
growing in tissues, was distinctly diminished—z.e., the
organism was in a condition to be much more readily de-
stroyed by the cells of the animal body, which appeared to
keep the upper hand throughout the struggle, very few of the
cells being destroyed, as is the case when virulent tubercle is
used, where although a number of the tubercle bacilli are un-
doubtedly destroyed, the degeneration of the proliferated cells
is so extensive that caseous nodules are formed, and the typical
appearance of caseating tubercle are presented in the inocu-
lated area. The second factor, though equally important, and
though recognized indirectly by all physicians, could not put
itself so fully in evidence as the first, and it was only after
Metschnikoff had made his beautiful observations on the daph-
nia and on the separating tail of the tadpole and the phagocyte
action of certain cells in these processes, that any direct evi-
dence of the bearing of the activity of the cells of the tissues
of the body on the destruction of micro-organisms within the
body could be definitely adduced. ‘Then, indeed, began the
narration of the history of the battle between the cells and
the bacilli. If the bacilli were weak, or were present in small
numbers only, the cells were invariably the victors ; if the
cells were degenerated, or were badly nourished, or if their
vitality was low, the bacilli proved themselves the more
powerful. How these results were brought about soon
became the subject of most violent controversy, and in the
controversy thus raised explanations of facts that had hither-
to puzzled scientific workers were projected and forced home.
Let us here, however, examine only those facts that appear
to have a special bearing on the subject of tuberculosis.
A perfectly healthy individual, placed under favourable con-
ditions as regards food, fresh air, and exercise, is never
attacked successfully by tubercle bacilli, the active, vigorous
tissue cells being perfectly competent to destroy any bacilli
that may make their way into the lungs, the pharynx, or the
intestine ; whilst even in cases of direct inoculation into a
wound, if the wound heals rapidly, no tubercular process
may result, the tubercle bacilli, as we have said, being
destroyed by the cells. It may be, indeed, that certain
secretions of the cells—z.e., those substances that bear the
same relation to the connective tissue cells that an enzyme
bears to the yeast-cells—also exert a deleterious action on the

TUBERCULOSIS. 231

bacilli in the healthy individual, and in a minor degree
even in individuals with weaker tissues ; the activity of the
bacilli is so interfered with or modified that they can be
readily attacked and devoured by the tissue cells, which, as has
long been known, have a most remarkable power of taking
up into their substance many effete materials and particles
of dead or inorganic matter. On the other hand, certain
excretions, by accumulating in the blood and in the lymph
spaces, may impair the activity of these tissue cells and so
render them less able—(1) to secrete their protective material,
and (2) to wage war directly against the bacilli; whilst, in
turn, the bacilli on their side, as we have already seen, secrete
a material that has a most injurious action on the tissue
cells, causing them to swell up and eventually to become
hyaline. . This material is only a poison when in large or
comparatively large quantities; in smaller doses it acts
in the first instance as an irritant or stimulant, stimula-
ting the protoplasm to exert all its powers against the
advancing bacteria, powers that are so strongly exerted (un-
less the conditions of nutrition and excretion are specially
favourable) that they are rapidly exhausted. It has been
observed that in those cases where phthisis was curable the
cure has been effected only by careful nutrition of the
tissues, and that as soon as they have been brought up to
a certain standard of health the disease has been checked, in
many cases permanently; on the other hand it has been
found that when the tissues have again fallen below far there
has been a fresh outbreak of the disease. These facts are in
themselves sufficiently interesting and suggestive, but as we
shall see they have a further important bearing on the
question of the curability of the tubercular phthisis.

It has been observed that a process of localization occurs
even when large caseous patches have been formed, and it
has been found that around these patches, just as around an
abscess, there is always erected a kind of barrier, made up
of vigorous connective tissue cells, small, round and larger
epithelioid cells; the blood vessels in this cellular zone
being comparatively numerous and of considerable size. We
have, in fact, in this arrangement of the blood vessels and
cells, a making of roads (the blood vessels) for the bringing
up and massing of forces (the active cells) around the
enemies’ camp (the tubercular or caseous mass with the con-

232 BACTERIA.

‘tained bacilli or spores), and by a process of close siege pre-
venting the organisms from making their way outwards, and
confining them entirely to their own territory, so that,
when they have utilized what food material there is in the
degenerated cells, they are no longer able to exist as vege-
tative bacteria, and only the spores remain—which may,
however, remain latent for a very long period awaiting a
favourable opportunity for another attack on weakened
tissues. These spores or hibernating germs are confined
within the same area, and the aébrzs with its contained
spores is gradually encroached upon by the surrounding
tissues until, eventually, if the mass is not large it may be
entirely absorbed, though, owing to the amount of fibrous
tissue that is formed by the attacking cells after their activity
is somewhat diminished, this process of absorption sometimes
goes on very slowly.

It was an easy enough matter, when the history of the
development of the tubercle bacillus was known, to kill the
organism outside the body, and Koch found that a very large
number of germicidal substances were capable of interfering
with its growth ; but unfortunately most of these germicides
also exerted an injurious effect on the tissues, so that what
was gained in one direction was lost in another.

In most other diseases in which preventive or curative
inoculation by means of vaccines—less virulent cultures of
the microbes—to accustom the animal to the action of the
poison and so enable it to resist the more virulent, has been
attempted, the aim has been to render the bacilli less, and the
cells more active, and to develop in these cells a special activity.

By accident, as he tells us, Koch found that by injecting
tubercle virus into the subcutaneous tissues of guinea-
pigs which had been previously inoculated with tuberculosis,
the tissue in which the tubercle bacilli were acting was
actually destroyed and an eschar or slough was formed
at the point of inoculation; whilst although the bacilli
were not directly destroyed, they remained embedded
in a mass of food material that was gradually but surely
used up to supply the needs of these bacilli, and
eventually they had to go into winter quarters. But
the products of the tubercle bacilli appear to act in
some way on the tubercles already formed; they do
something more than bring about complete degenera-

TUBERCULOSIS. 233

tion of the weakened cells. In the immediate neighbour-
hood of the young tuberculous tissue, z\¢., in those cells
that have been slightly affected by the products of the
tubercle bacillus it sets up a further reaction ; it stimulates
these cells, and at the same time causes a dilatation of the
vessels and probably also an increase in their number (this
latter may be only apparent); a larger amount of food
material is brought up for the nutrition of these cells,
excreted matter is more readily carried away both by vessels
and lymphatics, and in consequence of this, cells that have
been too far stimulated by the tubercle poison to recover,
die off, whilst those that are still capable of living, even
under the excessive stimulation, proliferate and help to form
the barrier between the dead mass and.the surrounding
normal tissues. In this way it would appear that in certain
cases of tuberculous disease, Koch has produced an imitation
of the natural process of cure. Bya process of combined
reasoning and experimentation, and basing his method of
procedure on the one that had been already adopted in con-
nection with the preparation of vaccines for other diseases,
Koch succeeded in obtaining a substance with which, in a
certain degree, at any rate, he was able to combat the
advance or to modify the tuberculous disease in animals.

As this advance in the treatment of tuberculosis marks a
most important point in the history of the disease, and in
order that there may be no misconception as to Koch’s exact
position, it may be well to give in his own words the descrip-
tion of the discovery, composition, and probable mode of
action of the remedy.

He says, in describing the observations, by the considera-
tion of which he was led to take the lines of experimentation
that ultimately led him to success: “If a healthy guinea-pig
be inoculated with a pure cultivation of tubercle bacilli, the
inoculation wound generally becomes glued over or sealed,
and appears to heal up during the next few days. It is only
in the course of from ten to fourteen days that a hard nodule
"is formed, which soon opens, forming an ulcerating spot which
persists until the death of the animal ; if an animal that is
already tuberculous be inoculated the course of events is
very different. The most suitable animals for this experi-
ment are those which have already been successfully inocu-
lated four or six weeks previously. In the case of an animal so

234 BACTERIA,

treated the small secondary inoculation wound becomes
sealed at first, but no nodule is formed ; a peculiar change
takes place at the point of (primary) inoculation. As early
as the first or second day the point becomes hard and dark-
coloured—a condition that is not confined to the point of
inoculation—and spreads around for about 0.5 to 1 centi-
metre. During the next few days it becomes more and
more clear that the epidermis thus changed is necrotic, and
finally it is thrown off, and a flat ulcerated surface remains,
which generally heals quickly and completely, without infec-
tion being carried to the neighbouring lymphatic glands.
Thus the inoculated tubercle bacilli act quite differently
on the skin of a healthy guinea-pig, and on that of a
tuberculous one. But this remarkable action does not
belong exclusively to living tubercle bacilli, it also be-
longs in the same degree to dead ones, whether they be
killed by low temperature of long duration, which I at
first tried, or by boiling heat, or by the action of certain
chemicals,

“ This peculiar fact having been ascertained, I followed it
up in all directions, and then further found that pure culti-
vations of tubercle bacilli thus killed, after they are ground
down and suspended in water, may be injected under the
skin of healthy guinea-pigs in large quantities without
producing anything but local suppuration. Tuberculous
guinea-pigs, on the other hand, are killed by an injection of
very small quantities of suspended cultures within a time
varying from six to forty-eight hours, according to the dose ;
a dose which is just insufficient to kill the animal being
sufficient to produce a widespread necrosis of the skin in the
region of the point of (primary) inoculation. If the fluid -
with its suspended matter be still further diluted, so that it
is scarcely turbid to the eye, the animals remain alive ; and
if the injections be continued at intervals of one or two days
a noticeable improvement in their condition soon sets in ;
the ulcer at the point of inoculation becomes smaller, and
finally cicatrization takes place. This is never the case
when such treatment is not resorted to. The swollen lym-
phatic glands become smaller, the condition as- regards
nutrition improves, and the progress of the disease is
arrested, if it is not already so far advanced that the animal
dies of debility.

TUBERCULOSIS. 235

“These facts formed the basis of a therapeutic method
against tuberculosis. But an obstacle to the practical em-
ployment of fluids containing in suspension the dead
tubercle bacilli was found in the fact that the tubercle
bacilli are not (readily) absorbed; they disappear, and
remain for a long time unchanged zz setu, producing
larger or smaller suppurating centres. It was clear, there-
fore, that in this method the curative effect on the tuber-
culous process was obtained by a soluble substance, diffused
so to speak, into the fluids that surround the tubercle bacilli,
and transferred without delay to the circulating fluids of the
body ; whereas that which has the pus-forming quality
seems to remain bound up in the tubercle bacilli, or at any
rate to be only very slowly dissolved or washed out. Thus
the only important thing that remained to be done was to
carry out the process that takes place within the body—
outside of it also—and if possible to extract and isolate the
curative substance from the tubercle bacilli. This problem
required long and continued experimentation, but at last I
succeeded, by the help of a 40 to 50 per cent. solution of
glycerine, in extracting the active principle from the tubercle
bacilli. My further experiments on animals, and finally on
human beings, were made with liquid thus obtained ; and
in this way, also, the liquid which I supplied to phy-
sicians and surgeons in order that they might repeat the
experiments, was obtained. Zhe remedy with which the new
therapeutic treatment of tuberculosts zs carrted out, 1s, there-
jie a glycerine extract of pure cultivations of tubercle

acills.

“Tn addition to the active principle there pass from the
tubercle bacilli into the simple extract all other substances
soluble in 50 per cent. glycerine, and therefore it is found to
contain a certain quantity of mineral salts, pigment, and
other unknown substances—extractives, &c. Some of these
substances can be readily separated, as the active principle
is insoluble in absolute alcohol, by which it can be preci-
pitated, not pure certainly, but in combination with such
other extractive matters as are also insoluble in alcohol.
The colouring matter, too, can be separated out so that it is
possible to obtain from the extract a colourless dry substance,
which contains the active principle in a much more concen-
trated form than does the original glycerine solution.

236 BACTERIA.

“This purification of the glycerine extract has, however, no advantages as
regards practical application, as the substances removed have no action on
the human organism, so that the purifying process would only involve un-
necessary expense. The constitution of the active principle can, as yet, be
only a matter of conjecture.

“Tt appears to me, indeed, to be a derivative of albuminous
bodies, and to be closely related to them, but it does not
belong to the group of so-called toxalbumens, as is proved
by the fact that it can withstand high temperature, and in
the dialyzer it passes quickly and easily through the mem-
brane. The quantity of active principle present in the
extract is in all probability very small. I estimate it at a

‘fraction of 1 per cent. Thus, if my assumption be correct,
we have to deal with a substance, the action of which on the
tuberculous organism, far surpasses that of the strongest
drugs known.

“ Various hypotheses may of course be formed as to the
specific mode of action of the remedy on tuberculous tissue.
Without in any way affirming that mine is the best possible
explanation, I imagine the process to be as follows: The
tubercle bacilliin their growth produce in the living tissues—
just as in the artificial cultivations—certain substances which
exert various but always deleterious influences on the living
elements surrounding them, the cells. Amongst these
substances is one which, in a certain concentration, destroys
living protoplasm, and causes it to undergo a transformation
into the condition called by Weigert, ‘coagulation-necrosis.’
The tissue having thus become necrotic, the conditions are
so unfavourable to the nutrition of the bacillus that it is
unable to undergo further development, and finally, in some
cases, it dies off. In this way I explain the remarkable
phenomenon, that in organs freshly attacked by tuberculous
disease—for instance, in a guinea-pig’s spleen or liver filled
with grey nodules—numerous bacilli are found, whilst bacilli
are rare or entirely absent when the enormously enlarged
spleen is made up of whitish substance in a condition of
coagulation-necrosis, such as is often met with in guinea-
pigs that die of tuberculosis. A solitary bacillus, however,
cannot produce necrosis at any great distance from itself, for,
as soon as the necrosis has covered a certain area, the growth
of the bacillus—and, in consequence, the production of the
necrosis-producing substance—diminishes, and thus a sort

TUBERCULOSIS. 237

of mutual compensation is set up, and to this is due the fact
that the growth of isolated bacilli isso remarkably restricted,
as for example, in the case of lupus, in scrofulous glands, &c.
In such cases the necrosis extends only over a part of the
cell, which then, in its further growth, assumes the peculiar
form of a giant cell. I thus follow in this statement of my
views the explanation of the growth of giant cells first given
by Weigert. Now if the necrosis-producing substance were
artificially added to that contained in the tissue surrounding
the bacillus, then the necrosis would extend further, and
thus the conditions of nutrition of the bacillus would
become much more unfavourable than is usually the case.
Then, not only would the more completely necrosed tissues
disintegrate, slough, and—where this is possible—take with
them the enclosed bacilli, carrying them outside the body,
but the growth of the bacilli would also be interfered with
to such an extent that they would die off much sooner than
they do under ordinary conditions. It is in calling forth
such changes that, to my mind, the action of the remedy
seeems to consist. It contains a certain amount of the
necrosis-producing substance, of which a correspondingly
large dose has a deleterious influence—even in healthy
persons—on certain elements of the tissues, probably on the
white blood corpuscles or cells closely related to them, thus
giving rise to the fever and the whole peculiar complex of
symptoms that supervenes. In tuberculous persons a much
smaller quantity suffices to cause, at certain spots—z.e.,
wherever tubercle bacilli vegetate, and have already impreg-
nated their surroundings with the necrosis-producing sub-
stance—a more or less extended necrosis of cells with the
production of the accompanying conditions that affect the
entire organism. In this way it is possible to explain—at
least for the present—in a provisional manner, the specific
influence which the remedy, in certain well-recognized doses,
exercises on tuberculous tissue, as well as the possibility of
increasing the doses in so remarkable a fashion, and, finally,
to explain the curative effect which the remedy undoubtedly
exerts where the conditions are at all favourable for its
exhibition.”

The substance to which the name of Tuberculin has been given

has been analyzed, and is found to be ‘‘a syrupy, slightly foaming
liquid (sp. gr. 1015?) of brown sherry colour, its aqueous solutions

238 BACTERIA.

showing a greenish florescence. In odour it resembles elder yeast or leaven,
combined with a sweet aromatic admixture, such as honey. If slowly
heated, the smell of yeast gives way to an agreeable odour resembling
fruits ; on further heating, the smell- becomes like that of fresh bread
crust,” but without the acid fruit odour. If the heating of the material is
continued, ‘‘ the smell assumes the empyreumatic character exhibited by
burning albuminous matter and carbonizing horny substances. Only an
extremely small quantity of ash (under 1 per cent.) was obtained. The
liquid shows a neutral reaction.” It was found to contain a small quantity
of mucine, indicated by a turbidity on the addition of dilute acetic acid,
which is increased on the addition of potassium ferrocyanide, indicating the
presence of albumen. Peptones are present in considerable quantities.
There is a slight reducing action obtained when the fluid is heated with
Fehling’s solution ; there was no reaction with acid bichromate of potassium,
so that acids and ptomaines are absent ; but it was assumed, as would now
appear incorrectly, that toxalbumens, or albumoses, globulins, or enzymes
must be the substances to which the material injected by Koch owes its
special properties,

Any description of the lymph other than that given by
Koch himself must be the result merely of guess-work, but
even guesses will sometimes afford indications as to the lines
on which researches are at present being carried out, not
only in connection with tuberculosis, but with several other
most deadly and wide-spread diseases. The peculiarity of
this method of treatment is, that it is not known to pro-
tect against an attack of tuberculosis, though Koch states
that he hopes his guinea-pigs will be protected from future
attacks ; it is used solely as a therapeutic agent to check or
stop the disease after it has once.obtained a foothold in the
body. Itis thus in principle more like Pasteur’s inoculation
against hydrophobia, for the inoculation is made after the
patient has been inoculated with the disease virus; and it is
also similar in certain points, though the principle is different,
to the protective inoculation obtained by Hankin, who, by
means of albumoses obtained from anthrax cultivations, has
been able to produce immunity against anthrax, though not to
cure, after that disease was once induced ; and that Dr. Cart-
wright Wood and I carried on with the pyocyanin products
in rabbits, to tide them over an attack of anthrax the bacilli of
which were introduced into the subcutaneous tissue shortly
before, or immediately after, the injection of the pyocyanin.
We, however, considered that the pyocyanin had not acted
directly on the anthrax bacillus, but that it acted by stimu-
lating the cells ; whilst Koch’s “lymph” acts (1) by destroy-
ing the tuberculous tissue, and rendering it unfit for the

|
|

TUBERCULOSIS, 239

nutrition of the bacillus, and (2) by setting up a localized
reaction in the neighbourhood of the bacilli, by means of
which the cells are so stimulated that they are able, as we
have already seen, to prevent the extension of the bacilli into
the surrounding parts.

Koch's fluid may not accomplish all that is expected from it ;
it may, in fact, be found that Koch has not completed his ex-
periments, but he has made a wonderful advance in our know-
ledge of the conditions necessary for the combating of micro-
organisms ; and has extended the observation of the earlier
workers at the globulines and albumoses who really opened up
the way for the advance of the numerous workers who have re-
cently come into the field. It would, however, take us beyond
the scope of the present work to say more about this marvellous
discovery of Koch's, Let it always be remembered that tubercle
destroys the tissues in which it grows, and that the treatment
by Koch’s method completes the process of destruction, if
this is not already accomplished ; so that, under the very
best conditions, phthisis can only be stopped, and, although

‘a comparative cure may be obtained, highly differentiated
tissues once destroyed can never be restored or replaced.

It will thus be seen that although Koch has selected a
special irritant material as that which it was found
necessary to separate in order to obtain the results that
he wished, and has obtained the sequence of specific (?)
stimulation of the cells, he has departed from the usually
accepted methods, in that he acts directly on the tissue cells,
and leaves the bacilli to die of starvation. It is not now a
case of the cells destroying or modifying the activity of the
bacilli; it is simply, in the. first instance, a cutting off, or
rather a rapid exhaustion, of the substances required for the
nutrition of the bacilli; the bacilli remain, and although
they may eventually undergo retrogressive changes, it appears
probable that their spores remain for some time, at any rate,
ready to break out should favourable conditions again pre-
sent themselves. Apart from this local action, however, the
fact cannot be ignored that where the tissues are not already
too far weakened, or the cells so imperfectly nourished that
they cannot react to stimulus, the liquid injected by Koch
may exert a general and specific action on the tissue cells
through which they are acclimatized, as it were, to resist the
poison ; so that it is quite possible that a partial immunity

240 BACTERIA.

as regards the tubercle bacillus may be acquired. It is
probable that the tuberculin acts as a stimulant on all cells,
and that to the increased metabolic changes that are set up
is due the general reaction that is described as following on
the exhibition of this material,

That this is not entirely a fanciful explanation may be
argued from an example. It is a fact, well known to those
who have charge of tuberculous patients suffering from
diseased glands—scrofulous glands, as they are called—that
so long as these glands remain uninjured and are subjected
to no stimulation, and so long as the nutrition of the patients
keeps fairly good, they remain as a rule comparatively free
from pulmonary phthisis and other forms of tuberculosis ;
whilst other members of the same family, existing under the
same conditions, both as regards hygiene and nutrition,
become affected with the commoner and more fatal forms of
the disease.

A further illustration may be taken from the difference
that exists between children of the lower classes and those
met with in other grades of society—children in a sick
children’s hospital frequently having every organ crammed
with, or almost replaced by, tubercular nodules and cavities, so
that it seems marvellous that the unfortunate children could
have remained alive at all ; whilst children better nourished,
but not previously affected by any form of tubercle, appear
to succumb very easily, when comparatively localized tubercle
has been developed. In these cases, however, it is usually
found that some vital organ such as the brain is affected.

It would appear that, in these cases, the tissues may de-
velop greater resistance in consequence of the circulation
in the fluids of the body of the soluble products; but
that when this acquired resistance is once lost, or is over-
come, the stimulation exhausts the cells, and they now
more easily fall a prey to the active tubercle bacilli. It
must, however, in this connection, be pointed out that the
products of the tubercle bacillus, or sterilized cultures of the
organism, when introduced subcutaneously into healthy
fowls or guinea-pigs, produce a condition which is spoken of
as ‘marasmus ;” and as early as 1879, Maffucci, as the result
of a series of experiments, concluded that these sterilized cul-
tures when left in the body, exerted such a marked influence on
the tissues, that they induced emaciation, atrophy of the liver

TUBERCULOSIS. 241

cells, and of the cells of the different parts of the spleen, and
that they also set up certain changes in the circulation, the
result of which was seen in marked congestion of the lungs,
kidney, spleen, &c. These experiments were no doubt sug-
gested by the similar changes that are met with in the
human subject in the course of tubercular disease, even in
those organs that are not directly affected by the tuberculous
infiltrations. Recently he has confirmed and extended his
former observations ; and it will be interesting to see whether
the conditions met with in patients injected according to
Koch’s method, are similar to those observed in animals
experimented on by Maffucci’s method.
How far the object of Koch’s endeavours will be attained
still remains to be seen, and his method has been, and will
be, put to most severe tests for it involves the question of
life or death to thousands, nay, tens and hundreds of thou-
sands. ‘There appears little doubt that in lupus (a tuber-
culous skin disease) the process has been checked, and, in
some cases, at any rate, it has not again broken out for a
considerable period after the treatment had been stopped.
There also appears to be pretty reliable evidence in favour
of his contention that there is an amelioration of the condition
of the patient and an improvement in the disease in certain
other forms of tuberculosis ; but the use of the remedy has
not been sufficiently prolonged to allow of our arriving at
any very definite conclusion, however favourable our opinions
may be. Virchow, the greatest pathologist of the age, has
found, in a number of cases that have come under his obser-
vation—a comparative small number when the enormous
number that have been injected is taken into consideration
—that the characteristic degeneration of the tissues of the
young tubercle is not always brought about, that the locali-
zation of the disease is not by any means perfect, that there
is a tendency of tubercle material that should be “thrown
off” to continue the infection and even increase its
rapidity of spreading, especially in the lungs, and that
in some cases the bacilli, instead of being rendered inert,
appear to take on greater activity, and to be carried in
the various currents in the body, even to parts situated at
some distance from the original tuberculous focus. We
must bear in mind, however, that almost all the cases that
up to the present have been brought to the post-mortem

17

242 ' BACTERIA.

table have been cases in which the disease was far advanced,
and in which Koch’s inoculative treatment has been sought
as a last resource by physicians and patients alike.

A most important point to be remembered in connection
with tuberculosis is that under favourable conditions it is an
extremely curable disease ; such conditions, however, must
be very far-reaching and must include a sufficient and
suitable supply of food to the patient, perfect oxidation of
the tissues, and facilities for the excretion of effete products.
The effects of climate, of exercise, of pure mountain air, free
ventilation in houses and of generally favourable hygienic
conditions, are all due to the promotion of the healthy
condition of the tissues and of the increased vitality of the
cells ; this vitality increasing their power of resistance and
enabling them to cope successfully with the bacilli and their
products with which they may be brought in contact. It
should not be left out of count, however, that we may, by
the use of suitable drugs, be able to exert an antidotal
influence by means of which, acting directly on or through
the cells, these cells may be put in a still better position for
resisting the attacks of the tubercle bacilli and their products.
This antidotal system of treatment, if it could be carried out
by means of drugs, should prove far more efficacious than the
use of any means of treatment that we have at present at
our disposal, especially if it could be combined with some
of these latter.

This is not the place to discuss the medical aspects of the
question ; but the above facts are mentioned in connection
with the biological problems associated with the action and
inter-action of the bacilli and of the cells of the body.

LITERATURE.

Banc.—Proc. Internat. Med. Congress, Copenhagen, vol. 1,
Path. Sec., p. 1, 1884.

BAuUMGARTEN.—Zeischrift f. Wissensch Mikroskopie, Bd. 1,
1884; Deutsch. Med. Woch., Bd. vil, p. 305, 1882.

Bo.itz.—Journ. Com. Path. and Therapeutics, p. 370.
Dec., 1890.

Boiiincer.—Baier Aerztl Int. Blatt., 1883 ; Centralbl. f. d.
Med. Wiss., Bd. xx1., p. 600, 1883.

CHEYNE, WaTson.—Practitroner, vol. XXX., p. 241, 1883.

CouNHEIM.—Uebertragbarkeit der Tuberculose. Berlin, 1877. -

TUBERCULOSIS. 243

Cornet.—Ztschr f. Hyg., Bd. v., p. 191, 1889; Ueber
Tuberculose, Leipzig, 1890.

FLtecr.—Ztschr f. Hyg., Bd. 1, 1882.

GarFFxy.—Mitth. a. d. k. Gesundheitsamt, Bd. 1, p. 126,
1884.

GERLACH.—Comptes rendus. Feb. 10 and 24, 1868.

HusBerMAAS.—Beitrag z. Klin. Chir. Mitth. a. d. Chir.
Klinik z. Tiibingen, Bd. 11, p. 45, 1886.

Joune.—Die Geschicht. der Tuberculose. 1883.

Kocu.—Berlin Klin. Woch., Bd. xv., p. 221, 1882; Mitth.
a. d. k. Gesundheitsamt, Bd. 11, p. 1, 1884; Deutsch.
Med. Woch., No. 10, 1883.

M’Fapyean AnD WoopHEap. Brit. Med. Assoc. Dublin,
Aug., 1887.

Marrucci.—Centralbl. f. Allg. Path. u Path. Anat., Bd. 1,
No. 26, Dec. 15, 1890.

MeErTscHNnikorr.—Annales de l'Institut Pasteur, 1888-9.

Nocarp.—Amer. Vet. Rev., N. Y., vol. 11, p. 258, 1884-5.

Nocarp anp Rovux.—Annales de J'Institut Pasteur, t. 1,
p. 19, 1887.

Paw.Lowsky.—Ann. d. l'Institut Pasteur, t. 11., p. 303, 1888.

Toussaint.—Comptes rendus, t. Xcll., pp. 281, 322, 350,
741, 1881.

VILLEMIN.—Etude sur la Tuberculose. Paris, 1868.

Watiey.—Lain. Med. Journ., p. 1088, 1888.

WEIGERT.—Deutschen. Med. Woch., No. 24, 1883 ; and No.
35, 1885.

Witurams, C. T.—LZance?, vol. 1, p. 312, 1883 ; Pulmonary
Consumption, 1887.

WoopHEAD.—Tabes Mesenterica and Pulmonary Tubercu-
losis. Reports from the Lab. R. C. P. Ed., vol. 1,
p. 179, 1889. :

Worzpurec.—Mitth. a. d. k. Gesundheitsamt, Bd. 1, p, 89,
1884.

WESENER.—Futterungstuberculose. 1885.

See also Comptes rendus et Mémoires de la Congress de la
Tuberculose. Paris, 1888-1890.

For the more recent controversy on Tuberculosis and Tuber-
culin, see Lancet and British Med. Journal.

The earlier literature is given very fully in CoRNEL and BaBEs
“ Les Bactéries.”

CHAPTER XII.
LEPROSY.

Distribution of Leprosy—Similarity to Tuberculosis—Description of Dis-
ease—Tubercular, Anesthetic, Mixed—The Leprosy Bacillus—Method
of Staining—Position—Leprosy Cells—Bacilli Resistant and Grow
Slowly—Cultivation Experiments Mostly Negative—Theories of Cause
of Leprosy.

Wiruin the last few years leprosy has, metaphorically speaking,
returned to life in this country. The occurrence of a case in
one of our market attendants hascreated morecommotion,and
has put in train a more complicated machinery, than phthisis,
with its thousands and tens of thousands of victims was for
long able to set going. Nevertheless, we can now afford to
think of leprosy as almost a thing of the past, as far as our
own country is concerned, although we still come across
traces of its sojourn amongst us in our Libertons, or Leper
towns, or Leptons, that indicate only too surely that this
disease was looked upon with the greatest dread by our
ancestors of the Middle Ages, who evidently took pains
to keep in their own regions, and within their own asylums,
the lepers of that period. With the exception of certain
isolated areas along the shores of Spain, and in Portugal,
in Norway, some parts of Sweden and Iceland, in Italy,
Roumania, and Hungary, in the Balkan Peninsula, and in
Greece where the disease is still endemic, leprosy is now
rarely seen in Europe; but in certain parts of Asia and
Africa it is still frequently met with. It is found doing its
fell work in the Sandwich Islands, in Mexico, in Cuba, in some
parts of Central America, in the north-east of South America,
and in the Argentine Republic, in the north-east and north-
west of Africa, in Guinea, and in Cape Colony, and in Mada-
gascar, along the shores of the Black Sea, in Persia, Arabia,
India, China, the Malay Archipelago, Japan, in Asia, and in
New Zealand.

LEPROSY. 245

From the whole nature of the symptoms, and from the
course of the disease, it was long considered to be a disease
somewhat similar to tuberculosis, and the discovery, by
Armauer Hansen, in 1880, of ‘a specific leprosy bacillus,
which was found to be present in enormous numbers in the
lymph channels of the skin in cases of leprosy, paved the
way, for the reception of Koch’s discovery of the tubercle
bacillus.

It was for some time supposed that the tubercular and
anesthetic forms of leprosy were different in their origin, and
that they might possibly be due to the action of different
bacilli, although it was of course known that in some cases
there was a kind of mixed leprosy. The fact that the two
diseases were the same was, however, strongly accentuated
when it became evident that the same kind of bacillus was
found in all three forms of the disease—the tubercular, the
anesthetic, and the mixed.

Before a description of the morphological characters of the
bacillus is given, the characters of the various forms of the
disease may be briefly indicated, in order that the patho-
logical changes produced by the bacillus may be more fully
understood, and that the bacilli may be followed and localized
in the tissues. In tubercular leprosy there usually appear
small irregular spots, sometimes brown, sometimes some-
what purple. These spots occur on the face or on the
limbs ; they gradually project so as to form more or less
marked tubercles, which vary very considerably in size.
There is irregular thickening of the tissues under the skin
of the face, which gives rise to a most curious facial expres-
sion, the patient appearing melancholy or jubilant according
to the folds affected ; the tissues about the various orifices
of the face, mouth, eyes, nostrils become swollen, and are
often studded with tubercles, the lower lip becomes heavy
and hangs down in a curious fashion ; these alterations giving
the face what is described as the “leonine” appearance.
Sometimes sensibility is interfered with, or it may be alto-
gether abolished. These tubercles are not confined to the
tissues immediately under-the skin ; they may also be found
in the submucous tissue of the mouth, larynx, and pharynx;
and small nodules may even be met with—especially where
the swelling is marked—around the eye in the soft tissue of
the eyelids. These nodules frequently ulcerate, especially in

246 BACTERIA.

the later stages of the disease. On the fingers the thickened
patches and ulcerations may extend so far and so deep that
part of the finger may be actually separated and thrown off,
first, however, becoming dry and black or mummified. The
patients may linger on in this condition for a considerable
length of time ; or the tubercles may disappear, leaving dis-
coloured patches, in which sensibility is entirely wanting, and-
which, in consequence, very rapidly undergo ulceration, usually
brought on by injury, the patient, unable to feel, allowing the
parts to be injured without making any effort to save them.
The second form—anzsthetic leprosy—commences in much
thesame way, except that the discoloured patchesaresomewhat
smaller, and may be red or brown ; in some cases the patches
have a peculiar glistening or silvery-white appearance. The
pigmented patches, which are often very widely distributed,
have, especially where they attain any very great size, a dull,
pallid centre, with a line of brown, or brown and red coloura-
tion, surrounding it. Some of the smaller spots are anzs-
thetic, whilst, in addition, there may be large areas in which.
sensation is markedly diminished or altogether abolished; the
hair falls out ; there is marked atrophy of the skin and the
cellular tissue immediately beneath, this giving rise to a
very wrinkled appearance of the face; and the patient, as in
the other kind of leprosy, becomes dull and heavy-looking,
as though he had lost all interest in external affairs. Here
also injury to the extremities is of very frequent occur-
rence, partly owing to the fact that the patient cannot feel,
but partly also, because the nutrition of the skin is markedly
interfered with. Other skin diseases, the formation of
vesicles, and so forth, are often met with. Either form may
gradually pass into the other, in which case we have what
are known as mixed varieties of the disease.

In order to demonstrate the presence of the bacillus in
one of the nodules in a case of tubercular leprosy, it is only
necessary to tie an india-rubber ring somewhat firmly around
the base of one of them until it becomes pale from the blood
supply being cut off, and then, with a needle-shaped lancet
or the point of a sharp knife, to make a small puncture,
from which a clear fluid exudes. In this may be found an
enormous number of bacilli. These bacilli resemble the
tubercle bacilli in a most remarkable manner, the only
difference being that they are, if anything, slightly shorter.

LEPROSY. 247

They may be stained by almost any of the methods that are
used for staining the tubercle bacillus, although here an

method that is used must be slightly modified, as the bacilli
characteristic of leprosy are rather more easily decolorized
by acids than are the tubercle bacilli, The Ziehl-Neelsen
method, with a contrast stain of methyl blue, gives most
admirable results, or Gram’s method may be used. The

Bacilli from Juice of a Leprosy Nodule. x 500.

bacilli so stained are seen to be from 4 to 6# in length and
.3-in breadth ; they are more constant in size than are the
tubercle bacilli, and, as a rule, are not marked by the curves
that are almost characteristic of that organism ; their ends
appear to be slightly pointed. It was at one time considered
to be beyond controversy that these bacilli contained spores ;
but more recent observations on these and other organisms
have led observers to the conclusion that what were at one

248 BACTERIA.

time considered to be spores are not spores at all, but are
appearances due to the alterations that have taken place in the
protoplasm during the processes of preparation and staining.
As it is not yet certain that the bacilli can be cultivated, little
evidence either for or against the spore theory can at present
be adduced, These bacilli, to be seen in their characteristic
form, should be examined as they lie in the thickened nodules
of the skin or in the mucous membrane of the mouth and
larynx, in the thickened nerves, in the lymphatic glands, in
the spleen, or in the liver. Ifa thin section be made of a
nodule of the skin, and the specimen be stained with fuchsin,
and decolorized with a mineral acid, and a contrast stain
of methylene blue be obtained (see Appendix), it will be
found that, throughout the section, branching lines or small
rounded points of bright red may be seen on a blue back-
ground ; these bright red areas are nothing but masses of
leprosy bacilli which fill the lymphatics of the skin, and, as
one would expect, interfere very seriously with its nutrition.
There seems to be a considerable difference of opinion as to
whether these bacilli actually invade the cells or whether
they lie free in the lymph channels ; there can be no doubt
that a very large number of them ultimately are seen as free
bacilli lying in these spaces, the difference of opinion being
as to the nature of the so-called “lepra” cells. There are
usually found in cases of leprosy a number of large proto-
plasmic masses, which are said to be multi-nucleated, and
are spoken of as giant cells, and in these are found numerous
bacilli. In younger nodules there are smaller masses of
protoplasm, in which nuclei and bacilli may be distinctly
seen. At the meeting of the British Medical Association,
held in Dublin in 1887, Dr. Unna, of Hamburg, placed
before the members his views on many of these so-called
lepra cells. He contended that the rounded outline was
due in many cases tothe form of the lymphatics, and he
showed in his specimens that the rounded section was equal in
diameter to the longitudinal section of one of these lymphatics
filled with bacilli ; that the longitudinal section proved that
we have here to deal simply with a lymph channel blocked
with leprosy bacilli, and therefore that the rounded masses
are merely transverse sections of these stuffed lymph chan-
nels. He also maintained that these so-called cells, with
their contained bacilli, were merely masses of these organisms

LEPROSY, 249

which were held together by gelatinous material ; and that
even where there were apparently globular masses in the
lymphatic channels, they were nothing but these zoogloea
masses, whilst a great number of the organisms were lying
entirely free, the nuclei of the so-called lepra cells again being
the nuclei of the walls of the lymphatics or of the lymph
cells that had become embedded in the gelatinous mass.
Many competent observers, however, maintain that the lepra
cells have a real existence, and there can be little doubt that
bacilli may be found in large cells squeezed from a leprosy
nodule,

One of the most marked distinctions between tuber-
culous and- leprous tissue is, that in tubercle the bacilli
are comparatively few in number, especially when they
are met with in the more chronic cases—the only cases
that could possibly be mistaken for leprosy; whilst in
leprosy the bacilli are almost invariably present in enor-
mous numbers in the lymph channels of the tissues in
which they are growing, on which they seem to exert com-
paratively little destructive influence, as they remain for a
considerable length of time in an almost quiescent condition,
setting up little change, but undergoing no retrogressive
changes themselves. Cornil and Babes record that in a
small fragment of a leprous nodule that had been left in
an envelope, forgotten for nearly ten years, they were still
able to stain the bacilli very distinctly ; and sections that
had been stained in picro carmine and kept mounted in
glycerine for a number of years, it was found, might
still’ be stained so as to bring the bacilli into prominence,
Then, too, leprosy affects the skin and nerves specially,
rarely the lungs and serous membranes. Tuberculosis, on the
contrary, affects the latter very frequently, and very rarely
the former. From the enormous number of bacilli that are
found in the lymph spaces, it can be readily understood that
when ulceration of a nodule takes place, these organisms
are to be found in large numbers in the blood and serum
that is discharged from the ulcerated surfaces. Although
this is the case it is a somewhat peculiar fact that this
organism is seldom found in the superficial layers of the
epithelium covering the nodule, although in the ducts of
sebaceous glands, and around the hairs of the skin, as Babes
demonstrated, the bacilli may be present in considerable

250 BACTERIA,

numbers ; they are seen in the internal sheath of the hair,
from which they may, in some few cases, pass through the
epithelium, and so to the surface; this is, however, a very
rare condition. Asa rule, leprosy bacilli are not met with
in the blood ; but in the febrile condition that occurs shortly
before death, bacilli have been described as being found in
the circulating blood taken at some distance from any nodule.
As the leprosy nodules may be found in all parts of the
body so also may the bacilli. In cases of anzsthetic leprosy
the bacilli may usually be readily demonstrated lying in the
dilated lymphatics of the thickened and nodulated nerves.
Here, too, as in tubercle, the lymphatic glands are distinctly
infiltrated. From its resemblance to tubercle, and from the.
fact that its specific bacillus.is found so constantly associated
with the disease, being most numerous in those positions
in which the leprous processes are most advanced, but being
present from the very commencement of the tubercle forma-
tion, it is evident that the bacillus bears a constant—probably
a causal—relation to the disease, and it was therefore supposed
that leprosy might be carried from one individual to another
through its agency ; that, in fact, the disease was a specific
infective disease and was inoculable.

Numerous experiments, made with the object of proving
this thesis, have, however, failed. (Babes and Klarindero
mention, in support of the contagious nature of the disease,
the case of a child that developed leprosy on the lips and
cheeks during the time that it was being suckled by a
leprous mother, and Dr. Castor and many others insist on
the communicability of the disease. A patient has recently
been reported to be dying from leprosy, the result of inocu-
lation in 1884. The patient was a condemned criminal in
Honolulu, who elected to be inoculated with leprosy by Dr.
Arning in place of being hanged). Even the inoculation of
fragments of leprous tissue gave rise in all recorded experi-
ments to no true leprosy, unless the patients were already the
subjects of the disease. The cultivation of the bacillus has
also proved to be a most difficult matter. Neisser observed
a number of small pellicles that appeared to shoot out from
small particles of tissue introduced into consolidated blood
serum, kept at a temperature of between 37° C. and 38° C.
Bordoni Uffreduzzi obtained growths from the marrow of
a bone in which there were a number of free leprosy bacilli ;

LEPROSY. 251

these appeared on serum (to which a quantity of glycerine
had been added) that was maintained at a temperature of
37°C. These he described as delicate, thin, slightly yellow
films with irregular borders ; on glycerine agar they are said
to have developed as small grey rounded isolated points
usually at the end of ten days or a fortnight ; secondary
cultivations, however, made their appearance at the end of
forty-eight hours, and after the first few cultivations the
organism could be grown on serum or on ordinary gelatine
and agar, but much more slowly than when glycerine had
been added. From the general description, and the im-
perfect staining obtained, some doubt must remain as to the
true nature of these bacilli. Babes also was able to obtain
cultivations on similar media, even from other organs; he
described the growths as being very like those of diphtheria ;
upon serum they appeared as pale yellow elevated plates,
glistening and waxy-looking and surrounded by a transparent
indented zone ; the cultures emitted a peculiar characteristic
odour. They developed all along the track of the needle,
especially in glycerine gelose ; the rods were elongated, but
there appeared to be a number of involution forms, and
many of the -bacilli were much more plump than usual, and,
‘bearing out Unna’s observations, they appeared to be sur-
rounded by a clear capsule somewhat similar to that met
with around Friedlander’s pneumonia organism. Babes was
not successful in making secondary cultivations, nor was he
able to produce the disease by inoculating with his cultiva-
tions any of the animals that he had in his laboratory.
Until these experiments are confirmed by other observers
they can scarcely be accepted as conclusive, as it requires a
much longer series of successful cultivations and more
careful comparison of the organism as it appears in the
tissues with that found in the cultures, than either Bor-
doni Uffreduzzi or Babes have made, to set the matter
at rest. Still they have indicated the lines on which future
work may be done, and we may anticipate that before long
Dr. Bevan Rake, Dr. Castor and others, may continue their
experiments with the enormous amount of material which
they have at their disposal and give to the world most im-
portant results.

None of the numerous non-bacillary theories as yet put
forward to account for leprosy appear to be sufficiently well

252 BACTERIA.

supported to be able to oust the bacillary theory from its
present position. Leprosy is found in all climates ; it is not
specially confined to the seashore, it occurs in regions where
fish diet cannot be resorted to, and where other articles of diet
such as pork and rice (to both of which have been ascribed a
causal agency in the production of this disease) are not used
at all. The only factor that is common to all forms of the
disease, and that is met with in every case, is the leprosy
bacillus, and in spite of the fact that we have not yet been
able to trace the method of contagion or infection through
the agency of this bacillus, we must, from what is known of
the presence and action of bacilli in other diseases, assign to
it the rd/e of leprosy producer, until much stronger evidence
than we have yet obtained can Be adduced in favour of any
other cause.
LITERATURE.

Authors already quoted. Cornil and Babes.
Arninc.—Virch. Arch., Bd. xcvi1, p. 170, 1884.
Bases.—Arch. de Physiol. July, p. 42, 1883; Comptes
rendus, t. XCVI, pp. 1246, 1323, 1883.

Bases AND KLaRINDERO.—Congrés Intern. de Dermatologie
a Paris, 1889.

Bevan Raxe.—Reports on the Trinidad Leper Asylum,
1888-90; Monats-Hefte f. Prakt. Dermatologie, Bd. vu.,
No. 12, 1889.

Borpont UFFrepuzzi.—Zeitschr f. Hygiene, Bd. m1., p. 178,
1888.

Fe.xin, R. W.—On the Geographical Distribution of some
Tropical Diseases, p. 36. Edinburgh, 1889.

Hansen.—Virch, Arch., Bd. Lxxix., p. 32, 1880.

Hirscu.—Handbook of Geographical and Historical Path-
ology, vol. 11., New Syd. Soc., 1885.

Lrtoir.—Memoires de la Soc. de Biologie, p. ror, 1885.

NerrsseR.—Virch. Arch., Bd. yxxxtv., p. 514, 1881.

Unna.—Brit. Med. Assoc., Dublin, 1887; Deutsch Med.
Woch., 1885-1886.

Vircuow.—Berlin Klin. Woch., Bd. xu., No. 18, p. 123.

CHAPTER XIII.
ACTINOMYCOSIS.

Nature of the Disease—Differences in Cattle, Man, Pig, and Horse—
Methods of Preparation and Examination of the Fungus—Microscopic
Appearances: of Fungus in Different Animals—Nature of Clubs—
Cultivation Experiments—History of Actinomyces—The Actinomyces
a Streptothrix.

A DISEASE that was for long very imperfectly understood,
and which has been described under very different names, is
Actinomycosis, which, in 1876, was first recognized by
Bollinger as being of parasitic origin. The disease mani-
fests itself in cattle, frequently in the jaws, where it is
known in various parts of the country as wen, osteo-sarcoma
or bony sarcoma, malignant tumour of the palate, or in
the tongue, where it may be recognized as “ wooden
tongue ;” the pharynx or the loose tissues under the skin of
the head and neck, or even the trunk, may also be affected.
In the pig, abscesses with running sores are most frequently
found in the milk gland and in the tissues around the
pharynx ; whilst in horses it is usually found as “ scirri:ous
cord,” a condition well known to veterinary surgeons, though
as Professor M’Fadyean points out, it may also be met
with in the tongue of the horse. This curious disease has
also been known to attack the human subject, in whom the
lesion resembles in its characters that met with in the pig,
the parasite giving rise, by its presence, to abscesses in the
lungs, to pus in the pleural cavity, and to similar conditions
in other organs; the bones, especially those of the spinal
column, are frequently attacked, and “cold abscesses” or
chronic gatherings form in those cases where sinuses from
which matter may escape are met with.

It is interesting to note in this connection that even in the
pig the lung is sometimes riddled with similar cold abscesses,
and that there is frequently breaking down of areas of

254 BACTERIA.

bone in the cervical and dorsal vertebra. Those who had
examined the condition only in the human subject and in the
pig describe the disease as consisting essentially of a sup-
purative process, whilst those who have examined ‘“ wooden
tongue”’ in cattle, and “ scirrhous cord ” of the horse, maintain
that the process, though sometimes leading to softening, is
essentially of the granuloma or new young tissue formation
type. These differences, however, appear to be due rather
to the nature of the tissues in which the growths occur than
to any difference in the character of the organism that
produces them, for it is found that in many cases in which the
bones are attacked in the cow the lesion takes on a suppura-
tive character. It was, however, the non-suppurative form
that was first described, and it will therefore be better to
examine it in the first instance. In “ wooden tongue” of
the ox, on superficial examination, firm hard points may be
seen scattéred over the surface, varying in size from that of a
millet seed to that of a split pea. On incising one of these,
it is found to be firm and fibrous at the periphery and especi-
ally in the small nodules ; the centre may be soft but not
purulent, or, rarely, gritty and almost calcareous ; in the larger
nodules little fibrous bands running through them form a kind
of net-work, and in the centre of each mesh is a similar
soft point. If this softened caseous point be removed with
the point of a knife and examined under the low power of
the microscope (x 50) it will usually be found to contain,
embedded in a small mass of cells, a small core, which by
transmitted light is yellow or brownish in colour, dashed
with a tinge of green. This little mass is seen to be com-
posed of a kind of star made up of a number of wedge-
shaped rays, the apices of the wedges meeting in ‘the
centre, the bases being rounded; looking down on the
centre of this cord it appears as though there are
rounded instead of wedge-shaped bodies (this is simply
because we are looking down on the rounded ends of the
wedges). If now one of the “ bony ” tumours, or one of the
tumours in the fibrous covering of the bone, where the
tumours are usually of larger size, be examined, it will be
observed that the fibrous net-work is exceedingly well marked,
especially as on section the softened caseous centre gives way
very readily : in the softened material similar “ rayed” or
star-like organisms may be found. In the material that is

ACTINOMYCOSIS. 255

discharged from the “cold abscesses” which are formed in the
pig or in the human subject, similar small yellow points
(which appear as green or greenish yellow grains, even
when examined with the naked eye) may be found on
examination under the microscope. The pus discharged
from these abscesses has an exceedingly characteristic appear-
ance; it is usually yellow or brownish yellow in colour,
is extremely granular, of a peculiar slimy consistence,
and contains the green points that may be said to be
specially diagnostic of the actinomycotic condition. The
small green points when taken from the cow are very
frequently somewhat gritty, this being due apparently to the
deposition of particles of lime in the core; but the particles
that come from the pus in the human subject are usually
soft and tallow-like, so that they can be readily flattened out
between two cover glasses. The appearance of the actino-
myces or “ray fungus” in cattle, as first described, was
so exceedingly characteristic that it was thought it could
not be mistaken for anything else, and the corresponding
condition in the human subject was for some time over-
looked, simply because the same typical appearances were
not always developed ; and it was only after some time that,
transition stages being found, first in cattle and then in the
human subject, the real nature of this fungus was thoroughly
understood.

On examination of the fungus under a high magnifying
power, when the sections have been properly stained, the
organism is found to be like the capitulum of a daisy, the
sterile flowers in the centre corresponding to the club-
shaped rays, and if we conceive of two of these heads of
flowers as placed base to base, or stalk to stalk, we may
obtain an idea of the appearance of the ray fungus as a whole ;
the organism in sections of course having the appearance
of sections through the little ball formed by the two heads.
The clubs, however, are not all simple, but in some cases
branch, sometimes dichotomously, sometimes irregularly,
compound clubs being thus formed.

In a tumour examined by Professor M’Fadyean, the transi-
tion stages between the forms sometimes found in the human
subject, and those most frequently seen in cattle, are met with ;
and as J have had the privilege of seeing Professor M’Fadyean’s
specimens I shall follow pretty closely his descriptions, as

256 BACTERIA.

I consider that the interpretation he gives of the appearances
presented is perhaps the most satisfactory that has yet been
published. The tumour from an ox was fleshy in consistence,
had a faint pink colour, and was studded with minute softened
pink points, and in each of these points was found an
actinomyces colony, at the margin of which, however, only
a few of the characteristic clubs could be found, the centre
being finely granular. On making sections of these tumours,
that had been hardened in alcohol, and staining by Gram’s
method (see Appendix), and examining under a high magnify-
ing power, Professor M’Fadyean observed that the colony
consisted of three distinct elements, though, in many
instances, the club-like bodies were absent. The first element
is a coccus about .5 in diameter ; these cocci are usually
arranged in chains consisting of ten or fifteen elements, a few
of them are usually found in the centre of the mass, but
immediately outside this they are exceedingly numerous, so
numerous indeed that they appear to be more like masses
than chains; as we approach the margin again the chains
radiate outwards and are very distinctly seen. Where they
are not very numerous, some larger cocci may be seen under-
going regular vegetative division, so giving rise to the forma-
tion of diplococci.

The second element is a thread-like leptothrix or clado-
thrix, a number of which, interlacing freely, form a kind of
felted net-work, especially in the centre of the colony. As
we pass outwards, however, they gradually assume a more
regular radiate arrangement, and near the periphery “ they
sometimes shoot out in a tendril-like manner beyond the
coccus heaps already described.” These threads vary very
considerably in length; sometimes they are divided into
short bacilli or even into cocci ; in other cases long threads
without any sign of division may be seen. The diameter of
these threads is usually greater than that of the cocci. They
are described “as some nearly straight, others gently curved,
and occasionally they show short nodules almost like a
spirillum.” Near the margin these threads sometimes
become branched just as in the case of the club-like forms
already mentioned. The club-like forms when met with
here appear to bear a definite relation to the threads ; they
are only found at the margin of the fungus mass ; they are
arranged radiately with the ends of the clubs outermost ;

ACTINOMYCOSIS. 257

they vary much in length, and are usually quite simple, “ but
some carried lateral buds, and occasionally two appear to be
carried by a common stalk.” These buds are best stained by
the Ziehl-Neelsen method; some of them exhibit a very
important relation to the leptothrix forms already described,
the thread appearing to be continued into the centre of
the club, the outer part of which is formed by a homo-
geneous, somewhat faintly stained material. This axial
thread may be divided into longer or shorter segments,
corresponding apparently to the bacilli and cocci forms.
They are usually most divided and are undergoing most
marked changes in the larger clubs, whilst there are also
small rounded bodies, which appear to be essentially of the
nature of cocci, surrounded by the same material that forms
the thickened club. In some of the smaller colonies of the
actinomyces Professor M’Fadyean describes cocci only, which
are usually arranged in short chains or in little groups.
They are embedded in masses of leucocytes, some being
actually within the cells, and appearing to be the points
from which the larger colonies start, the cocci being carried
by the leucocytes from point to point. Other colonies
contain only cocci and thread forms, the threads in this case
appearing to be developed from cocci, whilst those colonies
in which clubs are present appear to be in a still more
advanced stage of development.

By some observers the club-like forms have been described
as the spore-bearing parts of the organism, and are spoken of
as Conidia or Basidia, but it appears from the above case,
and from those that have been described in the human
subject, that the thickened extremity is due merely to a
kind of involution process, and occurs in the older thread-
like organisms as the result of a growth and swelling
of the outer sheath of the Cladothrix threads, this sheath
corresponding, in fact, to the gelatinous material which is
formed in zoogloca masses of other organisms, or to the
capsule first described by Friedlander which is formed around
the bacillus of pneumonia. When these clubs are once de-
veloped the central part may be only partially active, whilst
the periphery remains passive, but extremely resistant to the
attacks of the surrounding cells, which attack the club-
shaped masses very vigorously, the large thickened clubs
being frequently found in various stages of degeneration

18

258 BACTERIA.

within the large masses of protoplasm found in this
region.

In the human subject the felted network, the cocci and
the bacilli are usually most numerous; they are, in fact,
said to be typical of actinomyces in the human subject.
Here again the involution or club-forms are frequently
met with, especially in the pus that is discharged from
the abscesses of the lungs or of bone. The slimy pus
in these cases, however, appears to contain a considerable
proportion of the “ mucine,”’ that in cattle goes to form the
thickened sheaths above described. The process of evolution
of the “Ray” fungus is much the same in the human
subject as in the abnormal case already described as occurring
in the cow.

It is interesting to note that most of the experiments
that have been made on the cultivation of this organ-
ism have been attended with complete failure—a failure
that in some measure, at any rate, appears to be due to
to the fact that almost all experimenters have used for their
inoculating material only those colonies in which the club-
shaped organisms have become well developed. The first
attempt that was at all successful was made by Bostrém,
who, throwing aside the club-like processes, took for his
inoculating material the central network, selecting as far as
possible young growing colonies for his seed material. His
method of procedure was as follows: With the utmost
care he removed small colonies, which were at once in-
troduced into sterilized gelatine, with which a “plate”
cultivation was made. After a few days any points that
were found to be pure, ze. around which other ovgan-
isms were not growing, were removed from the gelatine
with a sterilized platinum needle, and were crushed be-
tween sterilized glass plates; with the platinum needle,
stroke cultivations were made on ox blood serum, and
agar-agar. A finely granular growth first made its appear-
ance along the line of inoculation; this gradually became
more marked, then small yellowish red nodules were seen,
around which delicate branched processes spread out ; these
yellowish masses soon began to run together, and at the end
of seven or eight days were covered with a delicate fluffy
white layer. This growth apparently went on best at the
temperature of the body, and on microscopic examination

ACTINOMYCOSIS. 259

of the structures, cocci, segmented threads, longer threads
and clubs were all found; threads and clubs alike in most
cases being characterized by a branching similar to that met
with in the fungus as it grows in the human body. Inocula-
tion of this fungus into the peritoneal cavity of rabbits and
other animals was usually attended with positive results.
Later, by attending to the same details as regards the nature
of the seed material, M. Wolff, J. Israel, and Babes have
all succeeded in cultivating this organism on agar-agar,
blood serum, and especially on the raw white of egg,
according to Hueppe’s method (see Appendix), and with the
cultivations so obtained actinomycosis has been produced
experimentally in animals. ”

From all these facts it will be gathered that actinomy-
cosis is the result of the activity of a living organism
introduced into, and existing as a parasite in, the animal
tissues; that the same organism may be found in animals
and in the human subject, that the club-shaped organism is
really an involution stage, and that the characteristic growth
is the mycelial thread-like mass which appears to develop
from the cocci. The fact that we are unable to cultivate
from the clubs alone, affords ample evidence that, in place
of being spore-bearing masses, they are merely encysted, or
thick-walled, involution forms.

, As to the mode of infection, it has been pointed out that
the actinomyces has been found lodged in the crypts of the
tonsils of the pig and of the human subject, and that the
parasite evidently leads an epiphytic life on barley and other
cereals. It may be introduced from without through
wounds, as in cases of scirrhous cord in horses, or through
inoculation, by means of accidental scratches of the skin, or
of the mouth and pharynx, and it is recorded that a case of
ptimary mediastinal actinomycosis in the human subject
was in one case supposed to be traced to perforation of the
back of the throat by a barley spikelet swallowed by the
patient. In pigs the mammary affection is thought to
be due to the entrance of the actinomyces into the teat
ducts.

In Denmark the farmers attach so little importance to
infection from animal to animal, that cows in which these acti-
nomycotic tumours are well developed are allowed to run
with the rest of the herd, at any rate until suppuration

260 BACTERIA.

commences; when this occurs, however, the animals are
usually slaughtered.

I may here summarize what is known of the history of a
colony in the words of M’Fadyean, who has given the subject
much careful study, and to whose authority in this matter
I attach much weight.

“y. It has its starting point in one or more cocci trans-
ported by the plasma currents, or by the agency of a carrier
cell (leucocyte).

“2, The cocci multiply by elongation and subsequent
fission. When undisturbed by the surrounding leucocytes
their growth and multiplication are after the manner of a
streptococcus, but frequently they become irregularly
grouped together (Staphylococcus heaps).

“3. By elongation, some of the cocci give rise directly to
short bacillary forms, and through these to long filaments.

“4. The further extension of the colony is effected by the
growth and multiplication of both threads and cocci. The
former multiply by segmentation into bacillary elements,
which may again elongate to leptothrix forms.

“5. The leptothrix filaments may give rise by close
segmentation to coccus forms.

“6, The formation of clubs and similar forms is evidence
of diminished vegetative power of the filaments (possibly also
cocci), in connection with which they originate.

“a, The growth of a colony may be arrested at any stage
by the agency of the animal cells (leucocytes), or by failure
in the supply of the necessary pabulum. Jn that event the
majority of the threads tend to develop clubs at their outer
ends (involution forms). The central cocci and the remainder
of the filaments then disintegrate ; but the clubs which offer
a greater (passive) resistance to the surrounding cells may
persist for an indefinite period.”

From what is here stated, and from what I have already
said, it will be evident that we have here to deal not with
an ordinary “mould” fungus, but rather with a form of
streptothrix that undergoes dichotomous division at certain
points. It must therefore be looked upon as possibly
one of the Cladothrix alge, or more probably as one of
the Schizomycetes in which the Cladothrix formation occurs,
and which has'been described as closely allied to the strepto-
thrix Forsteri.

ACTINOMYCOSIS. 261

LITERATURE.

The following works may be consulted :
Acianp.—Brit. Med. Journ., vol. 1, p. 1159, 1886 ; Trans.
’ Path. Soc., vol. xxxvul., p. 546, 1886.
BanGc.—Tidskrift for Veterinaer, 1883.
Botiincer.—Centralbl. f. d. Med. Wiss., No. 27, p. 481,
1877.
Bostrém.—Verh. d. Congr. f. Inn. Med. Wiesbaden, p. 94,
1885.
*CROOKSHANK.—Manuel of Bacteriology, London, 1890 ;
Medico Chir. Trans., vol. Lxxi1., p. 175, 1889.
DELEPINE.—Trans. Path. Soc. 1889.
IsrAEL, J.—Virch. Arch., Bd. cxxiv., Heft. 1, p. 15, 1878 ;
Bd. txxvitt., Heft. 3, p. 373, 1889; Bd. xcvi., Heft. 1,
p. 175, 1884. P
Joune.—Deutsch. Zeitschr. f. Thiermed.
*M’FapyEean.—Journ. Com. Path. and Therap., vol. IL,
p- 1, 1889.
PERRONCITO.—Giornale di Anat. Fisiol. e. Pathol. 1875.
Ponrick.—Die Aktinomykose des Menschen, Berlin, 1881 ;
Breslauer Aerztl. Zeitschr., 1882.
TrEvES.—Lancet, vol. 1., p. 107, 1884.
Wotrr.—Virch. Arch., Bd. xcu., Heft. 2, p. 252, 1883.
* Full references given.

CHAPTER XIV.
GLANDERS.

Glanders—Farcy—Clinical Appearances of the Disease—Chauveau’s obser-
vations on Glanders Poison—Loffler and Schiitz—Method of Demon-
strating the Glanders Bacillus—The Bacillus—Methods of Cultivation
—Glanders in Various Animals—Farcy in Man—Temperature relations
of Bacillus—Desiccation—Germicides.

ALTHOUGH one of the best known, and, from its anatomo-
pathological point of view, best described of all diseases with
which the veterinary surgeon has to deal, it was long before
most observers in the veterinary schools could be brought
to look upon glanders as a contagious infective disease.
It is found especially in the horse and the ass, but as in
the case of tetanus, it may also be encountered in other
animals. When it occurs in or under the skin it is known
as Farcy, and in this form is usually found in man.
The primary disease is usually in the respiratory passages,
the lungs and the skin, especially around the orifices of the
nostrils and mouth ; the parts secondarily affected may be
those in communication with the lymphatics from an in-
fected area, or we may have distinct metastatic abscesses (or
abscesses at some distance from the original focus of the
disease), the material in such cases being carried apparently
by the blood along the course of the blood vessels. In all
cases the intensity of the disease appears to be in direct
ratio to the number and rapidity of formation and softening
of the small nodules, the process, as we shall find, being
determined by the presence and activity of a specific bacillary
organism that has made its way into the tissues. The
small nodules, whether they occur in the skin or on
the mucous membrane, say of the septum of the nose,
appear as small grey, gelatinous-looking points about the
size of a millet seed. In spite of this gelatinous appearance
they are usually comparatively firm in consistence. Under

GLANDERS. 263

the microscope each of these points is found to be made up
of granulation tissue, consisting of small round cells very
like leucocytes or lymph corpuscles, a few larger nucleated
cells, with here and there fragments of disintegrating con-
nective tissue. After a time, as the nodules become rather
larger, the centre assumes a yellowish appearance, or small
Opaque points may be seen, and ultimately at these points we
have the tissue breaking down into soft, pulpy, or caseous,
purulent material. The small nodules met with in the
early stages of the disease, are usually surrounded by a deeply-
congested area, and if they occur in the nostril the mucous
membrane covering them is greatly congested, and there is
great discharge of water, or of very watery “ matter,” from
the nostril. As the nodules become softened in the centre,
ulceration of the tissues near the surface takes place, the
softened centre escapes, and a “ punched-out” ulcer is left.
These ulcers may gradually run into one another, and from
the fact that the mucous membrane is congested and thick-
ened, the loss of tissue seems to be very great—much greater
than it really is. Along the lines of the lymphatics, and in
the lymphatic glands communicating with the ulcerating
surfaces, there is great inflammation and induration, what are
known as “farcy-pipes ” being formed. Here just the same
processes are carried on as when the disease occurs in the
mucous membrane, in the lungs and in the glandsat its root,
the lymphatics being of course similarly affected. It will thus
be seen that glanders resembles, in a most remarkable man-
ner, certain other infective diseases already described, such as
leprosy, tubercle, actinomycosis, &c., and, as a matter of fact,
numerous observations were early made with the view of
proving, first, that this was really the case; and, secondly,
that the contagious element was a contagium vivum, and
probably a form of micro-organism. Chauveau, as early
as 1869, inferred from a series of interesting and most
ingenious experiments that the poison was particulate in
character, and was, very probably, present in the leucocytes.
In December, 1882, Léffler and Schiitz made known the
results of experiments by which they had been able to demon-
strate the presence of a bacillus which appeared to have
a distinct causal relation to the disease ; and about the same
time Bouchard, Capitan, and Charrin also described an
organism in glanders—an organism, however, the charac-

264 BACTERIA.

ters of which were somewhat different from those ascribed
by Léffler and Schiitz to their bacillus.

As I have from time to time had opportunity of verifying
a number of the points insisted upon by these latter, and as it
is now generally accepted that the Léffler-Schiitz bacillus
is the one really associated with the disease, this organism
may be briefly described.

The best method of demonstrating the bacillus is to take a small particle
of the softened central part of a nodule and squeeze it out between two
cover glasses ; all superfluous matter from the edge is carefully removed,
and the covers are then treated in the ordinary way, after which they are
stained in a mixture of concentrated alcoholic solution of methyl blue, one
part to three parts of a 1 in 10,000 liquor potassz solution; the cover
glass is rinsed for about a second in a one per cent. solution of acetic acid
which has been tinged to the colour of Rhine wine by the addition of a
watery solution of tropxolin. It is then quickly washed with distilled
water, then with absolute alcohol, cleared up with cedar-oil and mounted
in benzol- or xylol-balsam. Sections of a nodule that has been
hardened in absolute alcohol should first be placed for a few minutes in

- weak caustic potash solution, after which they may be transferred to the
stain and treated as above. An even better method of staining is to use
Ziehl Neelsen carbolic fuchsin or carbolic methylene blue, and then to
decolorize the tissues with distilled water, or with a two per cent. solution of
hydrochloric acid. Kiihne’s carbolic-methylene blue method may also be
be used for staining sections of tissues.

After preparation there may be seen minute rods from
2.5 to 5 in length, which are usually one-fifth to one-
eighth of their own length broad. They are always more
numerous where cell proliferation is going on most rapidly.
From the fact that these organisms do not take on any stain
at all readily, and also that they are very easily decolorized,
it is often an exceedingly difficult matter to distinguish
them from nuclei and nuclear detritus, both of which take
on staining material in much the same manner as the
bacilli, and it is only by the exercise of the greatest care
and by the use of the very best optical appliances that these
organisms can with certainty be distinguished in the tissues.
It is, therefore, all the more necessary to obtain pure culti-
vations of the glanders bacillus in order that its characters
may be accurately described, and to determine whether it
really plays an important etiological part in the production
of the disease.

The first successful attempt to cultivate this specific
bacillus was made by Schiitz in 1882. Adopting the strictest

GLANDERS. 265

precautions to prevent the entrance of extraneous organisms,
he took small particles of the grey translucent material
surrounding the caseous centres of some of the above
described: nodules from the liver, lung, spleen, and lym-
phatic glands of a glandered horse. These small particles
carefully broken down, were inoculated on fluid and solid
blood serum from the horse and from the sheep, and also
in broths made from the flesh of the dog, horse, fowl,
and ox, and even in various fruit and vegetable infu-
sions. For two days no indication of the presence of any
growth could be observed on any of these media, but on
the third day the fruit infusion became slightly turbid, and on
the gelatinized serum there appeared numerous small, clear,
transparent, yellow, slightly elevated drops, like drops of a
yellowish fluid that had been splashed on the surface of the
serum. In eight to ten days these became slightly cloudy or
milky.t On examining one of these small drops under the
microscope it was found to consist entirely of masses of short,
rod-like bacilli, similar to those already described as present
in the glanders nodules, giving the same colour reactions,
being perfectly distinct in this respect from the tubercle
bacilli, which, as regards size and general appearance, they
very closely resemble.

Similar bacilli were found in the fluid media, and were usually in
the form of pure cultures, that is, only this single kind of organism was
present. In some cases there were impurities, but these were so evident to
the naked eye that they could be detected at once, such impure cultivations
being thrown aside.

The bacillus is usually straight or slightly curved, is
rounded at one end, and if any difference at all can be
observed between it and the tubercle bacillus, it is slightly
shorter and perhaps thicker than that organism, especially

t In order to obtain growths on sterilized potatoes some of the nasal
discharge from a glandered horse should be mixed with from 100 to 10,000
parts of boiled distilled water, and_a few drops of this mixture run over
the surfaée of the potato. Three days after inoculation there appear spots
of an amber-like growth; these points, as they increase in size, become
redder and more opaque, the colour deepening until it becomes like ‘‘ copper
oxide.” The manner of growth, according to Loffler, is quite characteristic ;
there are only two which are at all like it, the bacillus of blue pus (the
growth of which has a yellowish-brown tinge on potato but none of the
amber transparency and a peculiar pearly iridescence), and the cholera
bacillus.

266 BACTERIA.

when it is grown ina fluid medium. It exhibits no movement.
With pure cultivations, obtained in this way, and propagated
for several generations outside the body, Léffler and Schiitz
succeeded by careful inoculation in producing typical glanders
in various animals, the artificially infected animals pre-
senting on examination exactly the same appearances as the
animals that had been naturally infected, and from the nodules
artificially produced the typical glanders bacillus could again
‘be cultivated and passed on to other animals, where they, in
turn, gave rise to the usual symptoms of the disease.

In making these experiments they came across a most
interesting point. Inan old horse which they inoculated, and
that was apparently quite healthy at the time of the opera-
tion, they observed that the disease remained perfectly
localized, and that the ulcers very early evinced a marked
tendency to heal. The animal, however, was killed, and on
making a post-mortem examination it was found that it had
already, in all probability, been affected with glanders,and that
this had been running its course for some considerable length
of time, as not only were there old scars on the septum
nasi, but there were old caseous masses scattered through
the lungs. We have here an exactly analogous condition
to that recently observed by Koch in the case of guinea-
pigs already affected with tuberculosis, on which he made the
observations that led him to the discovery of his protecting
fluid ; this animal had been “protected”! against the general
outbreak of glanders which usually results from an artificial
inoculation by a previous chronic attack of the disease.

In order to illustrate the method of procedure in such cases, we may give
a short resumé of one of Loffler and Schiitz’ series of experiments. Two
horses were inoculated—one twenty years old from the eighth cultivation,
and the other two years old from the fifth serum cultivation that had
originally been taken from a /guinea-pig which had in turn been inoculated
from a fourth generation (also grown on serum), originally taken from
another animal. Both these horses were inoculated on each side of the
neck and on the breast ; and the younger animal was also inoculated in the
posterior nares. This was purposely omitted in the older animal, in order
to see whether the ulcers would occur in the nasal mucous membrane if it
were left intact, as far as direct inoculation was concerned. At the end
of a few days both horses had been attacked ; they ate badly ; diffuse boggy
swellings made their appearance at the points of inoculation; they were stiff
in the joints, the hair was ruffled, and on the eighth day farcy pipes corres-
ponding in their distribution to the course of the lymphatic vessels and glands
of the affected area could be distinctly felt under the skin. At this time

GLANDERS, 267

the swellings at the points of inocilation had ulcerated, and were dis-
charging an opaque, greenish-yellow fluid. On the twelfth day an ulcer
about the size of a shilling appeared on the skin of the forehead; its margins
were thickened, and it was so deep that it extended down to the bone ;
there was a discharge from both nostrils, at the margins of which it dried
into thin yellow crusts or scales; then small ulcers with indurated and
thickened margins appeared on the nasal mucous membrane in both
animals ; in fact there were here-all the characteristic features usually associ-
ated with glanders, These two animals died within twenty-four hours of
each other, and typical glanders lesions were found in the tissues after
death. Guinea-pigs inoculated with material from these cases exhibited
similar characteristic symptoms and lesions ; they died in from fifteen to
fifty days, and on post-mortem examination it was found that the
characteristic macro- and microscopic appearances were present in all of
the lesions.

In the study of the specific infective diseases, it has been
found that certain animals are especially susceptible to
one specific virus, whilst others are but little affected, and
that in the case of a second virus, things may be exactly
reversed—the non-susceptible animal remaining entirely
immune, or, in place of a constitutional disease being set up,
only small local lesions making their appearance. It is
therefore necessary for experimental purposes to determine
in all cases what small and easily-kept animals are susceptible
to a given disease, and what are unaffected or are only
slightly affected by the virus of that disease.

In the special case of glanders it is a matter of very great importance,
from the diagnostic point of view, that the guinea-pig is very susceptible to
the disease, the natural virus or pure cultivations of the glanders bacillus
setting up a typical disease which eventually brings about the death of
the animal. Similar inoculations into a rabbit produce merely a slight
rise of temperature, some local irritation accompanied by swelling, and per-
haps by slight ulceration, without any further constitutional or general
symptoms. Having found that this is the case as regards these two ani-
mals where the inoculations are made from animals that are undoubtedly
glandered, it is evident that in a doubtful case of glanders, strong proof for
or against the specific nature of the disease may be obtained by inoculating
rabbits and guinea-pigs with material from the doubtful sources ; if the
guinea-pig dies with characteristic symptoms, and the lesions remain local
in the rabbit, there is strong presumptive evidence (it might almost be
.said, definite evidence) that the suspected case is one of glanders

In connection with this immunity (complete or partial) or
susceptibility of different animals to this disease, it should be
pointed out that the human subject may be inoculated either
through wounds or scratches or through the application of

268 BACTERIA.

the nasal discharge of a glandered animal to the mucous
membrane of the nose or mouth. There are, undoubtedly,
cases recorded of glanders occurring in the human subject,
but these are not so numerous as they might be if it were
possible to put all those cases described as acute or chronic
bood poisoning under their proper heading. An old friend of
mine, the late Dr. Howard Bendall, in a Thesis. presented
for the degree of M.D. in the University of Edinburgh,
1882, described a case of acute farcy in man, and collected the
records of 68 similar cases,a number that might now be
very considerably added to. Of all the cases of acute farcy,
47 in number, only 6 were cured ; whilst of 21 more chronic
cases no fewer than 15 recovered or were partially cured: the
acute cases run a very rapid course, the duration of the disease,
however, varying from 4 to 47 days, the average course of
the disease being from 2 to 3 weeks. In chronic farcy the
patients died in from 50 days to 14 months, whilst of those
that recovered, the disease lasted as long as two and a half
years. It has now been proved beyond doubt that this
disease of farcy in man is due to the action of the same
bacillus that is found in the glanders of the horse, this
minute organism having been found both in the blood and
in the contents of pustules taken from a man affected with
farcy.

Cattle are completely immune against glanders as regards spontaneous
infection, and only localized ulceration, which rapidly heals, follows inocu-
lation. The goat is somewhat susceptible to the disease, though it
appears to occupy a position between cattle and the horse in this respect.
Sheep are fairly susceptible, but the disease runs its course very slowly,
and appears to resemble the chronic farcy in man. Lions, tigers,
and cats may all become affected, the disease in such cases running a
very rapid course. Dogs react to the poison very much as do rabbits.
So markedly is this the case that it has been suggested that the glanders
bacillus might be attenuated by passing it through the dog before it is inocu-
lated into horses and asses. It should be pointed out, however, in connec-
tion with all these experiments, that if a very concentrated virus, such as a
pure cultivation of the bacillus, especially in considerable quantities, be
inoculated even into a rabbit, a generalized disease may be set up, whilst
a weaker virus, such as that contained in the discharge from the nasal
mucous membrane of a horse, will produce nothing but localized symptoms.
(This element of quantity can never be ignored in making experiments
with bacteria of any kind.) Field mice are extraordinarily susceptible
to the glanders poison, whilst white mice and house mice are quite exempt.
The pigeon appears to be the only bird that is at all susceptible to the
disease,

GLANDERS. 269

We have already spoken of bodies that looked like spores
in these bacilli, but from the fact that the glanders virus,
both in fluids and in tissues, loses its vitality after fifteen days’
drying, it must be assumed that the organism does not form
endospores similar to those that are found in bacillus subtilis,
for example. The bacillus grows best at a temperature of
37° C., it will not grow at 20° C., nor at 45° C. at the other
extreme. At 22° C. it commences to grow slowly, whilst
at 25°C. it flourishes most luxuriantly, although it rapidly
loses its virulence when cultivated for several genera-
tions outside the body. Commenting on this fact, Léffler
points out that glanders is essentially a disease of hot
countries, where the comparatively high temperature appears
to be extremely favourable to the development of the bacillus
outside the body, especially in such materials as fodder,
manure, and stable refuse generally.

We have interesting evidence of this in statistics collected by Krabbe,
who gives the following proportion of horses affected with disease per
annum per 100,000 horses in the following countries: Norway, 6; Den-
mark, 8.5; Great Britain, 14; Sweden, 57; Wurtemburg, 77; Russia, 78;
Servia, 95 Belgium, 138; the French Army, 1,130; the Algerian Army,
1,548. ,

As already mentioned, desiccation for twenty-one days is
usually quite sufficient to prevent the multiplication of the
bacillus when placed in nutrient media. Consequently it
may be possible, by proper ventilation, to diminish the mor-
tality from this disease even in the warmest countries."

Another agent which helps greatly in preventing the mul-
tiplication of this bacillus is putrefaction, as the organisms or
products developed during that process appear to interfere
very markedly with the growth and multiplication of the
glanders bacillus. The most satisfactory of all disinfectants,
however, is heat, and it has been proved experimentally that
a temperature of 55° C., continued for ten minutes only,
is quite sufficient to destroy the bacillus, and with it the
infective power of the virus, from the fact that no spores,
probably, are formed to perpetuate the species. A spray of
steam would therefore, in all probability, be the most

t Léffler, however, was able to kill with a virus that had been dried on
silk threads for eighty-nine days, and Fraenkel states that the organisms or
their spores withstand drying.

270 BACTERIA,

serviceable and the most available of all disinfecting agents,
or perhaps, better still, a thorough washing out of the in-
fected stalls with boiling water. Chlorine and carbolic acid
are both capital disinfectants. A 2 per cent. solution of
carbolic acid applied for twenty-four hours kills the glanders
bacillus, and a solution of 4 per cent. applied to the nasal:
discharge for a single minute renders it perfectly innocuous.
AI per cent. solution of permanganate of potash,'0.23 or even
a 0.16 solution of chlorine water, or one-fifth per mille solu-
tion of corrosive sublimate, will also render the bacillus
perfectly innocuous within a couple of minutes. By means
of any of these the process of disinfection may be carried
on in stables in which glanders has occurred, with the
greatest ease and with absolute certainty.

LITERATURE.

The following works may be consulted :
-Benpat, Howarp.—Graduation Thesis Univ. of Edin. 1882.
BoucHarD, CAPITAN AND CHARRIN.—Bull. de l’Acad. and
Rev. de Méd. Francaise. Dec. 30, 1882.
CuavuvEeau.—Coniptes rendus, t. pxvur., No. 14, 1869.
FLEMING.—Manual of Vet. Sanitary, Science and Police, vol.
L, p. 509, 1875. oe :
GRUNWwALpD.—Oesterr. Monatsschr. f. Thierheilkunde, No. 4,
1884.
IsraEL.—Berl. Klin. Woch., No. 11, 1883.
KrasBE.—Deutsch, Zeitschr. f. Thiermed, Bd. 1, Heft. 4,
p- 286, 1875.
*LOrFLER.—Arbeit a. d. k. Gesundheitsamt, Bd. 1, p. 141,
1886.
LOFFLER AND ScHiirz.—Deutsch. Med. Woch. Dec., 1882.
*M’FapDvYEAN AND WoopHEaD.—Reports National Vet.
Assoc. 1888. ; :
Scutirz.—Journ. Comp. Med. and Surg., vol. vir., No. 1, p.
196, 1886.
VuLPIAN AND Boutry.—Bull. de ’Acad. de Med. 1883.
WassiLierF.—Deutsch. Med. Woch., Bd. 1x., No. 11, p. 135,
1883.
WEICHSELBAUM.—Wien. Med. Woch., No. 24, p. 754, 1884.
* Full references given.

CHAPTER XV.
ANTHRAX.

The Bacillus Anthracis—Early Observations—Pollender—Davaine—Koch
— Pasteur—Methods of Examination—Appearances of Bacillus under
Different Conditions—Spore Formation—Non-spore Bearing Bacilli—
The Vitality of the Bacillus and of the Spores—Cultivation Experi-
ments— Cover-glass Preparations—Inoculations into Animals—Methods
of Infection—Anatomical Characters of Malignant Pustule—Animals
Affected—Spores not formed in the Living Body—The Disposal of
Anthrax Carcases—Various Disinfectants—Pathogenic and Sapro-
phytic Anthrax—Buchner’s Experiments on Anthrax Bacillus and
Bacillus Subtilis—Hueppe and Wood’s Experiments.

ANTHRAX, or splenic fever, is perhaps the best known of all
the specific infective bacillary diseases. The Bacillus An-
thracis, compared with other pathogenic organisms, is of
very considerable size; it is from 5 to 20p long, and 1 tol. 5p
broad. It multiplies with very great rapidity in the blood of
certain animals, and may be very easily cultivated outside
the body ; in consequence of these features it was the first
organism that was proved definitely to be associated with a
specific disease, and it was certainly one of the first to be
recognized as occurring in both animals and in man.

In 1849, Pollender, and in 1850, Rayer and Davaine
described these organisms as occurring in the blood of animals
that had succumbed to splenic fever; then again, in 1857,
Brauell, examining the blood of a man affected with anthrax,
found this same bacillus.

Later, as already described, Pasteur’s wonderful experiments
on fermentation were published, and these led Davaine, in
1863, to commence a series of observations on anthrax, which,
carried on until 1873, gave everything but absolute proof that
the anthrax bacillus was the actual exciting cause of this malig-
nant disease. This proof, however, was not supplied until 1876,
when Koch, who had then been working at the subject for

272 BACTERIA,

some time, furnished most rigorous proof of Davaine’s hypo-
thesis. At the same time he was able to give much additional
information as to the development, mode of production, and
general life-history of the organism—information that could
only have been obtained by a long continued and careful
study of the organism outside the body. :

The following year Pasteur also succeeded in cultivating

Photo-micrograph of Anthrax bacilli in a preparation of the fresh spleen pulp of a
cow that had died from splenic fever. x 1000. .

this organism as a saprophyte and from that time up to the
present new facts have constantly been garnered, and our
knowledge of the biological history of this organism has
been greatly extended.

To observe the organism, a drop of blood is taken from
the spleen of a cow (or of any other animal that has died of
anthrax), and spread out between two cover glasses ; these are

ANTHRAX. 273

then separated, and one of them is at once lowered on to a
drop of three-quarter per cent. salt solution, the other being
set aside to dry, after which it may be gently heated in the
flame of a spirit lamp and then stained in a water solution
of methylene blue, well washed in water and alcohol, and
mounted in a drop of water or glycerine. In the unstained
specimen there will be found lying between the red blood cor-
puscles a number of short rods of the size above mentioned,
each of which has slightly rounded ends; sometimes also
there may be seen a delicate transverse mark running across
the middle, this being especially well marked when the rods
are longer than usual. The centre of each rod in the stained
specimen appears to be quite homogeneous, and is usually
deeply stained ; around this deeply stained portion is a kind
of sheath which remains unstained, or is only slightly tinged
by the colouring reagent. In some cases there is also at the
point of junction, on each side of the transverse line, a some-
what oval area slightly stained, so that when a number of
these rods are placed end to end without being separated
they have very much the appearance of a finger with the
joints slightly enlarged, or of a bamboo cane with its charac-
teristic thickenings placed at almost regular intervals. Both
rods and threads are perfectly motionless. In other cases, in
place of a mere transverse line, there is the appearance pre-
sented of two bacilli that have only recently become separated
from one another, still close together, however, and often so dis-
posed that they enclose an angle. In the coloured prepara-
tion the ‘same thing is observed. It is now seen that where
the rods obtain any considerable length they are distinctly
segmented, each chain being divided into a number of short
rods, and at regular intervals at the points of segmenta-
tion there is usually a slight swelling, although as yet there
is no trace or evidence of any spore formation.

In cases of wool-sorters’ disease, which is very frequently
accompanied by pleurisy, these organisms may be found in
the fluid that accumulates in the chest as long threads,
which may be grouped together in a kind of network or
felt, the individual threads of the bundles often reaching
an enormous length without there being any appearance of
segmentation.

A similar growth of long threads has also been obtained by
cultivating the bacilli, from the blood of the guinea-pig

19

274 BACTERIA.

affected with anthrax, for two or three hours in aqueous
humour, the threads then become elongated, the marks of
division are not nearly so distinct, and the threads remain
homogeneous. It is possible, therefore, that the appearances
met with in the fluid in the chest are due to the fact that the
organisms have been allowed to remain at rest in the
exudation, and that a process of cultivation has been going

Photo-micrograph—Modified Anthrax bacillus—longer threads leptothrix form. x 1000, |

on. In preparations made from the blood it is found that the
corpuscles are somewhat irregular in shape, that they are
running together in little irregular masses, and that the
bacilli are much more numerous than the blood corpuscles,
As regards the size of the bacilli it may be observed that the
rods vary not only in length (in the same animals), but that
they are of different breadths according as they develop in

ANTHRAX. 275

one species or in another, those found in a guinea-pig being
thicker than those seen in the mouse or sheep, whilst those
in the rabbit are thinner than any of those above mentioned.

If one or two of these bacilli be placed in a moist cell, in
what is called a hanging drop of nutrient broth, and the
temperature be kept at about 37° C., the organism may be
kept under observation under the microscope during the whole
course of its development. First, the short rods so increase
in length that they may ultimately cover a whole field of the
microscope, the protoplasm at the same time becoming
granular ; then a large number of very minute points make
their appearance, the hyaline appearance is gradually lost,
and the threads become quite opaque. A little later, if the
observation be continued, it will be found that instead of a
single thread, little bundles of the same long threads make
their appearance, in which there are found, at pretty regular
intervals, the highly refractile bodies with well-defined
margins and sheaths which have already been described as
spores. When the spores occur in long threads there is
usually a slight enlargement at the point at which the spore
is situated, and this, with the bright shining point, gives to
these spore-bearing threads an appearance that is described
as being like a chain of pearls. In many cases the spores
make their appearance in the shorter rods.t_ In place of the
regular forms above described there are others, or involution
forms, which are found to present themselves when the
organism is grown under unfavourable conditions ; for ex-
ample, irregular moniliform threads are usually met with
where the temperature is too high or too low, the soil is
exhausted and so on.

It has been found by Lehmann, Hime, Buchner, Behring,
and Roux that anthrax bacilli may be obtained that do not
give rise to spores. This appears really to be due to an
interference with the vitality of the protoplasm, as asporo-
genous organisms are best obtained by acting upon them
with antiseptics, be these chemical or physical. Roux, for
example, was able to obtain asporogenous bacilli, and bacilli
that remained asporogenous for several generations, simply

* The development of the spore has already been described, as most of
the descriptions of the setting free and development of the spores are taken
from observations made on this species and described by Koch, Buchner,
Prazmowski and others.

276 BACTERIA.

by immersing them for some time in 1,000 parts of nutrient
fluid, to which one part of carbolic acid had been added.
Under these circumstances the growth of the organism is
not completely prevented, but no spores appear to be de-
veloped. It cannot multiply at a temperature below 16° C.
nor above 45° C., 30° to 37° C. being the optimum tempera-
ture. It is distinctly zrobic in its growth, and cannot
develop unless it can obtain a pretty free supply of oxygen.

It has already been stated that the organisms which occur in
the blood are homogeneous, and it is a well-known fact that
spores are never developed in the bacillus that grows in the
blood, the multiplication there being entirely vegetative in
character, and being due to fission or division of the rods as
they increase in length. The same thing holds good even in
the dead body so long as it remains intact ; when once, how-
ever, the blood is allowed to come to the surface and in con-
tact with oxygen, spores are very rapidly formed within the
bacilli. This spore formation will not take place below from
24° to 26° C. (Koch says 18° C.), and then only in the
presence of oxygen, so that they can best be seen in those
bacilli that are cultivated on the surface of such nutrient
substrata as agar-agar, solidified blood serum, and potato, or
in fluid media through which a pretty constant stream of
oxygen is allowed to pass.

In this respect the spore formation of anthrax bacilli ap-
pears to agree with the sporulation of yeasts, which, it will be
remembered, takes place best on a surface of plaster of Paris
that is constantly kept moist and well supplied with air.

Anthrax bacilli as distinguished from the spores are very
readily killed. The ordinary putrefactive processes that
are undergone in the decomposition of carcases in which
these organisms have been present during life, especially if
the air be excluded, cause the death of these bacilli in about
a week, The temperature of boiling water maintained for a
few seconds kills the bacilli, but, according to Klein, boiling
for ten minutes is not to be relied upon to kill the spores
although Koch states that at 100° C. the spores are killed in
five minutes. The bacilli are killed by a two minutes’ ex-
posure to a one per cent. solution of carbolic acid in water,
whilst the spores may remain alive for more than a week in
a similar solution. ‘They must be kept for nearly a week in
a three per cent. solution, and twenty-four days in a five per

ANTHRAX, 277

cent. solution if these reagents are to have any lethal effect
on them. Carbolic acid in oil—five per cent. solution
—had little more, if any, effect than pure olive oil. The spores
remain alive, for an indefinite period almost, in a five per cent.
solution of olive oil. Sulphurous acid vapour in the propor-
tion of one to one hundred of air kills the bacilli in half an
hour, but the spores resist the same antiseptic for seventy-two
hours. Corrosive sublimate in one per mille of water suffices
to kill spores by simply wetting them.

‘These tests of the vitality of the spores, however, were
made by inoculating them into gelatine after they had been
treated with the antiseptic. If, in place of inoculating into
a nutrient medium of this nature, the spores are introduced
into the circulating blood of a living animal, it has been found
by Klein that it requires much longer exposure and much
stronger solutions to hinder the development of the spores
into bacilli and prevent the production of anthrax.

We have already stated that the organism cannot con-
tinue its growth at any temperature above 45° C., but it may
still remain alive up to about 60° C. if this temperature be
not continued for too long a period, the spore-bearing bacilli,
though themselves killed at this temperature, leave their
spores, which will withstand a temperature of 100° C. if
continued only for a short time, and start into life and into
active vegetative growth when again placed under suitable
conditions. The only other physical condition that appears
to be fatal, or at any rate injurious, to anthrax spores is
strong sunlight ; this appears to deprive them in whole or
in part of their powers of further development in a most
remarkable manner, always causing distifct attenuation of
their pathogenic virulence before completely destroying
them.

The best way of maintaining cultures of anthrax bacillus is
to take a drop of anthrax blood, sow it on a potato or agar-agar, ,
allow it to grow there for several days at about 30° C. until
spores are well developed, triturate a small quantity of the
growth with some distilled water, place a number of silk
threads which have previously been sterilized by heat in this
mixture, and then dry them carefully, cut into short lengths,
and keep them ina stoppered bottle or in a plugged test tube.
From these threads cultivations may be made on almost any
artificial nutrient medium. In addition to the media already

278 BACTERIA.

mentioned, starchy materials, vegetable infusions, hay and
meat infusions, and even sterilized alkaline urine, may be used,
the only thing that appears to interfere with their growth being
an acid reaction. Even this, however, may be present to a
slight degree if other conditions are favourable; for example,
the anthrax bacillus grows readily on potato, which gives a
slightly acid reaction. On gelatine plates colonies grow and
are visible to the naked eye, on the second or third day, as
small white points which gradually spread outwards, and as
they come to the surface cause a slight liquefaction of the
surrounding gelatine; théy are then seen as small white
masses with wavy margins lying in a clear space, formed by
the liquid gelatine. When examined under the low power
they appear as round dark-green points with an irregular
outline ; on the second or third day this irregularity
becomes much more marked and, as Fliigge describes it,
when it reaches the surface of the gelatine “the dark
remnant of the deeply-placed colony can only be seen in
the middle. Around this centre, however, there is a light-
brown or light-grey shimmering mass, which consists of
numerous wavy, curling bundles, recalling the appearance of
locks of hair or snakes on the head of a Medusa. Ultimately
individual threads, or bundles of threads, branch off from
the irregular periphery, and grow over the gelatine in various
directions. At the same time the gelatine is liquefied over a
small area; the colonies, which have now a diameter of 2
to 4 mm., begin to float and break down at their margins
under the action of the fluid formed.”

An exceedingly good method of obtaining a permanent record of the
appearance of these colonies, is to lower a cover glass on to one of them
and then to raise it carefully without sliding it over the surface, so as to
take an imprint of the colony upon the glass; the surface of the colony
adheres to the glass and a thin layer is removed. This, when stained
with fuchsine or methyl blue and mounted in Canada balsam, gives an
exceedingly faithful ‘‘impression” of the appearance of one of these masses.

The puncture cultivations of the bacillus anthracis pre-
sent most characteristic appearances. Along the track of
the inoculating needle there appear delicate feathery rays,
which pass for some distance into the gelatine ; small lateral
rays are given off from these, and the whole of the young
growth has a peculiar feathery appearance. These rays are
always longer near the surface, and gradually become shorter

ANTHRAX. 279

and fade off as the lower part of the track is reached. After
a time the gelatine begins to liquefy, and slowly the feather-
like mass sinks to the bottom of the liquefied medium,
eventually forming an opaque white layer, which is found to
consist of bacilli in different stages of degeneration. The
upper layer of the gelatine, though perfectly clear, is now
quite fluid. This process of liquefaction comes on first along
the track of the needle, near the surface, and gradually
extends downwards until the whole needle track has dis-
appeared. We have then in the tube, the upper liquefied
gelatine, then the opaque layer of bacilli, and lastly the
clear solid gelatine below. If agar-agar be used in place
of gelatine, much the same appearances are presented ;
there is a smooth glistening greyish-white surface growth,
and the feathery rays appear to get more and more solid as
the growth continues, but no liquefaction takes place. On
solidified blood serum the colonies grow as greyish-white
layers on the surface, and the medium is very slowly liquefied.
On sterilized cooked potatoes the bacillus grows asa creamy-
white somewhat parchment-like granular mass, which rises
above the surface for some little distance but never extends
far in a lateral direction ; it has a peculiar dry appearance.
The growths on potatoes, as on other media, take place at
the ordinary temperature of the room. This organism can
be very readily inoculated into certain animals, with results
that may be looked upon as very definite ; for example,
if with a needle the point of which has been dipped into
the spleen of a cow that has died from anthrax a mouse be
pricked at the root of the tail, it will die in from seventeen
to eighteen hours, enormous numbers of bacilli being found
in its blood. It has been observed that in the case of some-
what larger animals, such as the guinea-pig, death does not
take place till a rather later period ; for instance, a guinea-
pig setoned with a silk thread containing spores of anthrax
bacillus will die on about the second or third day. If the
animal be examined after death, it will be found that near
the seat of inoculation there is usually very little to indicate
that this was the point at which the infective material was
introduced. Should, however, the inoculation be made into
the abdominal wall, there is usually marked cedematous
swelling of the peritoneum; there may be small hemor-
rhages into the subcutaneous tissue, and there is usually

280 BACTERIA,

some emphysema; the abdominal muscles are pale, moist,
and are evidently in a condition of cloudy swelling, or in
some cases there may be in the muscles, near the point of
inoculation, hyaline degeneration. The spleen is enlarged,
soft, and pulpy, and appears to contain a very large quantity
of blood ; itis dark in colour. The liver also is changed ; it
has a half-boiled look, and contains a considerable quantity
of blood. The lungs are bright red and the cavities of the
heart are distended. As already stated, the blood and lymph
from the tissues contain the bacilli in very considerable
numbers. If sections of various organs and tissues are
afterwards made, it will be found that the bacilli are most
numerous near the capillary vessels or in those small
venules that arise from the capillaries. They are found
in all parts of the spleen and in the small venules of the
liver. They may also be found in the small capillaries of
the glomeruli of the kidney.

Another method of infection, especially met with in wool-
sorters’ disease, is by the air passages, a condition that was
most carefully described by Greenfield in. this country, and
has since been made the subject of careful observations by
Buchner in Germany. It appears that the spores are inhaled
along with dust from hair, wool, &c. ; they develop in the
air passages of the lung, make their way through the alveolar
membrane or through the walls of the bronchi, and so into
the lymph channels and blood vessels of the lung. In
some cases, however, they appear to " operate” from
the air channels themselves, and, multiplying in these
positions, give rise to a kind of pneumonia. The air vessels
become filled with sero-fibrinous exudation, in which the
bacilli may develop most rapidly, the walls of the alveoli
become cedematous ; the bacilli make their way from these
points into the circulation, and general anthrax is set up.
Pleurisy, with effusion into the thoracic cavity, as already
mentioned, may also’ develop in these cases, and the walls
of the bronchi appear to be invaded by the bacillus. It has
been found that if the bacillus is taken into the alimentary
canal the acid contained in the stomach usually destroys it..
If, however, resistant spores make their way into the alimen-
tary canal, they may pass untouched through the stomach
and so into the alkaline contents of- the intestine. At the
body temperature they are then under very suitable conditions

ANTHRAX, 281

for development into the vegetative forms ; and as they are
developed. they make their way, especially through damaged
epithelial cells which they gradually push to one side, into
the deeper layers of the intestinal wall, into the lymph
channels, or directly into the blood vessels, so giving rise, in
many cases, to a general infection.

Susceptibility of animals to infection through the intes-

Photo-micrograph of Anthrax bacilli in the blood vessels of the mesentery of a mouse,
* At one or two points the bacilli may be seen lying in the substance of some of the
white blood cells (phagocytes). x 375.

tinal canal varies very greatly, the guinea-pig, which is very
susceptible to the disease conveyed by inoculation, withstand-
ing a very large dose. of the spores when given by the
stomach, whilst sheep and cattle, both of which are more
resistant as regards inoculation, are comparatively easily in-
fected through the alimentary canal. I have stated that at —
the point of inoculation in animals there is usually no evi-

282 BACTERIA,

dence at all that it has been the point of entrance of the
bacilli, but in the human subject there very frequently
occurs at the point where the bacilli enter a wound, a
marked local reaction, the result apparently of an effort
on the part of the tissues to prevent the further advance
of the bacilli. There is first irritation at the point of in-
oculation, this usually occurring in from one to three days
after the inoculation ; about a day later a minute vesicle
surrounded by a zone of inflammatory redness and swelling
makes its appearance. The serum in this vesicle becomes
brown, and gradually a- kind of mortification is set up;
other vesicles, forming a ring, appear around the original
point ;- these in turn become brown or black, until
gradually an extensive black scar is formed. If these
little pustules, with the surrounding tissue, be freely
excised and the wound well cauterized with strong carbolic
acid, there may be no return of the disease ; in fact, if the
removal be free enough, this result is most certainly ob-
tained ; but if the inflammation be allowed to go on
unchecked, there is gradual extension of the vesication,
sloughing, and blackening, until a very considerable area
is affected, the bacilli sooner or later making their way into
the blood vessels and giving rise to general anthrax. In the
vesicles, and also in the tissues around the point of inocula-
tion, anthrax bacilli can usually be found in considerable
numbers, the tissues are somewhat ‘cedematous, and there is
an increase of leucocytes in the inflamed area. When the
blackening commences there are usually found along with
the bacilli, or in some cases in the centre of the ulcer. re-
placing them, chains of micrococci ; these are seldom seen
in the very early stages, but in the later stages they are
almost invariably present.

The animals most susceptible to this disease are sheep,
mice, rabbits, guinea-pigs, and, according to Fliigge, horses,
hedgehogs and sparrows, all of which die or are very
ill when the organism is inoculated directly into the sub-
cutaneous tissue. Most birds are not readily inoculated, and
for a long time it was found impossible to kill fowls by means
of anthrax. White rats, old dogs, and amphibians are ex-
ceedingly resistant to the disease in almost any form ; cattle,
too, which are readily infected through the alimentary canal,
are but slightly susceptible to anthrax introduced by direct

ANTHRAX. 283

inoculation into the tissue. It has been found, however, that
by reducing the temperature of the fowl and by raising that of
a frog, these animals may be infected by inoculation, and they
have even under certain conditions been killed. The usual
method of infection in man, of course, is by the skin, and it is
found that anthrax usually affects those who attend on or
slaughter animals that are subject to this disease, so that those
usually affected are butchets, grooms, and others engaged
in similar employments. During the time that I was acting
as physician to the Western Dispensary in Edinburgh, which.
was situated near the public abattoir, several cases of localized
poisoning and two of general anthrax poisoning came under
my charge within a comparatively short time. ‘These were all
cases of butchers, who having slight scratches on their hands
or arms, had been infected by the blood of animals that they
had killed which were suffering from splenic fever. T'anners,
skin-dressers, and wool-sorters are also specially liable to this
form of the disease, though the latter class are also very
frequently affected through the lungs. In certain regions
where the disease seems to be almost endemic the spores of
the bacillus may make their way into the alimentary canal
of the human and other subjects, through the medium of
food or water.

It has already been stated that the anthrax bacillus within
the body of an animal is incapable of forming spores, but in
those cases where the bacilli can make their way from the
lungs into the saliva, from the mouth, or from the lower
part of the alimentary canal, whence they are discharged
with the secretions, spores may be readily enough formed.
Consequently an affected animal must always remain a centre
of infection, and even the dung from a diseased animal may
contain a large number of spores which, when scattered over a
field, are sufficient to infect whole herds or flocks. The only
way in which to get rid of the infection in such a case is to
burn the animal at once, or to bury it deep down in the
earth. This latter method of disposing of the animal should
only be resorted to where it is possible to bury it six or seven
feet deep. It is a well-known fact that the organism cannot
form spores where oxygen is absent, and at any tempera-
ture below-12° C., and below this temperature the bacilli are
incapable of existing for any length of time. At a distance
of six or seven feet from the surface the temperature of the

284 BACTERIA,

ground is usually below 12° C., so that animals put below
this depth, even if swarming with bacilli, cannot be
looked ‘upon as centres of infection; the organisms, no
longer able to form spores, soon lose their virulence and
die out altogether. In order to get rid of any spore-
bearing bacilli, all discharges or blood that may have
escaped at the place where the animal lay after death
should be carefully disinfected with 5 per cent. carbolic acid.
It was for long thought that by cultivating the anthrax
bacillus through a great number of generations on ordinary
nutrient media it could gradually be converted into the
hay bacillus or the bacillus subtilis — a very similar
organism, but one that has no pathogenic properties—and
Buchner, in a published series of experiments, claimed that
he had obtained this conversion, and that in the same way
he could again turn back the wheel from the hay bacillus to
the pathogenic form. These results, however, have not been
repeated, although many have tried similar experiments ;
but Hueppe and Wood, by using a species of earth bacillus,
which, in its morphological characters and method of growth
on nutrient media, appears to be almost identical with
anthrax bacillus (but possesses no pathogenic properties),
have found that it must be very nearly related phylogene-
tically, from the fact that when inoculated into animals it
acts as a kind of vaccine, and renders the animal immune to
an attack of anthrax.

It will be well, however, to take up this and other ques-
tions relating to anthrax in connection with vaccination and
immunity.

LITERATURE.

The following works may be consulted:

Botiincer.—Centralbl. f. d. Med. Wiss., No. 27, p. 417,
1872.

BraveELu.—Virch. Arch., Bd. xt, Heft. 2, p. 121, 1857, and
Bd. xiv., Heft. 5, 6, p. 432, 1858.

Bucuner.—Nageli’s Untersuch. i Nied. Pilze ; Eine neue
Theorie i. Erziel d. Immunitaét gegen Infectionskran-
kheiten Minchen, 1883; Virch. Arch., Bd. xc1., Heft.
3, p. 410, 1883.

CHAUVEAU.—Comptes rendus. 1880-1885.

DavaineE.—Comptes rendus, 1863-1866.

ANTHRAX. 285

GREENFIELD.—Proc. Roy. Soc., vol. xxx., p. 557, 1880.

Jousert.—Comptes rendus, t. XCV1., p. 641, 1883.

Kien.—Rep. Med. Off. Local Gov. Board, p. 207, 1882.

Kocu.—Beitr. zur. Biol. der. Pflanzen, Bd. 1, p. 277,
1876; Mitth. a. d. k. Gesundheitsamt, Bd. 1, p. 49,
1881.

*M’FaDYEAN AND WoopDHEAD.—Reports Nat. Vet. Assoc.,
1888.

PasTEuR.—Comptes rendus, t. LXXXIV., p. 90, 1877, t. XCVI.,
P- 979, 1883 ; Rev. Scientifique, Jan. 20, 1883.

PoLLENDER.—Casper’s Vierteljahrsschrift, 1875.

PRaAzMowsKI.—Biol. Centralbl., Bd. rv., No. 13, p. 393, 1884.

Rocer et Davaine.—Bull. de la Soc. de Biol. de Paris,
1850.

ToussainT.—Comptes rendus, t. LXXxIV., p. 503, 1877.

See also various papers in Centralbl. f. Bakt. u. Parasitenk ;
Annales de l'Institut Pasteur ; Cornit and Bases Les Bac-
téries ; HuEpPE Joc. cz#. and L6rFLer, Vorlesungen iiber Die
Geschichtliche Entwickelung der: Lehre von den Bacterien,
Leipzig, 1887. :

* Full references given.

CHAPTER XVI.
TETANUS.

Tetanus a Specific Infective Disease—A Wound Fever—Organism found
by Nicolaier in the Soil taken from Streets and Fields—Experiments
on Animals—Symptoms of Disease—Pure’ Cultivations Obtained—
Description of Organisms-—Characteristic Shape—Spore Formation—
Organism Anzrobic—Cultivations—Kitasato’s Method of Cultivating
the Organism—The Bacillus found only at the Seat of Inoculation—
Wide Distribution of Spores—Bossano’s Examination of Earth—
Vaillard and Vincent’s Observations—Tetnaus Bacillus a Facultative
Pa eee under which Tetanus is Contracted —Poisoned

rrows.

TRAUMATIC tetanus, or convulsions resulting from poison-
ing associated with an open wound, was for long sus-
pected to be the result of some condition similar to septic
or hospital fever; that it was an infective disease was
recognized by many of the older surgeons, and attempts
were made at a very early date to treat it as a traumatic
infective disease. That this poisoning was the result of
the activity of an organism, which made its way to a wound
and there flourished and gave rise to the characteristic pro-
ducts and symptoms, was not the result of direct experiment
made with the object of finding out such an organism,
although attempts were not wanting to demonstrate its
presence in the wounds and along the course of the nerves
in cases of tetanus. All efforts, however, proved un-
successful until after an organism obtained from other
sources had been obtained and described, and an artificial
tetanus had been produced.

In 1884 Nicolaier, working with soils obtained from the
streets and from the fields, found that these when inoculated
into certain animals produced effects different from those
produced by soils taken from cultivated gardens and from
woods. ‘The former he found, when a small particle was
placed in a little pocket under the skin of mice, rabbits,

TETANUS. 287

and guinea-pigs, gave rise to symptoms which he described
as tetanic in character. In from two to four days after
the inoculation the hind-quarters of the animal became
paralysed, first the one near the seat of inoculation, then the
other ; then rigidity came on followed by loss of motion ; the
forelegs were in turn affected, then the neck, and at length
the whole:of the body became rigid, and sometimes curved as
in tetanic convulsions occurring in the human subject. On
examination after death there was found, at the point of in-
oculation, a small abscess, in the pus of which were several
species of micro-organisms. One of these, when obtained
pure or practically pure, if inoculated into another animal
produced exactly the same symptoms. Nicolaier was not
able to obtain any perfectly pure cultivations, but he
described an organism which was afterwards separated by
Kitasato and by Tizzoni and Mdlle. Cattani independently.
This organism, although never directly demonstrated in the
earth that produced the disease, is constantly found in the
pus of the abscess, in the walls of this abscess, and even in
the immediately surrounding tissues. It occurs as long
delicate threads scarcely thicker than the bacilli of mouse
septicemia with slightly rounded ends; like some other
organisms, especially those met with in putrefactive pro-
cesses, they give rise to spores which are usually developed
at the end of the shorter rods into which the long threads
break up ; this spore, forming the head of what is called
the drum-stick-shaped bacillus, is usually large and may be
seen as a clear mass causing enlargement of one end of the
bacillus. The spore develops best at the temperature of the
blood, and under favourable conditions is completely formed
about thirty hours after multiplication has commenced ;
at the temperature of the room this does not occur for about
aweek, although the organism itself developes readily enough
at thistemperature. The rods are motile. Owing to the fact
that the organism is anzrobic and that the presence of
oxygen interferes very greatly with its development (it is
said that oxygen kills it altogether), it proved a somewhat
difficult matter to obtain perfectly pure cultivations, although
when once it had been recognized that the organism was
anerobic, plate cultivations were readily enough obtained

For further description of the disease see ‘‘ Micro-Organisms,” Dr. C.
Fliigge, New Sydenham Society, 1890,

288 BACTERIA.

by keeping the plates on which the nutrient medium
was spread in an atmosphere of hydrogen. When puncture
inoculations are made in tubes of gelatine to which grape
sugar has been added, there is no growth along the part
of the track of the needle near the surface, but in the
deeper part away from the air, there is a moderately
luxuriant growth which appears in the form of a central

Specimen from pure culcure of the Tetanus Bacillus, with enlarged
spore-bearing “drum-stick” ends. x 1000.

streak, from which numerous spikes pass laterally into
the surrounding medium. The culture at this stage has
very much the appearance of a spruce-fir, the lower branches
(z.e., those in the deeper part of the gelatine where the
access of oxygen to the organism is entirely cut off)
being longer and more distinctly marked than those nearer
the surface. Later this characteristic appearance is lost,
the organism invades the whole of the nutrient medium,

TETANUS. 289

forming a kind of cloud; the gelatine becomes softened,
and there is emitted a peculiar fusty smell which appears to
be almost characteristic of this organism.

Most of the earlier experiments were somewhat unsatis-
factory, and considerable doubt was cast on this organism as
a producer of tetanus from the fact that in many cases pus that
was known to contain this specific bacillus, and even old pure
cultivations of the organism, failed to set up the characteristic
symptoms when inoculated in the usual manner. More
recent observers, however, have pointed out that the tetanus
bacillus, like many others of the septicaemia group, is virulent
only so long as it is grown under anzrobic conditions. This
is especially the case in those organisms in which there has
not been time for the spores to develop; so that when
grown in the presence of free oxygen (when that is possible),
or when exposed to the air after the growth has been com-
menced under favourable conditions, and has gone on for
some time, but before there has been time for the formation
of spores, the organism rapidly loses its virulence, or, as we
have seen, dies off altogether. Experiments made by inocu-
lating the pus from tetanic patients often gave entirely nega-
tive results. Here it was evident that the failure was due,
in many cases at any rate, to the fact that the pus with its
contained organisms had been exposed to the air for some
time, and the bacilli had been compelled to grow under
conditions unfavourable to the retention of their specific viru-
lence, before they were used for purposes of inoculation. In
such organisms, as we should expect, it is found that spores
are not seen, or they are very imperfectly developed, and it
may be in the case of the older cultures, where these
spores are developed into the young bacilli, that these,
not having attained their full resisting power, die off very
readily in the presence of oxygen. By paying attention to
this point it.has gradually been proved, almost beyond doubt,
that the tetanus that may be produced in white mice or
guinea-pigs by the inoculation of small particles of garden
earth, is of the same nature as the tetanus that is produced
by the inoculation of the pus from a primary wound which
has apparently given rise to tetanus in the human subject.
Further, now that the conditions under which the organism
exists have been studied, and its anzrobic character
recognized, pure cultivations have. been made, and it has

20

290 BACTERIA.

been proved that the disease may be produced without
fail if certain definite precautions are taken before the
inoculation be made. One great difficulty connected with
the obtaining of pure cultivations was, of course, that
under the conditions favourable to the growth of the
tetanus organism, other anzrobic bacteria would also
take the opportunity of developing. Some only of these
other organisms, however, give rise to the formation of
spores, and this occurs at a later date than in the case of the
tetanus bacillus. Kitasato, very ingeniously, made this fact
the stepping-stone to the cultivation of a pure growth of the
spore-bearing tetanus bacillus. As we have already seen,
spores of many bacteria can readily withstand a high tem-
perature (in some cases of even 100°C., if this is not continued
for too long a time, and they will withstand for a con-
siderable period the action of a temperature of 80° C.), whilst
the vegetative or fully developed forms are killed off very
rapidly at a comparatively low temperature. Kitasato’s
method of procedure was as follows : From the immediate
neighbourhood of the suppurating wound of a patient who
had died from tetanus, he took a small fragment of tissue,
and placing it under suitable nutrient conditions, z:e., in the
specially prepared gelatine at a temperature of little over
30° C., and in an atmosphere of hydrogen, he obtained
a very luxuriant growth of anzrobic organisms ; amongst
these he observed that the drum-stick-shaped organisms
developed their spores at a much earlier period than any
of the others that were growing in his cultures. As soon
as these spores made their appearance he raised the tem-
perature to 80° C., with the result that all those bacteria,
in which spores were not already developed, were very
rapidly destroyed ; the tetanus dacz/dz were also destroyed
(that is, the vegetative forms were destroyed), but the sfores
still retained their vitality, and on being transferred to
suitable nutrient media, and placed under other suitable
conditions, they “‘ hatched ” out into the vegetative form, and
a pure cultivation of the tetanus bacillus was obtained. It
is a rather curious fact that here, as in the case of diphtheria,
the organism seems to be localized at the actual point of
inoculation, for although, as we have seen, the bacilli, how-
ever numerous in the pus and in the walls of the abscess, and
in the infiltrated tissues immediately around the abscess,

TETANUS. 291

are confined to a well-defined and localized area, and the
most careful researches, conducted with the help of both
cultivation and histological. methods, have hitherto failed to
enable any observer to demonstrate the presence of these
organisms in the internal organs. It would appear, then,
that as in the case of septicemia and diphtheria, the poison
is manufactured by the organisms at the site at which they
are actually introduced, and that from this point it is absorbed
into the body, and is carried to the special tissues on which
it acts. Brieger, indeed, was able to separate from the
limb of a patient who had died from tetanus an exceedingly
virulent basic poison or ptomaine which he speaksof as tetanin,
whilst he also found a very poisonous proteid substance,
tetano-toxin, which he called a toxalbumen, a substance of
which we shall have to speak in a later chapter.

At first sight it appears somewhat extraordinary that the
development of the tetanus bacillus should be so extremely
localized, and that the localization should be confined to a
position so near the surface—z.e., to the surface of a compara-
tively open wound, to which the oxygen of the atmosphere
might at first sight appear to have easy enough access.

When, however, we come to think more carefully of the
conditions that prevail in the wound, this does not seem
quite so extraordinary. In the first place, there is, covering
it, a layer of pus which, as is well known, has little power of
holding oxygen in solution, any small quantity that is there
being gradually taken up by the leucocytes that escape to the
free surface or into the abscess, and utilized by them for their
own purposes, Beneath this, especially in the later stages
of infiltration, and before the capillary vessels of the granula-
tion tissue have begun to form, or are fully formed, the supply
of oxygen must necessarily be comparatively small, and any
that is present is promptly taken up by the active wandering
and proliferating connective tissue cells. Here then we have
the conditions under which the tetanus bacillus is enabled to
flourish ; there is a condition of anzrobiosis, or oxygen famine.
Beyond this, however, where the infiltration is not so great,
and where the vessels are considerably dilated, the red blood
corpuscles are constantly bringing up their fresh supplies of
oxygen to the tissues, and in consequence the fluids at some
little distance from the wound contain more oxygen than
there is near the surface of the wound, and the conditions

292 BACTERIA.

are consequently much less favourable for the continued
existence of the tetanus bacillus.

The spores of the tetanus bacillus seem to have a remark-
ably wide distribution. Originally they were only cultivated
from garden soil, but successful inoculations have since been
made with such material as the sweepings of a hay-loft, and the
dust that had accumulated on the furniture of horses. The
specific bacillus has been found on the grime on a man’s hand,
and on imperfectly cleansed surgical instruments. Tetanus
is said to be specially associated with the horse, but the more
recent observers insist that this is simply because the horse is
susceptible to the action of the bacillus and its poison, and
because the germs have such a widespread—in fact, an almost
universal—distribution. How universal this distribution is
may be gathered from the fact that M. Bossano, who was
able to obtain the soil from forty-three different regions in
various parts of the globe, got positive results with twenty-
seven of them. With the soil from these forty-three places
he inoculated a number of animals, introducing a small por-
tion, about the size of a pea, into a little subcutaneous pocket
of a white mouse or a guinea-pig, and with the soil obtained
from twenty-seven of these places tetanus was produced in
from two to four days. He says of the soil obtained from
England, that from Bath produced tetanus in two out of
three white. mice inoculated, both of them dying in about
two days. Soil from Portsmouth did not contain tetanus
bacilli, whilst that from Plymouth and from Manchester
caused the death of some of the animals that were inoculated
with it, all the characteristic symptoms of tetanus being
developed. Speaking colloquially, a worker at the Brown
Institute told a friend that they grew the tetanus bacillus in
the garden there. From his experiments Bossano concluded
that soils which contain much organic matter, almost in-
variably contain tetanus bacilli, and that latitude, climate,
and special meteorological conditions, have far less influence
on its development than defective drainage, imperfect hy-
gienic conditions, and the degree of cultivation of the soil.
It appears, however, that our’methods of cultivation are not
yet perfect, for it is an undoubted fact that failures to pro-
duce tetanus with pure cultivations are of very common
occurrence, even in the hands of those best fitted to carry on
experiments of this kind. So markedly is this the case that

TETANUS. 293

Chantemesse and Widal at one time thought that the pre-
sence of other organisms was necessary in order that the
tetanus bacillus might act, and it has been suggested that
zrobic organisms which have great avidity for free oxygen
must also be present in order to allow of the development
of the full virulence of the anzrobic tetanus bacillus. It is
a fact, whatever may be the explanation, that the tetanic
organism soon loses its virulence under cultivation, especially
when it is grown “ pure.” ;

Recently, however, Vaillard and Vincent, as the result
of a series of most careful observations, have arrived at the
conclusion that it is usually the tetanus poison — which
they compare to snake poison—and not the organism
itself that gives rise to the tetanic symptoms in animals
that are infected experimentally. They also find that until
the organism has grown in artificial culture media for
some time it has not the power of setting up disease, a
fact that was accounted for when they found that no poison
was developed until some time after the organism had begun
to grow, the production of the poison appearing to go on
simultaneously with the formation of a peptonizing enzyme.
Even spores, when injected alone, could not set up tetanic
symptoms, but when these were injected along with other
organisms such as lactic acid bacillus, or even with lactic acid
itself, with bacillus prodigiosus, or into a bruised wound, or
where they were injected along with a quantity of their
own poison, tetanus was invariably set up. The tetanus
organisms form their poison slowly, and in healthy tissues
they are rapidly destroyed by the tissue cells long before
they have time to form sufficient poison to produce the
nervous symptoms of tetanus ; whilst in the cases above
mentioned the tissue cells are so engaged in removing the
other foreign matter, or are so paralyzed by the action of
the lactic acid, or the small portions of tetanus poison, that
they are not able to contend on equal terms with the tetanus
bacilli, which being under favourable conditions grow rapidly,
give rise to the formation of the special poison, and the
patient succumbs. This remarkable poison in doses of 25
millegrammes is quite sufficient to kill a rabbit, or the 25th
of a millegramme to kill a mouse.

The tetanus bacillus is a facultative saprophyte, the nature
of the wounds through which tetanus is inoculated bearing,

294 BACTERIA.

out in a most remarkable fashion the experimental proofs
that have been already adduced. A horse which, in the stable
and in the field, always collects a certain quantity of earth
on his skin and in his hoofs, may be easily. inoculated ; he,
in turn, may readily inoculate a man or another animal by a
kick with the sharp iron of his dirty shoe. Gardeners, agri-
cultural labourers, and all who work with horses or in the
soil, bear on their hands a virus which only needs a bruise or
a cut, but especially the former, to allow of its setting up the
characteristic symptoms of tetanus. Soldiers, during a cam-
paign, when their garments and equipments are soiled from
contact with the ground during their camping and bivouack-
ing are always more liable to the disease than when they are
hurt accidentally during times of peace. It is also pointed out
that in warm countries where people are in the habit of sleep-
ing out in the open air and on the ground, tetanus is very
frequently met with as the result of comparatively slight
wounds, whilst amongst children during the years they
crawl: on the ground, or play in gardens or in fields,
tetanus is always more common than in later life, when: the
parts that come in contact with the ground are usually pro-
tected by shoes and gaiters. Many of these facts are well
known to savage tribes, whose powers of observation and
opportunities for experimentation are of a very high and
extended order, and we find that Dr. Ledantec, in an interest-
ing account of the poisonous arrows used by the inhabitants of
Santa Cruz, of the Solomon Isles, and of the New Hebrides,
speaking of the deaths from this cause of Bishop Patteson
and Commodore Goodenough, with their companions, refers
to the fact that they were all attacked by tetanus, or lockjaw.
He gives a short description of these poisoned arrows. They
are about three feet in length ; the shaft is made of a reed, then
comes a middle portion composed of hard wood, and lastly a
point which is usually composed of a fragment of human bone,
which is carefully sharpened to a very fine point, and is so
fixed that it readily snaps off on the slightest shock. Witha
sticky substance obtained from an incision made in the
bark of a tree, the point composed of the fragment of bone
is smeared. This fluid, on exposure to the air, becomes
thicker, and of a more viscid consistence. Thread is then
wound in a spiral direction round and round the sticky
point. A quantity of soil from the edge of a mangrove

TETANUS. 295

swamp is taken in a cocoanut shell, or some similar vessel,
and into this the arrow-head is plunged. It is then carefully
dried in the sun, after which the thread is removed, when
a roughened point covered with a film of dry mud and
dust is left. In this mud there are probably both septic
vibrios and tetanus bacilli, the former, however, are rapidly
killed by exposure to the sun, whilst the tetanus bacillus of
Nicolaier, which, as we have seen, developes a well-formed
spore at one extremity, may remain active for months and
even years, although, as the savages well know, the poison
gradually becomes more and more attenuated, until old
arrows are known to become entirely inoffensive, except as
mere mechanical weapons of warfare or hunting.

Stanley, speaking of the hunting appliances and offensive
weapons of the pigmies whom he encountered during his
African wanderings, makes a similar interesting observation,
to the effect that these small specimens of humanity use arrows
so poisoned that the slightest scratch with one of them pro-
duced one of two things, tetanus (or convulsions), which is
evidently due to the action of a poison similar to that above
described, or death, accompanied by peculiar gangrenous
sloughs at the seat of the wound, which is very probably
associated, so far as we can see, with malignant cedema or
“black quarter,” a condition that was also described by
Nicolaier as resulting from the inoculation of soil collected
from gardens and forests. These pigmies are described as
dwellers in the dense forests of that part of Central Africa
through which Stanley travelled.

LITERATURE.

The following works may be consulted :

ARLOING ET Lifton Triprer.—Arch. de Physiol. Normale et
Path., t. IIL, p. 235, 1870. _

BeELFANTI u PescaroLo.—Centralbl. f. Bakteriol. u. Parasi-
tenk, Bd. v., No. 20, p. 680, and No. 21, p. 710; Bd.
vi., No. 10, p. 283, 1889.

Bonome.—Fortschr. der Med., Bd. v., No. 21., p. 690, 1887.

Bossano.—Comptes rendus, t. Cvil., p. 1172, 1888; Re-
cherches Expérimentales sur l’Origine Microbienne du
Tétanos, 1890.

BRIEGER.—Bericht. d. deutsch. Chem. Gesellsch., Bd. xrx.

296 BACTERIA

BRIEGER UND FRAENKEL.—Berlin Klin. Woch., pp. 241,
248, 1890.

CarLE E Ratrone.—Giornale della R. Acad. de Med. di

_ Torino. March, 1884.

CLarkE.—Trans. Roy. Irish Acad. 1789.

CHANTEMESSE ET W1DAL.—Les Bactéries, Cornil and Babes,
t. 1, p. 567, et seq., 1890.

Faper (Knup).—Om Tetanus som Infektionssygdom.
Copenhagen, 1890.

Fiiigce.—Die Micro-Organismen, Leipzig, 1886, Tran.
W. Watson Cheyne, New. Syden. Soc., 1890.

Kirasato.—Kongress f. Chirurgie, 1889.

Kirasato unD WEyYL.—Zeitschr. f. Hygiene, Bd. vu, pp.
41, 404, 1890.

Kocu.—Mitt. aus d. k. Gesundheitsamte, Bd. 1., 1881.

KussmauL.—Deutsch. Arch. f. Klin. Med., Bd. 11, Heft. 1,

p. 1, 1873. :
Lepantec.—Annal de l'Institut Pasteur, t. 1v., Nov. 25,
1890.

MarsHat Hatt.—On the Diseases and Derangements of the
Nervous System. London, 1841.

Nicotarer.—Beitr. zur. Aetiol. des Wundstarrkrampfes.
Dissert. Géttingen, 1885. :

Nocarp.—Recueil. de Méd. Vét., t. 1v., No. 18, p. 617, 1887 ;
Arch. Vét., 1882.

Rretscu.—Comptes rendus de l’Acad. d. Sc., t. CVIL, p. 400,
1888. .

Rose.—Ueber d. Starrkrampf., 1870. ;

SaNCHEZ-TOLEDO AND VeEILLON.—Arch. de Med. Expérim.
Bd. 11, p. 709, 1890. :

SaxTorPH.—Clinisk Chir. 3dje. Del. Copenhagen, 1878.

Simpson, Sir J. Y.—Works, vol. 1., 1871.

Tizzoni AND CaTTANI.—Riforma Medica. April, 1889.

VaAILLARD ET VincenT.—Annal. de l'Institut Pasteur.
Jan., 1891.

VERHOOGEN ET, Baert.—Premiéres Recherches sur la
Nature et l’Etiologie du Tetanos. Bruxelles, 1890.

VERNEUIL.—Soc. de Chir., t. x1, pp. 327, 438, 1885.

WIEDERMANN.—Zeitschr. f. Hygiene, Bd. v., p. 522, 1889.

CHAPTER XVII.

DIPHTHERIA.

Diphtheria an Infective Disease—The Organism found in the False Mem-
brane in its Deeper Parts—Method of Staining the Bacillus—Characters
of the Bacillus—Involution Forms—Cultivation Methods—Appearance
of Colonies—Nutrient Media—Results of Inoculation Experiments—
Klein’s Bacilli differ somewhat from Léffler’s—Streptococci found in
Diphtheria Poison—Extreme Virulence—Resemblance to snake-bite
poison—Toxicity—Predisposing Conditions—Conditions fatal to the
Bacillus—Roux and Yersin’s Observations—Fraenkel’s Observations
—Attenuated Diphtheria Virus—Increase of Virulence.

ALTHOUGH it has long been known that diphtheria was
an extremely infectious disease, it is only within compara-
tively recent years that any reliable information as to the
nature of the specific infective poison has been forth-
coming. Even when the organic nature of other specific
infective poisons had been practically proved many difficulties
still remained to be overcome; in most other cases where
the disease could be proved to be the result of the vital
activity of a micro-organism, such organism could usually
be found in a pure condition, or greatly predominating
over all others, in the blood, in the internal organs, or in
some special fluid in the body. This was found not to
be the case in diphtheria. Most careful and elaborate
researches were entered upon, but it was found impos-
sible to demonstrate any special organism as occurring in
the blood, in the lymph, or in any of the organs of the
body. The only position in which any could be found was
in the false membranes (composed of fibrinous lymph and
_altered epithelial cells) that are found in the throat, and it
was at once surmised—a surmise that was afterwards found to
be correct—that the poison which gives rise to the constitu-
tional symptoms must be formed at the point at which the
micro-organisms are found; and that, being of an exceedingly
diffusible nature, it is thence absorbed and carried to various

298 BACTERIA,

parts of the body, giving rise to heart failure, or to certain
forms of paralysis, partly through its action on the nervous
system and partly owing to its interference with the
nutrition of the various tissues of the body. Here again,
however, those who were studying the subject were con-
fronted with another difficulty ; there were so many forms
of ‘micro-organisms that found in this false membrane a suit-
able subsoil on which to grow, it was almost impossible
with the methods then at command to separate them and so
to obtain pure cultures, in order that the specific and bio-
logical characters of each might be investigated.

In 1875 Klebs found in the false membranes a small bacillus
with rounded ends, and with, here and there, small clear spaces
in its substance, a bacillus that was not readily stained, that
grew luxuriantly in broth, and which, inoculated into animals,
gave rise to a peculiar dirty fibrinous-looking slough at
the seat of inoculation. He found, however, that in certain
cases this bacillus was absent, the predominating organism
then seeming to be a micrococcus arranged in masses or in
short chains. This, when cultivated in broth, gave rise to
the formation of chains of considerable length. As a result
of thése observations he described diphtheria as occurring in
two forms, one form resulting from the action of one organ-
ism, the second being caused by the other.

These researches were continued by other workers, and
Formad, in America, came to the conclusion that the rod-
shaped bacillus had little to do with the disease, but
that the streptococcus, or chain-forming micrococcus, was
the real exciting cause. Matters remained at this stage
for some time—in fact, until Léffler took up the subject.
After examining a number of cases of diphtheria, he found
that, although there are numerous organisms in the false
membranes or diphtheritic patches, these were mostly near
the surface, and many of them were simply the organisms
that were usually found in the mouth now growing under
more favourable conditions of nutrition. He found, however,
that in the deeper layers, or at the inner margin of the layer
of exudation, the Klebs bacillus might almost invariably be
found. It was more deeply situated than any of the others,
and was always most numerous in the oldest part of the
membrane. This was in cases of pure diphtheria, In the
so-called diphtheritic sore throats met with in other diseases,

DIPHTHERIA. 299

especially in scarlet fever, the streptococcus appeared to be
the predominant and characteristic organism.

These rods described by Klebs and Léffler vary much
in length, but they average from 3 to 6; they are straight
or slightly bent, one end or both sometimes being a little
swollen. There may be deeply stained or bright glistening
points in the protoplasm, though these are not usually met
with. Babes indeed ‘describes spores as occurring in the
diphtheria bacillus, but, as is mentioned later, these are not
real endospores but consist merely of altered protoplasm.

In order to stain the bacillus it is only necessary to remove a small
fragment of the false membrane by means of a piece of absorbent cotton
wool tied firmly to a pair of forceps or to a pen holder; from this it is trans-
ferred to a scrap of blotting-paper, and thence to a cover glass, where it is
broken down as finely as possible, heated over a flame in the ordinary
fashion, and stained with Loffler’s alkaline methylene blue, or by Gram’s
gentian violet method (washing thoroughly with water before attempting to
exe): or bya method adopted by Roux and Yersin, who use a blue, com-
posed of equal parts of aqueous solution of violet dahlia and methyl green,
with water added until a clear, but not too deep, blue is obtained. A drop
of this is placed on the slide, the cover glass on which the fragments are
dried is inverted and lowered on to it, the superfluous fluid is removed with
a piece of blotting-paper, and the organism is examined at once.

The vital characteristics of the organism may be used in separating it
from false membranes, even where contamination from the organisms of the
mouth and pharynx has occurred ; and it is recommended that in order,
wherever there is any doubt, to be absolutely certain of the diagnosis of
diphtheria, cultivations on blood serum should be made.

The specific diphtheria bacilli appear to be stained more
readily and more deeply than any of the organisms that
usually accompany them. They occur in small groups, as
short, straight, or curved rods, with ends sometimes pointed,
sometimes curved ; they are never absent from cases of true
diphtheria in the early stages, and in some cases the
membrane consists of an almost. pure cultivation of the
bacillus. In older cases, however, the organisms do not
stain so equally ; many pear-shaped and club-shaped bacilli
are present, and, in some very old membranes, it is difficult
or impossible to distinguish any characteristic bacilli, the ac-
companying organisms becoming more numerous, especially
as the surface becomes fcetid and softened. In such cases the
specific organisms can only be found entangled in the
deeper fibrinous net-work.

Roux and Yersin hold that microscopic examination gives the most precise

390 BACTERIA.

information, even in the case of dried false membranes sent from a distance
wrapped in linen or blotting-paper. They also hold that where improve-
ment is taking place, the specific bacilli become less numerous, the other
microbes increasing in number, and that this may be followed day by day
with a microscope, the course and prognosis of the disease being indicated
by the changes that are met with. They also hold that at the beginning of
a case of diphtheria it is possible to predict a favourable issue from thé
presence of a small number only of the specific bacilli, and a large number
of other forms, and they also believe that some of the micro-organisms met
with under these conditions interfere with the growth and activity of the
specific bacillus. >

The Klebs-Léffler bacillus cannot be cultivated outside the
body on peptone meat gelatine, as it will not grow sufficiently
rapidly at the room temperature to outpace the putrefactive
organisms which accompany it ; it was, therefore, found
impossible to make plate cultivations in order that the
different forms might be separated.

By mixing the membrane with boiled distilled water and
allowing drops of the mixture to trickle over the surface of
solidified blood serum, Léffler succeeded in obtaining pure
cultivations. Weyssokowicz, using agar instead of gelatine
for making plate cultivations, and incubating at 35° C., was
able, on a small percentage of his plates, to obtain pure
cultivations of the characteristic diphtheria bacillus ; most

- of his plates, however, were overgrown with putrefactive
and other non-pathogenic organisms.

To obtain a somewhat purer inoculating material, put a scrap of mem-
brane between blotting-paper, or on a cover glass in a test tube, allow it to
dry, and then heat toa temperature of 98° C. for a whole hour—a tempera-
ture which few ordinary organisms can withstand for even a less time than
this—so that, when the dried material from a cover glass so treated is
inoculated on solidified blood serum, a very pure cultivation is usually
obtained.

Roux and Yersin adopted a simpler method. They used a
platinum needle beaten out at the end to form a kind of spa-
tula ; on this they took a particle of the false membrane froma
case of diphtheria and then made stroke cultivations on the
surface of the solidified blood serum, using the same needle
without re-charging for some half-dozen tubes. When these
are incubated at from 33° to 35° C. the bacilli rapidly make
their appearance ; they are visible at the end of twenty hours
and have a characteristic appearance in forty-eight hours.
This rapid growth is very characteristic of the diphtheria

DIPHTHERIA. 301

bacillus, which appears to be the only organism of all those
found in the membrane that can form colonies visible
to the naked eye in twenty hours. Such colonies grow as
small rounded greyish white points, the centre of each of
which is more opaque than the periphery; they spread
rapidly, form greyish rounded discs, and continue to develop
so quickly that they are very evident before the other
organisms have begun to form a colony at all visible to the
naked eye. From these points, inoculations on to blood
serum may be made.

Probably the best nutrient material for the diphtheria organism is that
recommended by Léffler ; it is composed of three parts of blood serum,
one part of neutralized broth, to which has been added 1 per cent. of pep-
tone, .5 per cent. of common salt, and 1 per cent. of grape sugar. Ordinary
blood serum comes next, and then agar-agar jelly. On agar plates the
colonies situated in the substance are coarsely granular, dark brown, and
somewhat rounded or oval; although where several colonies have run to-
gether they may give rise to somewhat irregular outlines. The superficial
colonies are much lighter in colour, are not so dense, and have an irregular
scalloped border.

These cultivations are found to be made up of bacilli
similar to those described by Klebs; they are not quite so
long as the tubercle bacillus, but are rather thicker, the
extremities, which are more deeply stained than the central
portion, are often slightly enlarged. In older cultivations
the rods are not uniformly coloured, and there may be seen
in the substance of the protoplasm bodies which somewhat re-
semble spores. It was soon noticed that with this inequality
of staining, certain other changes in the organism might be
met with ; so-called involution forms make their appearance.
In these the bacilli are cut up into small rounded masses of
protoplasm, some of which have a less diameter than that of
the bacillus, whilst cthers may be considerably larger and are
oval in shape. These involution or degenerate forms soon
make their appearance where the conditions for the growth
and development of the bacillus are unsatisfactory, and it is
supposed that many of the failures to find the typical rod-
shaped bacilli in old diphtheritic membranes are due, in some
measure, at any rate, to the occurrence of these involution
forms in the later stages of the disease, where the membrane
is invaded by putrefactive and other organisms which inter-
fere with the growth of the specific bacillus. It is also

302 BACTERIA,

found that the diphtheria bacillus has a tendency to lose
its virulence on cultivation.

It was at first concluded that the bright, strongly refrac-
tile particles and deeply stained granules were more or
less perfectly developed endospores, and that some of the
involution forms might possibly be arthrospores. Although,
however, the organisms remain alive and potentially active
after being subjected to drying for a considerable time, a
moist temperature of 58° C. is quite sufficient to kill them
off, which would scarcely be the case were these bodies true
spores.

The bacillus, as we have seen, grows freely on meat fluids that have an
alkaline reaction, but as it grows it first renders these media slightly acid,
but later, if there is free access of air, the fluid again becomes alkaline.
The acid reaction is always most marked in those cases in which there is
glycerine in the cultivation medium. The organism grows ‘in vacuo,”
but more slowly than in air; the exclusion of air, however, interferes with
the formation of acid, so that under these conditions the bacillus may retain
its vitality for a period of six months, or even longer. Examined in a fluid
medium it is found to be quite motionless,

Although Léffler was able to produce some of the symp-
toms of diphtheria ‘by the inoculation of this bacillus on an
excoriated mucous membrane, he was not satisfied that this
was the real causa causans of the disease, and it was left for
Roux and Yersin to demonstrate the intimate causal relation
that actually exists between this organism and true diphtheria.
They repeated Loffler’s experiments of inoculating the bacilli
on the damaged mucous membrane of rabbits, guinea-pigs,
and pigeons, and in all cases they found that characteristic
diphtheritic patches were produced. Injected under the skin,
the bacillus causes swelling at the point of inoculation, and the
animal dies with symptoms of acute poisoning. In some cases
there is found congestion and effusion into the serous cavities ;
in others there is evidence of fatty degeneration of the liver,
similar to, but more acute than that met with in cases
of diphtheria in the human subject. A very important
point determined by them was, that if death did not take
place too rapid’y characteristic diphtheritic paralysis usually
supervened. The bacillus in these cases was found only at
the point of inoculation, and even in the most congested
organs it could not be demonstrated either in the blood
channels or in the lymph spaces, whilst it often disappeared

DIPHTHERIA. 303

even from the seat of inoculation during the latter stage of
the disease ;—another fact that helps to explain Loffler’s
inability to find the organism in certain of his cases.

From all this it is concluded that the local symptoms of
diphtheria are due to the action of a specific bacillus on a
weakened mucous membrane or on a wounded surface ; that
once having gained a footing it gives rise to an acute in-
flammatory process, probably by the direct action of the
poisonous material that it forms on the cells and on the
blood vessels in the immediate neighbourhood ; this caustic
action is so intense that the epithelial cells undergo de-
generation,—the fibrinous lymph and leucocytes which are
exuded also become more or less rapidly degenerated—and
give rise to the grey false membranous patches that are so
characteristic of true diphtheria, When the growth of the
organism is rapid, and where the area of surface attacked
is extensive, the amount of poison developed may be very
great indeed, and where this latter is greater than can be
dealt with in the inflammatory area, owing to the rapidity
with which it is produced by a large number of organisms,
especially when they are situated deep down in the
tissues, there is rapid absorption of the poison, but not of
the bacilli, into the system, and the characteristic constitu-
tional symptoms of the disease are set up. We must thus
distinguish carefully between the local action of the bacillus
and its products, and the toxic constitutional effects of these
products.

Additional proof that these products are the active agents in
the causation of the disease was found in the fact that from
pure cultures of the diphtheria bacillus there may be separated,
by means of Chamberland’s porcelain filter, a special chemical
substance, or series of substances, which, after being proved
quite free from bacilli and injected under the skin of animals,
gives rise to all the constitutional symptoms and lesions of
the disease that follow the inoculation of the bacillus itself,
the only feature wanting being the false membrane, which
usually does not make its appearance. Animals into which
small doses are injected are frequently attacked by diph-
theritit paralysis.

Of course it has been objected that the diphtheria pro-
duced in animals is not necessarily the same thing, nor is it
necessarily due to the same organism, as the diphtheria of

304 BACTERIA,

man. Léffler himself has examined the diphtheria of a
calf, and he acknowledges that it is not even related to the
diphtheria of the human being.

Klein considers that cats are especially susceptible to
diphtheria, and that they may act as the intermediate hosts
for the development of the diphtheria bacillus between two
human patients, whilst he also holds that cows may be
inoculated, especially on the udders, with diphtheria bacillus,
the disease manifesting itself in the form of small pustules.
He considers that milk from such cows may readily become
the agent by means of which the disease is spread. Eminent
veterinarians, however, are strongly opposed to this view,
and it can yet scarcely be maintained that Klein has fully
proved his point, although he seems to have obtained strong
evidence in support of his position. As he is now working
most carefully at the subject, however, it may be well to
suspend judgment until he publishes a full report of his
observations and experiments.

Klein’s bacilli, however, undoubtedly differ in certain

‘ essential points from the bacillus described by Loffler ; most

important of all in the fact that whilst Loffler never suc-
ceeded in obtaining growths of his bacillus at the ordinary
temperature of the room, and was not able to grow it in
gelatine, those that Klein describes as occurring in the
pustules on the ulcerated udder of the cow grow luxuriantly-
in gelatine at the ordinary temperature of the room.

Klein describes a second bacillus as growing slowly upon
gelatine, but this he does not consider to be so important as
the more rapidly growing one.

Young rabbits and guinea-pigs are the animals with
which most experiments have been made. It has been found
that rats and mice enjoy almost perfect immunity from
the disease, a fact which has recently been utilized by
Behring in connection with the production of an immunity
against diphtheria in rabbits and guinea-pigs.

We shall have to wait for further light on these points,
but from what we have already seen, young rabbits, guinea-
pigs, and young dogs, may undoubtedly contract diphtheria,
or something very similar to that disease ; both local and
toxic symptoms resulting from the introduction- of the
poisonous material. It must be borne in mind that there
are usually several kinds of bacteria present, that the compli-

DIPHTHERIA. 305

cations of the disease are numerous, and that it undoubtedly
occurs as a complication in other diseases. But when all this
is taken into consideration the fact must still be accepted
that careful clinical observation and experimental investi-
gation have been made by many thorough workers, and that
these workers have assigned to a special bacillus the power
of giving rise to at least one form of diphtheria. It is
possible that the streptococcus may play an important part
in certain cases of diphtheria, but this has not yet been
actually proved ; whilst the evidence in favour of the specific
infective Klebs-Loffler bacillus is now almost overwhelming.

As regards the streptococci that are found in this disease, it appears that
these organisms so fully described by Klebs, Formad, and Prudden and
Northrup, and later by Léffler and Babes, when inoculated give rise to
local inflammation, and.even to inflammation of joints, &c.; in no case,
however, do they appéar to give rise to the formation of a false or a fibrinous
membrane. No doubt they play a part in the preparation of the tissues
for the diphtheria by weakening them so as to enable them to offer less
resistance to the action of. the specific organism. These streptococci
may actually make their way into distant organs of the body, and it
has been found possible to make pure cultivations of them from such
organs. These, then, differ very markedly, as regards their distribution in
the body, from the diphtheria bacillus, which grows only at the point of
inoculation, manufactures all the poison in that position, the poison only
being carried.into the body; the bacilli remaining z# sétw, multiply
during the advance of the disease, and degenerate as soon as the tissues
‘begin to obtain the upper hand, which they do in all cases where recovery
occurs, and even in some cases where the patients afterwards die from the
effects of the absorption of the specific poison.

The diphtheria bacillus, having been obtained pure, was
naturally seized upon by Loffler, then by Roux and Yersin,
and, lastly, by Brieger and Fraenkel for their experiments on
toxalbumens ; the way having been paved somewhat by
Hankin’s discovery of albumoses in certain anthrax culti-
vations.

The diphtheritic poison is always most active when alka-
line ; during the acid period already mentioned, the toxic
power is very considerably diminished. Moreover, if an
acid be added to a virulent alkaline filtered liquid, its
poisonous activity is immediately diminished ; but, curiously
enough, this property is immediately regained when the
fluid is again neutralized by the addition of a fixed alkali.
So virulent is the alkaline liquid that one-fifth of a cc. of ’
the filtered fluid, from a cultivation of the diphtheria bacillus

21

306 BACTERIA.

that has been allowed to grow for forty-two days, proves
fatal to a guinea-pig in about thirty hours. Larger doses,
from 4 to 20 cc. injected into dogs of: various sizes kills
them in from fourteen to twenty-six hours. In doses of
2 cc. it proves fatal in four to six days. In all cases the
symptoms are those of more or less acute poisoning, re-
sembling septic poisoning in some respects and phosphorus
or metallic poisoning in others. <A dose of less than I cc.
of the filtered liquid injected into a dog of middle size causes
a temporary paralysis, very similar to the post-diphtheritic
paralysis of the human subject. It is a curious feature that
when paralysis occurs in a rabbit, death invariably ensues ;
but in both the pigeon and the dog, especially in the latter,
recovery may frequently follow this condition just as in the
case of the human being. Sheep are susceptible to the
action of diphtheritic poison, but rats and mice are unaffected
by it. The effects on other animals have been already
mentioned. a

It is always a difficult matter to determine what is the
nature of an organic poison. In the first place it is produced
in such small quantities that it is difficult to obtain
sufficient to determine, even by chemical analysis, its exact
nature. Further, it is so unstable that it may become
completely altered by the various reagents that have
to be used in separating it out from the mixture in
which it occurs. A rise of temperature beyond a certain
point is fatal to its activity, and it undergoes various oxida-
tions under the least provocation. Roux and Yersin consider
that this special poison has certain features in which it
resembles the diastases. Thus, when heated in sealed
tubes over a water bath to 58° C. for a couple of hours,
the toxic activity is diminished at least seven-eighths of its
original power ; whilst very large doses of the filtered poison
that has been heated to 100° C. may be introduced into the
veins of a rabbit, or under the skin, without producing any
immediate effect, although symptoms are produced later
which can only be due to the action of this material as a
modified diphtheritic poison. Like diastase, the diphtheritic
poison is rapidly modified by sunlight in presence of air, but
if air be excluded the diminution in toxic activity brought
about by exposure to sunlight is comparatively slight.

On evaporating a filtered diphtheritic bacillus culture 2”

DIPHTHERIA. 307

vacuo over sulphuric acid at a temperature of 25° C., a sub-
stance is left which is soluble in water, and possesses marked
toxic properties. It is insoluble in strong alcohol, and may
be precipitated by it from a watery solution in greyish white
flakes. It passes slowly through a dialyzing membrane.
The addition of lime water and a solution of phosphoric
acid to the filtered culture liquid causes an entangling
precipitation of the poison, phosphate of lime appearing to
hold it more tenaciously than any other substance. The
filtered fluid thus treated loses its toxicity, whilst the gela-
tinous precipitate inoculated into an animal kills with the
utmost certainty, though perhaps on account of the slightly
insoluble nature of the substance formed, somewhat more
slowly. To form some idea of the virulence of the poison
produced by these diphtheritic organisms, Roux and Yersin’s
calculations that 1 cc. of the active liquid evaporated
wm vacuo leaves 1 centigramme of dried residue, may be
accepted. Deducting from this the weight of the ash and
the portion soluble in alcohol which has no toxic action,
there remain four-tenths of a millegramme of organic
material, of which only a small proportion can be diph-
theritic poison ; even this quantity, however, is sufficient to
‘kill eight guinea-pigs, two rabbits, or one medium-sized dog.
If the latter does not succumb to the poison it remains ill
for some time. Like snake-bite poison, however (which
Waddell has. shown to be weakened on being exposed to
peptic digestion, a change that is ascribed to the breaking
down of the albumose), it may be taken into the stomach
in much larger quantities without giving rise to any very
serious effect. This poison, when injected into the veins
or into the subcutaneous tissue, appears to act specially
on the walls of the blood vessels, giving rise to vascular
dilatations, minute hemorrhages, and the small cedematous
patches so characteristic of certain forms of the disease.
It can scarcely be too strongly insisted on that the activity of
a poison formed by a micro-organism is not at all the same
thing as virulence, which must be defined, according to
Roux and Yersin, as the power that a pathogenic organism
possesses of continuing to live and carry on its functions in
the tissues of the animal or human host. For example, one
might take a young culture of the diphtheria bacillus in which
the bacilli are vigorous, but in which the quantity of poison

.

308 BACTERIA,

developed is as yet small; inoculating this into an animal it
would be found that death would take place at the end of a
certain period, and that, on examination at the point of
inoculation, numerous bacilli, evidently the result of vege-
tative growth of the micro-organism within the tissues at
this point, might be demonstrated ; whilst on the other hand,
if an old cultivation (in which the organisms are weak, and
in which many involution forms are present, but in which
there is a large quantity of poison which has been developed
by the micro-organisms during their period of activity, and
which from standing has become diffused into the liquid) be
inoculated, death will be produced very rapidly, but no
organisms can be found at the seat of inoculation ; there is
set up, in fact, a true toxic condition. If the residues left on
the filter from the above cultures were inoculated, it would be
found that in the case of the young culture, death of the
animal would take place at very much the same period as
when the unfiltered culture was inoculated ; whilst in the
case of the old culture the period at which death takes place
is very much delayed; in the one case, the organisms
are active, although deprived of the poison which they form ;
they can live in the tissues, and can produce fresh poison ;
whilst the older organisms and involution forms, no longer
able to develop in the tissues, and deprived by the filtration of
the poison they produced whilst they were active, sometimes
do not cause the animal to succumb even at a late date.

This virulence, then, is associated with the power of a microbe to-develop
in the body of a living animal, a power which may be considerably increased
by passage of the organism through a series of suceptible animals, the
bacillus acquiring a more and more parasitic habit in each successive host.
Researches on micro-organisms are of no value to medicine unless they
throw light directly or indirectly on the cause of disease, and so enable
the physician to combat its advance. At first sight it would seem that in
the case of the diphtheria bacillus there is, on account of the extreme activity
of the poison, little hope of rendering the tissues of an animal resistant to
its action, as even very minute doses produce marked poisonous effects. On
the other hand, however, we have, from the nature and position of the
development of the poison, indications as to treatment and also as to
prevention of the disease.

The indications as to prevention are of course similar to
those for other micro-organismal diseases ; if we know the
natural history of the diphtheria bacillus, we know at what
point it is most vulnerable, the conditions favourable for its

DIPHTHERIA, 309

development can be removed, and unfavourable conditions
substituted ; whilst, as regards treatment, it is evident that,
because of the energetic toxic action of the material formed
by the organisms, diphtheria should be attacked as early as
possible. If sufficient time be allowed to the bacillus to
form a large dose of the poison, it is useless to remove the
false membranes, as, though the bacilli may be then destroyed,
sufficient poison may have passed into the system to cause
the death of the patient, “for in diphtheria, contrary to what
occurs in most other infective maladies, the infection is not
produced by the invasion of the tissues by a microbe, but
by the diffusion through the organism of a toxic substance
prepared on the surface of a mucous membrane altogether
outside the body, so to speak.”

The bearing of recent researches on the prevention of
the spread of an outbreak of diphtheria can only be fully
understood when some of the facts that they brought to
light are enumerated. It was found, for instance, that the
presence of the diphtheria bacillus in the mouth is not
necessarily followed at once by the appearance of the diph-
theritic membrane, and it appears that these bacilli can exert
little or no injurious effect where the mucous lining of the
throat, larynx, &c., remains sound and unaffected by minor
diseases. When once, however, we have such conditions as
inflamed tonsils or inflammation and ulceration of the mucous
membrane, the diphtheria bacilli find a soil ready prepared
for their reception, and typical diphtheritic symptoms are the
result. That such ulcerated sore throats, inflammation of the
tonsils, and similar conditions usually precede outbreaks of
diphtheria, has for long been a well recognized clinical fact ;
these experiments give the explanation of it, whilst they also
afford indications as to the mode of treatment. Antiseptic
throat washes, not merely gargles, plenty of fresh air, and good
nourishing food, are what are required. Kill the germs as
far as possible by means of the antiseptics, and strengthen
the tissue cells by plenty of oxygen, and by promoting the
excretion of effete products, by food and exercise, so that the
cells shall be able to form their protective products and shall
also be able to play their part as phagocytes when called
upon to do so. Another important point to be borne in
mind is that the disappearance of the bacilli from the mouth
is not simultaneous with the removal of the false membrane,

310 BACTERIA,

and Roux and Yersin have found that the specific bacterium
may persist in the mouth for several days (in one case four-
teen days) after all traces of the membrane have disappeared,
and they give the good practical advice that diphtheritic
patients who are becoming convalescent should not be
allowed to associate with their school-fellows, play-mates or
families, for at least a fortnight after the membrane has
disappeared ; and that it is quite as important to wash out
the throat freely three or four times a day with disinfecting
lotions as that the clothes and bed linen should be
thoroughly disinfected. As regards the tenacity of life
exhibited by these bacilli, it is found that at the ordinary
temperature of the room these organisms retain their
vitality for a period of at least six months, and pro-
bably considerably longer. As the temperature rises this
period is gradually diminished, for we find that at 33° C.
the organisms succumb in about five months; whilst at
39° C. they are are found to be no longer capable of living
and of multiplying when introduced into solidified blood
serum or glycerine-agar. When deprived of air and pro-
tected from the light, even when kept at the ordinary
temperature, they may continue capable of germinating,
when introduced on to a suitable soil, for a period of
thirteen months. If they are dried and are kept at the
temperature of the room, they are killed in four months, at a
temperature of 33° C. they are killed in three months, and
at a temperature of 45°C. in four days. If a fragment of
the false membrane containing bacilli be removed, wrapped
in sterilized paper, or linen, and be carefully protected from
the action of light, cultivations may be made from it at any
time during a period of five months. If, however, instead of
keeping it dry and in the dark, fragments of these mem-
branes are exposed to the light and moistened and desiccated
alternately, the virus is destroyed much more rapidly. From
all this, and from the fact that the bacillus is destroyed by
moist heat at 58°C., it is evident that by far the best method
of disinfecting clothes, the floor, the walls, and furniture, is
by the use of a liberal supply of boiling water ; for although
a temperature of 98° C. (dry), continued for an hour is
necessary to destroy the vitality of the bacillus, moist heat
at a very much lower degree (acting only for a minute
or two, according to the temperature) is sufficient to dis-

DIPHTHERIA, 3rl

infect everything on which it is allowed to act. In the case
of the cholera bacillus we have already pointed out that
under certain conditions it is capable of producing a much
more violent form of disease than in others.

Roux and Yersin have been able to demonstrate that the virulence of the
bacillus of diphtheria undergoes marked modifications even during the
progress of an attack of diphtheria in the same individual, and they believe
that the condition of the patient is modified not only by the alterations in
the number of the bacilli, but also by their virulence at different stages.
They find, for example, that in the early stages of the disease (except in
very rare cases) the number of bacilli is comparatively small, but that as
the disease advances the number of virulent bacilli present also increases
rapidly, this being tested both by microscopic examination and by cultiva-
tion and inoculation experiments ; whilst, on the other hand, as the case
approaches cure the bacilli that can be isolated are not only fewer in
number, but those that are cultivated are not nearly so active, for when
inoculated into animals they produce neither such marked constitutional
symptoms nor such severe local reactions. It is indeed believed that
the virulent and non-virulent bacilli represent a difference in degree of
virulence only, and not a morphological or specific difference, that the
difference is one of degree and not one of kind, and that the pseudo-
diphtheritic bacillus described by Léffler and Hoffman is really only the
organism undergoing a kind of saprophytic phase which is interpolated in
the life of the parasitic bacillus.

Roux and Yersin obtained an imperfectly attenuated virus first by
keeping the dried membrane for a considerable time, when they found
‘that cultivations from them of bacilli, in which were present all the typical
appearances and morphological characteristics, when inoculated into
animals had lost their virulence. They found that they were able to
diminish the virulence of the diphtheritic organisms growing in broth
by passing currents of air at 39.5° C. through this medium, and that if
the process was continued too long the bacilli were completely deprived
of life. It would appear that this was simply an interference with the
vitality of the organism which was deprived of one function after another
until it was killed altogether. They found it impossible, however, by
these methods to graduate the attenuation, and although they proved
that the virulent bacillus alone could elaborate the toxic material, and then
only under favourable conditions, the virulence became modified as the
conditions were altered, the activity of the toxine being also modified ;
these conditions could not be controlled except in a very rough and in-
adequate fashion.

Recently Fraenkel has found that by heating cultures of
the diphtheria bacillus to a temperature of 65° or 70° C. and
injecting from 10 to 20 cc. of such cultures into guinea-pigs,
after an interval of fourteen days subcutaneous injections of
even the most virulent diphtheria poison had no effect. If
however, the animals were again injected within the
prescribed period of fourteen days they succumbed to the

312 BACTERIA.

diphtheria poison. On the other hand, the diphtheritic virus
applied to an abraded mucous membrane even after fourteen
days was capable of producing typical local symptoms. These
points, if confirmed, indicate that the immunity against
diphtheria may not be so easily acquired as in some other
diseases, or, if acquired, it must be through the application
of methods different from those hitherto described.

Another feature that is brought into special prominence
by the recent researches on diphtheria is, that the attenuated
diphtheria bacillus requires the fulfilment of certain con-
ditions before it can again acquire the virulent form, one of
these being that it shall be allowed to grow on the surface of
the fauces, or outside the body altogether, in the presence
of certain organisms, such as the streptococci found in
erysipelas, or those streptococci that occur in the throat
affections of scarlet fever, measles, and similar diseases. It
is quite possible that other organisms have the same effect,
and that the attenuated diphtheria bacillus growing outside
the body may become so virulent that it is capable of pro-
ducing a very grave form of diphtheria, but from what
occurs in outbreaks of diphtheria—in which the first cases, as
a rule, are mild, successive cases becoming gradually more
and more severe until an extremely fatal form of diphtheria
attacks susceptible individuals—it would appear that the
growth on mucous surfaces of this bacillus, along with these
streptococci, is specially favourable to the development of the
virulent form of the organism. :

. These experiments exemplify in a most remarkable manner the use that

bacteriological investigations have been to medicine, and Roux and Yersin
sum up the practical outcome of their researches as follows :-—‘‘ The best
method of arresting the spread of diphtheria is to recognize the disease
as early as possible; consequently a precise diagnosis should be made: by
microscopic examination of the false membranes, and this should be con-
firmed by cultivations on blood serum.” As the former takes only a few
minutes, and as the latter gives results in twenty-four hours, both these
methods are available in private practice or where patients can be sent to
an observation ward.

‘¢ Active diphtheritic virus can remain in the mouth for a long time after
the malady is cured. Consequently diphtheritic patients should only be
allowed to resume their ordinary mode of life when they are no longer
bearers of the bacillus.

‘‘ Diphtheritic virus retains its virulence for a long time when kept ina
dried state. It is therefore necessary to disinfect in a steam sterilizing
apparatus the linen and all articles that have been in contact with diphtherj-
tic patients.

DIPHTHERIA. 313

‘The attenuated virus of diphtheria is widely distributed and it readily
regains its virulence. It is therefore necessary at the very commencement
of simple forms of throat disease, and of those associated with measles
and scarlatina, to practice careful and frequent swabbing of the throat with
antiseptics.” :

LITERATURE.

Works that may be consulted :

Bazses.—Virch. Arch., Bd. cxrx., Heft. 3, p. 460, 1890.

BEHRING AND Krrasato.—Deutsche. Med. Woch, Bd. xvi,
No. 49, p. 1113, Dec., 1890.

BRIEGER AND FRAENKEL.—Berlin Klin. Woch., Bd. xxvil.,
No. 11, p. 241, and No. 49, p. 1133, 1890.

CHANTEMESSE AND WIDAL.—Semaine Med., May 14, and
Bull. de l’Acad. de Méd., June 5, 1890.

GAMALEIA.—Annal. de l'Institut Pasteur, p. 609, 1889.

GRANCHER.—Bull. de l’Acad. de Méd. 1890.

K.ess.—Beitrage zur Kenntniss der pathogenen Schizomy-
ceten ; Archv. f. exp. Path. u. Pharm, Bd. rv., Heft.
I, 2, p. 107, and Heft. 3, p. 207, 1875; Verhand. des
Congresses f. inn. Med., 11., Abtheilung, Wiesbaden, p.
143, 1883

Krerw.—Centralbl. f. Bakt.u. Parasitenk, Bd. vir., No. 25,1890,

Kouisko anD Pattaur.—Centralbl. f. Bact. u Parasitenk,
Bd. v., No. 22, p. 735, 1889.

L6rFLer.—Mittheil. a. d. k. Gesundheitsamte, Bd. 1, p.
421, 1884; Centralbl. f. Bakt. u. Parasitenk, Bd. 11., p.
105, 1887; Deutsch. Med. Woch., Bd. xv1, No. 5, p.
81, and No. 6, p. 108, 1890. |

OrrtaL.—Deutsche. Arch. f. Klin. Med., Bd. vin, Heft. 3,
4, Pp. 242, 1871. :

PrRuDDEN AND NortHRup.—lInternat. Journ. of Med. Sci.
Vol. xcvut., p. 562, 1889.

Roux anp Yersin.—Annal. de l'Institut Pasteur, t. m., No.
12, p. 629, 1888 ; t. m1, No. 6, p. 273, 1889 ; t. 1v., No.
7) P- 385, 1890.

Rurrer.— rit. Med. Journ., p. 202, July 26, 1890.

TaLamon.—Progrés Médical, pp. 112, 498, 1881.

WanbbELL.—Scien. Memoirs by Med. Officers of the Army
in India, Part Iv., p. 47, 1889.

Woop anp Formap.—JMed. Times and Gazette, Phil., Dec
4, 1880 ; Bull. Nat., Bd., Health, No. 17, 1882.

ZARNIKO.—Centralbl. f. Bakt. u. Parasitenk, Bd. vr., No. 6,
p- 153, No. 7, p. 177, and Nos. 8, 9, p. 224, 1889,

ny

CHAPTER XVIII.
HypDRopHosiA.

Pasteur’s Experiments—Attempts to Demonstrate Micro-organisms—
Hydrophobia does not arise Spontaneously—Disease not Confined to
Man or Canine Animals—Pasteur’s Early Experiments with Saliva
Unsuccessful—Successful Experiments—Symptoms of the Disease—
Position in which Virulent Material is Found—Different Animals
Differently Affected—Alteration of Virulence—Method of Preparing
Inoculation Material—Description of Experiments—Joseph Meister
the First Patient Treated—Method of Treatment now Adopted—
Treatment of Wolf Bites—The Time Factor in the Disease—Rationale
of Inoculation Method—Filtered Virus Non-Virulent—Method of
Inoculation—Description of Departments, Apparatus and Methods of
Working in the Pasteur Institute.

By some it may be objected that in a work on micro-organ-
isms the subject of hydrophobia can scarcely be legitimately
considered. When it is remembered, however, that the
methods adopted and the principles involved in the study of
the production of this disease are in many respects the same
as those concerned in other diseases in which bacteria
undoubtedly play the part of causal agents, and when, too,
it is remembered that Pasteur’s researches on the productian
of immunity against the attack of this disease were carried
on under the full conviction that in hydrophobia the excit-
ing cause is a poison, the most probable source of which is a
vegetable organism, one has, I think, ample excuse for the
introduction of this subject. When further we take into
consideration the extreme interest of the subject, both from
a scientific and from a practical point of view, any remaining
objection should at once fall to the ground. To treat the
subject in a purely historical or chronological order, it would
be necessary, in the first instance, to describe Pasteur’s experi-
ments ; but in order to clear the ground it may be well to
give a short résumé of the attempts that have been made
to find, cultivate, and inoculate a specific organism that might
be causally associated with rabies, Pasteur, Chamberland,

HYDROPHOBIA. 315

and Roux all made most careful search for organisms in the
various tissues of animals affected by hydrophobia, and,
although they at first imagined that they had been successful,
they ultimately came to the conclusion that the small round
micrococcus-like bodies that they found were not associated
with the disease or had only an indirect relation: to it. In
1884 Gibier was able to demonstrate in the medulla of
animals suffering from hydrophobia small rounded refractile
bodies, which looked like micrococci aggregated into small
masses. He demonstrated their presence by mashing down
the medulla in sterilized distilled water, and then adding
two or three times the quantity of water, and examining
some of the opaque fluid thus obtained, under the microscope.
Those organisms which remained in fragments of the cere-
bral substance were perfectly motionless, whilst those that
were free in the fluid exhibited a certain motility. No such
bodies could be obtained, by a similar method of treatment,
from healthy brains. Although these bodies resembled
micro-organisms in many respects, they could be only faintly
tinged with the aniline colours, and it was never found
possible to make cultivations into any nutrient media with
which experiments were made. In 1885 Fol brought before
the French Academy of Sciences some observations made on
sections of the cord taken from rabid animals prepared by
Weigert’s hzematoxyline method.t

He found in specimens so prepared, small granules re-
sembling micrococci, not only in the lymph spaces of the ~
neuroglia but even between the axis cylinder and its medul-
lary sheath, the cavities in some cases being of very consi-
derable size, and containing these small granules in large
masses. Each granule is perfectly distinct and takes on a
deep violet stain, they are about .2# in diameter, are some-
times arranged as diplococci, but seldom or never in chains
of any considerable length. Fol believed that he was
able to cultivate these organisms, but only directly from
the cord itself, and he describes a slight cloudiness of the
medium (broth) as making its appearance after the intro-

* Weigert’s method. Cords hardened in bichromate of potash and then
transferred to sulphate or acetate of copper solution. Sections coloured with
solution of 1 part heematoxyline crystals, 90 of water, and 10 parts of
alcohol, then decolorized by a solution of 2.5 of ferrocyanide of potassium,
2 parts of borax, and 100 parts of water.

316 BACTERIA.

duction of small fragments of the spinal cord. This cloud
is thrown down at the end of the fourth day, and the pre-
cipitate when inoculated into a healthy animal produces a
modified form of hydrophobia, modified, however, only in that
there is a longer period of incubation than in the ordinary
disease. He found on treating a thin film of this fluid, dried
on a cover glass, as he had treated the sections of the cord,
that small groups of micrococci might be demonstrated, and
he concluded that he had cultivated the specific organism.

Cornil and Babes appear to doubt the accuracy of these
observations ; they have never been able to make out any
other granules than those which are invariably met with in
sections of healthy nerve tissues that have been stained by the
Weigert method. Babes, however, claims that he has been
able to demonstrate in the brain and in the cord of rabid
animals groups of rounded micro-organisms, with a diameter
three to four times as great as that of the organism described
by Fol; these are stained 2 sztz by Loffler's alkaline
methylene blue solution, which gives them a peculiar rose
colour.

They may be cultivated on blood serum at a temperature
of 37°C., on agar-agar, and upon nutrient gelatine made with
an extract of the brain of a rabbit. In cultivations the
organism grows slowly, appearing as a faint grey spot at the
end of several days. The growths spread best in the deeper
part of the gelatine. A pure culture of the second or even
of the third generation when inoculated into animals occa-
sionally produces hydrophobia, but in most cases the cul-
tures have no pathogenic properties, and it must therefore
be concluded that the microbe has either lost its virulence,
or that it is not the actual cause of the disease. From what
has been learned of the causation of other infective diseases
it is quite conceivable that there exists in addition to this
micrococcus some hitherto undemonstrable element, which
along with the organism is capable of producing the disease.
For example, in preparations of the brain and of the cord,
another microbe is also occasionally met with. This second
special micrococcus can only be coloured by using Gram’s
staining method, and by leaving the sections for a consider-
able length of time in the staining reagent. It affects speci-
ally the surface of the brain, and is there found in cells
which “ frequently also contain fatty and proteid granules.”

HYDROPHOBIA. 317

These latter correspond to some of the cocci described by
Gibier.

From Babes’ description of the larger organism growing in the deeper
parts of the gelatine, and also in vacuo, it is evident that the organism is
anzrobic ; other organisms (micrococci) grow on the surface in the form of
grey, yellowish, dry streaks, when they are in the form of large cocciarranged
in chains, or as curved spindle-shaped bacilli. In all these forms the
micrococci or rounded points within the bacilli are stained pinkish with
Loffler’s methylene blue; whilst the intermediate substance and the bodies
of the curved bacilli take on ablue tinge. Similarly, spindle-shaped curved
organisms have been found in the cerebral substance of rabbits and guinea-
pigs suffering from rabies; whilst curved thick motile bacilli have been
found in the blood of rabbits during the stage of fever that precedes the
regular nervous symptoms of rabies; the inoculation of such blood, how-
ever, seldom produces rabies; in fact, blood from almost any rabid animal
a few or none of the symptoms of ordinary rabies. Ptomaines

ave been described as present in the nerve centres taken from rabid
animals, and it is claimed that with the separated poisons, symptoms
analagous to those of true hydrophobia have been produced, but the experi-
ments, although probably accurate, are as yet too few in number to allow
of their being very seriously discussed.

It is, however, doubtful whether the real causal agent has yet been
observed. It appears to be quite as probable that one of the lower animal
protozoa, coccidia, psorosperms or the like, may be the real cause of the
disease as that a bacterium stands in this relation to hydrophobia. In
the same way it has been suggested that the acute exanthemata from
which organisms have not as yet been in most cases successfully cultivated
may have a similar causa causans.

It was very early determined that hydrophobia or rabies
never arises spontaneously ; the actual date at which the
implantation of the disease took place could easily be traced,
as it was found that in the human subject the outbreak of
the disease always bore a definite relation to some such
injury as the bite of a rabid dog, wolf, or cat, or it might be,
to the licking of an abraded surface by an apparently
healthy animal, especially a dog which afterwards was known
to develop symptoms of hydrophobia.

This disease is not confined to man and the animals men-
tioned. It has been observed that rabbits, deer, guinea-pigs,
and even horses may be similarly affected. Although. the
disease had been described most carefully from its clinical
point of view, and although the saliva of a rabid animal was
supposed to contain a specific infective material which was
probably the cause of rabies, Hydrophobia or Rage, it was
not until 1880 that Pasteur set himself to study the virus of
this terrible disease. His first experiments were made with

318 BACTERIA.

the saliva from a child in whom the disease was developing
as the result of the bite of a mad dog. He took a little of
the saliva from this child and introduced it into a small
pocket under the skin of a healthy rabbit ; he found the
animal dead at the end of a couple of days. Taking some
of the saliva of this animal, he treated another rabbit
in a similar manner with a like result, but he also dis-
covered that the blood of the first rabbit when introduced
under the skin of another rabbit also produced the disease
in a most virulent form, so virulent indeed that the old
question of septic versus other specific infective organisms
again cropped up, and it was suggested that the animals
had died of septic poisoning due to the presence of ordinary
septic organisms in the saliva, and therefore in the blood
of the animal that had first been inoculated with the
saliva. The fact, also, that the symptoms of hydrophobia in
many cases resemble so markedly those of another form of
blood poisoning—tetanus or lockjaw—and the extremely
rapid course that it ran in the animals inoculated, made it
a matter of extreme difficulty to determine the exact nature
of the symptoms, and rendered it impossible for Pasteur to
say whether he was dealing with hydrophobia or with some
other form of specific infective poisoning. He found, how-
ever, that it was speciallyin the later stages of the disease
that hydrophobic symptoms became tetanic in character.
After the bite there may be no symptoms at all for a month
or six weeks, or even in some cases for twelve months, during
which time the poison lies latent in the system, or though
active, gives rise to no symptoms. This period is known,
technically, as the period of incubation. At the end of this
incubation period the wound first of all becomes slightly
uncomfortable ; there is itching, and the heat becomes almost
intolerable, especially as this is usually accompanied by a
sharp stinging pain ; the patient becomes feverish and very
thirsty ; the face is pallid and has a peculiar anxious expres-
sion, the muscles of the face being drawn and restless, and
gradually this expression amounts to one of actual terror or
horror. On the second or third day the patient becomes
much more excited, is restless in every sense of the word,
and a very peculiar feature is that he has a characteristic
habit of giving a suspicious side glance as though con-
stantly looking out for some hidden danger ; then as the

HYDROPHOBIA. 319

fever advances a rambling delirium supervenes ; the thirst
increases, but along with this there is great difficulty in
swallowing—especially fluids—and after making one or
two attempts to swallow, the very sight of water suggests
such horrors that thirsty as the patient is, he is anxious to
avoid it. Then muscular tremors are noted, these become
more and more marked and violent spasms are easily
stimulated as in tetanus. A sharp sound, a touch, a bright
light, or even a breath of air, may give rise to violent muscular
convulsions, and eventually the patient is slowly suffocated
as in tetanus. One-can well imagine that to a man of
Pasteur’s temperament and mode of thought such a terrible
disease as that, the symptoms of which I have just sketched,
would just be the subject that would fascinate him. Here was
a problem surrounded by difficulty but of such a character
that the very difficulties invited success; whilst if success
were attained he would have the satisfaction of feeling that
he had done something more to alleviate human suffering—
more even than he had already accomplished ; hence the
preliminary experiments we have already mentioned. In
order to convince himself that the disease which he had
produced in the rabbit was really hydrophobia he must
obtain the poison in a perfectly pure condition ; as he had
already found that Galtier’s experiments as regards the blood
of rabid animals were incorrect, he determined to repeat the
same experiments with fluid taken from the cavities of the
brain and spinal cord; here again he was successful, for
although Galtier had been unable to produce the disease
with such fluid, Pasteur found that by taking a few drops of
the cerebro-spinal fluid and introducing it under the outer
membrane of the brain (chloroforming a rabbit and removing
a small round disc of bone from the skull cap, then replacing
the bone and stitching up the wound, which healed almost
immediately) hydrophobic symptoms were rapidly developed.
This fluid from the central nervous system of a hydro-
phobic rabbit contained no septic organisms, and as the
disease was rather more slowly developed after the inocu-
lation of such virus (although it could be induced with
absolute certainty) than when saliva was used, he discontinued
the use of the latter, the inoculations being now made from
animal to animal and always with the cerebro-spinal fluid.
Fragments of the brain and spinal cord introduced under the

320 BACTERIA. .

dura mater (the outer skin or covering of the brain) produced
similar effects. Nerves were also found to contain the virus,
but the saliva, as we have seen, and the salivary glands
introduced in the above manner produced a much more
virulent and rapid form of the disease than the other tissues
and fluids mentioned: quite as virulent a form, in fact, as
when the animal was actually bitten by a rabid animal.
Finding that these tissues and fluids taken from a rabid
animal varied in their virulence, and knowing that in the
case. of anthrax virus the virulence may be diminished or
increased by inoculating into an animal of another species,
he made another series of experiments, as a result of which
he found, that although virus taken from similar positions,
‘say, the cerebro-spinal fluid, had always the same action in
the same species, when the fluid was taken from an animal
of a different species it was weaker or stronger as the case
might be. Thus in a dog the virus is of constant strength,
and inoculations made from dog to dog kill the animal with
the same incubation period, the same symptoms, and practi-
cally in the same time. When inoculated from the dog to
the monkey, however, the virus becomes less virulent ; it
is said to be attenuated or weakened, the attenuation be-
coming more and more marked in successive inoculations
from monkey to monkey ; the course of the disease becomes
longer and longer, until eventually there may come a time
at which the virus, when introduced under the skin or into
the cranial cavity, is not sufficiently active to cause the death
of this species. If this attenuated fluid be now inoculated
into a rabbit, a dog, or a guinea-pig, it still remains com-
paratively weak for a time, through successive inoculations
on these animals—z.e., at first it does not kill, then it kills,
but only after a considerable time; but gradually the
virulence returns, until at length it reaches its original level
of malignancy ; whilst, if the successive inoculations are
made in rabbits with primary fluid from either the dog or
the monkey, the virulence may become so exalted that it is
considerably greater even than that of the virus taken from
the street dog, which at one time was supposed to be the
most virulent form except that of hydrophobic wolves, which
has always been known to be specially fatal ; the virulence

is doubled as the inoculation period is reduced to about one
half.

HYDROPHOBIA, 321

Having found, then, that the virus could be intensified or
modified, and that different animals were affected in a
different degree by the same virus, Pasteur set himself to
work out the question whether it was possible so to alter the
resistance of an individual that a virulent hydrophobic
material would have little or no effect on the tissues. The
only way in which this could be done appeared to be by the
introduction of an attenuated virus into the animal that was
to be rendered immune, as in the case of anthrax, and so to
accustom the tissues to the presence of the specific poison,
rendering them better able to resist a stronger poison ; he
thought, in fact, that by successive inoculations of stronger
and stronger poisons he would be able gradually to “ accli-
matize” the tissues to the presence of even the strongest
virus, and so enable them to deal with it successfully, probably
by converting it into innocuous proteid materials, so rendering
it harmless to the delicate and highly organized cells of the
nervous system. In his earlier experiments he obtained an
attenuated virus by inoculating a monkey, from which he
took material from the central nervous system with which
to inoculate a series of rabbits; each rabbit supplied a
slightly more powerful virus with which to inoculate another
one of the series. He thus obtained inoculation material of
all degrees of virulence. With material from each rabbit
in the series he inoculated twenty dogs, each one receiving
a stronger and stronger dose each time it was inoculated.
Out of the twenty dogs so treated only about three-quarters
were protected against virulent hydrophobia ; but this for a
first series of experiments was a most extraordinary success,
and so satisfied and delighted Pasteur that he was encouraged
to continue his research ; eventually the results he obtained
were even more remarkable.

Having observed that the cords of rabbits that had been
dead for some time contained a less virulent poison than the
cords of freshly-killed animals—this being especially the case
in dry weather—he adopted a method based on this observa-
tion, by means of which he was able from the same cord to
obtain inoculating materials of very different degrees of
virulence, this varying according to the length of time that
had intervened between the death of the animal and the use
of its cord for protective injection. He proceeded as follows :
Having sterilized a glass flask plugged with cotton wool by

22

322 BACTERIA,

dry heat, he filled it ta the depth of three-quarters of an
inch, or an inch with some hygroscopic material such as
solid potassium hydrate ; it was then ready for use.

A rabbit was previously inoculated in the following
manner: The skin over the part of the skull covering the
brain is carefully saturated with a 5 per cent. solution of car-
bolic acid. (This serves two purposes: first, it purifies the
skin through which the knife is to pass, and, secondly, it
renders the skin and the tissues beneath perfectly insensible
to pain, so that the rabbit will remain perfectly quiet while
the operation goes on; it does not apparently suffer even
discomfort, and I have seen a rabbit going on eating whilst
the operation was being performed. I have also seen the
operations performed as in the Pasteur Institute, while the
animal was under the influence of chloroform ; but I have
never yet seen it done without either a local or a general
anzesthetic being administered). An incision is made through
the insensitive tissues so as to give either a cruciform incision
or a semi-circular flap, the soft tissues are dissected from the
bone, and then with a small tube with teeth at the end, and
a sliding pin in the centre (a trephine), a little circular disc
of bone is removed, as far as possible without injuring the
external covering membrane of the brain.

With a subcutaneous injection syringe, carefully purified
by means of some chemical germicidal reagent, or heat, a very
minute quantity of the cerebro-spinal fluid, taken from an
animal that has succumbed to the disease, is then injected
under the dura-mater (the membrane above mentioned),
which is immediately beneath the bone of the skull. The
disc of bone is then replaced ; the wound is closed by means
of a couple of stitches; a pad of cotton wadding, carefully
purified by heat, is used to dry the skin as much as possible ;
after which a little of the same cotton wadding is used as a
dressing ; this dressing is kept in position by a free ap-
plication of flexile collodion, the two together forming an
air proof shield, through which no organisms from the
external air can make their way to the wound, which, as
a rule, heals up most perfectly in less than a couple of
days. The operation does not appear to affect the animal
so treated in the slightest, and until the seventh day it
appears to be as lively as any of its companions ; it then
gradually begins to lose the power of the muscles, first

HYDROPHOBIA. 323

in the hind legs, then gradually in the muscles through-
out the body ; other nervous symptoms appear, the animal
becomes unconscious and comatose, and about the tenth day
after inoculation it dies. The cord is taken out as soon
after death as possible, and great care is exercised to prevent
organisms, septic, putrefactive, or any others, from finding
their way to the surface of the cord, which as soon as re-
moved is suspended by a sterilized silk thread in the flask,
the air of which, having been rendered extremely dry by the
potash, now absorbs a very large proportion of moisture
from the cord, and prevents it from undergoing putrefactive
change. Originally the cord was left in this flask from twenty-
four hours to fifteen days, the material from the cord that had
been left for fifteen days having almost or completely lost its
virulence, the one day cord remaining nearly as virulent as
a cord that had undergone no desiccation.

On the 26th of October, 1885, Pasteur described this
method to the French Academy of Sciences. He showed
that by inoculating animals on ten successive days with
fragments of different cords, each beaten up with twice its
volume of sterilized bouillon, commencing with the weakest
virus, and continuing until he had used an emulsion from
the cord that had.been exposed only two or three days to
the dried air, and kept pretty constantly at a temperature of
17° or 18° C., they were protected against hydrophobia, even
when extremely virulent virus was afterwards injected sub-
cutaneously, or into the membranes of the brain. Of fifty dogs
so treated (no two exactly in the same way), every one was
refractory to the disease in proportion to the theoretical
degree of protection that had been given ; such protection
lasting apparently for at least two years, and probably more.

Having obtained such success with dogs the next step
was to protect patients who had already been bitten by
mad dogs or wolves. The first human being so inoculated
against hydrophobia was a little boy, Joseph Meister, aged
nine years, who, on the 4th of July, 1885, was bitten so
severely on the arms and legs by a mad dog, that it was
with difficulty the poor child could walk. He was at-
tended to by a doctor who cauterized the worst of the
wounds with carbolic acid, but not until twelve hours after
the child had been bitten. As the dog was undoubtedly
mad, and as there was little chance of the survival of the

324 BACTERIA,

child in the ordinary course of events, Pasteur resolved,
after consulting with Professors Vulpian and Grancher, who
agreed to share the responsibility, to treat the boy as he had
treated the dogs that he had already been successful in pro-
tecting. During the following ten days he made thirteen
injections :—

2on the 1st day with emulsions made from cords that had been exposed
to the air in the flask for 14 and 10 days respectively :

2 on the 2nd day +5 3 11 and 9 days respectively
I x 3rd day ” % 8 days
I » 4th day ” ” 7 days

and so on until the roth day, when he inoculated with the cord of a rabbit
that had died on the same day, z.¢., the cord in which the rabic virus still
retained its full virulence.

For every injection that was made into the child, a corre-
sponding one was made into a test rabbit, and it was found
that the five rabbits inoculated with the first five injecting
materials, had no hydrophobia, whilst the other eight
succumbed to the disease ; the period at which the animals
succumbed being gradually shortened as the cords exposed
to the dry air for the shorter times were inoculated. It
was remarkable that although the later vaccines proved
fatal to rabbits, in the patient, prepared by the previous
inoculations, they did not produce the slightest discomfort,
he never had the faintest symptom of hydrophobia, and
now, five years later, we are told that the boy is still alive
and well. Since that time an enormous number of patients
have been inoculated, and it certainly appears from statistics,
given monthly in the “ Annales de l'Institut Pasteur,” that
the percentage of deaths after inoculation has been much
lower than in those patients left without the anti-rabic
treatment.

Babes, in order to make the virus as constant as possible,
finds that it is advisable to make a mixture of cords from three
or four different days (z.¢., cords that have been exposed to the
drying process for different periods), and to inoculate at least
twice a day, or more'\ frequently in serious cases, such as
those in which there are wounds about the face, or where
the wounds are inflicted by a mad wolf; the period of
treatment now also is longer, in addition to which much

HYDROPHOBIA, 325

larger quantities of the protective material are used, especi-
ally in severe cases.

Take as an example of this method of treatment, one given by Cornil
and Babes in their work on bacteria, that of a child severely bitten about
the face, which only came under treatment four days after it had been
bitten.

On the Ist day it received injections of 2 grammes of emulsion, made up
of cords that had been exposed 13, 12, 11, and Io days respectively.

On the 2nd day 2 grammes _,, ar 10, 9, 8, and 7 days 55
” 3rd day ” ” ” 75 6, 5,4) days ”
» 4th day 14 grammes ,, ee 4 and 3 days a3
>, 5th day ae < - 3 and 2 days 3
» 6th day 2 grammes _,, ss 8 and 7 days a
” 7th day or) ” ” 7 and 6 days ”
” 8th day » 2 ” 6 and 5 days ”
» 9th day ” » 95 5 and 4 days ”
»» loth day 14 grammes,, 3 4.and 3 days a5
», 2th day 2 grammes ,, 55 8 and 7 days. 35
” 13th day 2” ” ” 7 and 6 days ?
» 4th day 33 ae sy 6and 5 days 33
” 15th day ” ” ” 5 and 4 days ”
3» 16th day 14 grammes,, ” 4 and 3 days 3
1, 18th day 2 grammes ,, 5 8 days
» Igthday ‘a is 9 7 days
s, 20th day 33 8 9 6 days
», 2ist,22nddays.2 ,, ‘5 5 days

» 23rd,24thdays.,, ,, ‘5 4 days
»» 2§th,26thdays.,, ,, 5, 3 days
On the 11th and 17th days there was no treatment.

At Bucharest, where wolf bites are of frequent occurrence, the treatment
may last for more than a month, four and five injections being made on the
first few days, and as much as 12 or 13 grammes of the emulsion being
injected per diem ; the whole series of attenuations being gone through in
mixtures of three each in three days. Then the same process is gone
through with mixtures of two attenuations, and lastly, the emulsions from
single cords throughout the whole series are injected. For instance, on the
Ist day there are injected 4 grammes of an emulsion, made from the cords
of 13, 12, and 11 days; then 4 grammes from the 12, 11, and 10 days;
3 from the 11, 10, and 9 days; then 2 from the 10, 9, and 8 days; the last
injection on the 3rd day consisting of 1 gramme of the 3, 2, 1 days, or the
strongest virus ; then on the 4th day similar injections are made of the most
virulent virus ; on the 5th day 16 grammes of the weaker virus from the
12th to 9th daysare injected ; and on the 30th day 4 grammes of the strong
virus (2 days) are injected. This treatment is modified for special cases,
but this is an example of the treatment for very severe bites.

Bujwid adopted a somewhat different process, he injected 2 grammes
daily of the mixture of 12 and 10 day cords on the first day ; 8 and 7 day
cords on the second day ; 6 and 5 cords on the third; 4 and 3 on the

326 BACTERIA,

fourth, and then again 12 and 10 cords on the fifth, and so on through a
third series, the whole treatment lasting 14 or 15 days.

Cornil and Babes have collected some most interesting statistics, which
we may here quote. In the Institut Pasteur in 1886, out of 2,682 cases
treated, there was a mortality of 13.4 per 1,000; in 1887, in 1,778 cases,
a mortality of 11.2 per 1,000; in 1888 7.7 per. 1,000; and in 1889 5.4
per 1,000. In St. Petersburg, in 484 patients treated, the mortality was
26.8 per 1,000; in Odessa, in 1,135 treated, 17.1 per 1,000; in Moscow,
out of 107 treated, 34 per 1,000; but out of 500 inoculated with stronger
virus, and for a longer period, the mortality was only 13 per 1,000. ° At
Warsaw, of 297 people treated by the milder method, 80 per 1,000 suc-
cumbed ; whilst of 370 inoculated by the intensive method, none succumbed
to hydrophobia. At Charkow, of 233 treated, there was a mortality of
38 per 1,000; at Turin, of 502 treated in cases where the dogs were un-
doubtedly rabid, there was a mortality of 25 per 1,000; at Bucharest, out
of 310 patients, a mortality of only 2.9 per 1,000; at Naples, of several
hundreds treated, the mortality was only 15 per 1,000; and at Havannah,
where there were 170 patients, only 6 succumbed.

The factor of time between the bite and the commence-
ment of the treatment plays a most important part, and it
has been found that there is always the greatest success
obtained in the cases of those patients who submit them-
selves for treatment within two or three days of being bitten.
One of the most convincing proofs of the efficacy of this
inoculation that has been given, is that recorded by Babes.
Thirteen men and thirty animals, cattle, horses, pigs, and dogs,
he states, were attacked by rabid wolves; of the thirteen men
so attacked, twelve came to Bucharest for treatment, and all
of them recovered except one whose head was fearfully torn
and lacerated by the fangs of a wolf; the thirteenth man,
who would not present himself for treatment, died of
hydrophobia. <A very significant fact was that every one of
the thirty animals succumbed to typical hydrophobia. The
patient who died succumbed ten days after the completion
of a very intense and prolonged treatment (thirty-two days).
The other patients were then subjected to a further treat-
ment during six days of mixtures of two cords from the
twelve days down to the one day ; and as we have seen they
all recovered.

It is interesting to note in connection with Galtier’s
early observations, which were at first supposed to be dis-
proved by Pasteur, that they were confirmed by Nocard
and Roux, and by Vestea and Zagari, who have proved
that the injection into the blood of rabic virus does not
always produce hydrophobia, and that in certain ruminants,

HYDROPHOBIA. 327

such as the sheep and the goat, the intravenous injection of
rabic virus, although it does not produce hydrophobia,
protects these animals against -rabic infection. It is now
generally acknowledged that when properly performed, the
inoculation of even very large quantities of virus may be
safely carried out, and as it is found that greater success
is obtained where these larger quantities are injected we may
look forward to still greater improvement in the treatment of
this disease, and to even further diminished mortality.

This process of inoculation in hydrophobia brings up a
phase of the vaccination question that has not yet been fully
developed but one that appears to be destined to cast an
important light on some of the questions relating to im-
munity. Pasteur’s explanation of the results he obtained
does not appear to be entirely satisfactory. JI am inclined
to think that the explanation advanced by Wood and myself,

that the treatment consists essentially in causing the tissues °° '

to acquire a tolerance before the microbe has had time to
develop, is more in accordance with facts. The tissue cells
are acted upon by increasingly active virus, each step of
which acclimatizes the cells for the next stronger virus, until
at length when the virus formed by the micro-organisms
introduced at the time of the bite comes to exert its action,
the tissues have been so far altered or acclimatized that they
can continue their work undisturbed in its presence ; and
treating the micro-organisms themselves as foreign bodies,
destroy them. When the cells are suddenly attacked by a
strong dose of the poison of this virus they are so paralyzed
that the micro-organisms can continue to carry on their
poison-manufacturing process without let or hindrance, but
when the cells are gradually, though rapidly, accustomed to
the presence of the poison by the exhibition of constantly
increasing doses they can carry on their scavenging work
even in its presence, and the micro-organisms are destroyed,
possibly even before they can exert their full poison manu-
facturing powers. Some such explanation as this would
account for the interference with the course of the disease
even after the patient has been bitten. The micro-organism
is localized, it takes some time to form its poisonous products,
and whilst this is going on the whole of the nervous and other
tissues are being gradually acclimatized by the direct applica-
tion of small quantities of the poison artificially introduced.

328 BACTERIA.

It is a most remarkable fact that although no micro-
organisms can be found in the virus, filtration through the
Pasteur filter keeps back the effective part of the virus,
whilst heating to 100° C., or even ‘a prolonged heating to
80° C., destroys the activity of the virus; further, an alcoholic
extract of the emulsion does not contain any substance that
will confer immunity, nor will it, when inoculated, produce.
any symptoms of rabies.

‘That the patients themselves have great faith in the
treatment, we have ample evidence in the fact that so
many present themselves for treatment. It was an ex-
tremely interesting sight to see the members of the poly-
glot crowd in the waiting-room brought in, one by one,
for inoculation in the Pasteur Institute. As the patient’s
name was called out, one assistant standing at a table on
. which was arranged a row of little conical glasses, each
covered with a paper cap, and containing an emulsion of a
cord of a certain length of exposure, filled or partly filled
a Pravaz syringe with emulsion from one of these glasses,
according to the stage at which the treatment had arrived
(determined by refererice to the case book). He handed the
syringe to the operating doctor, who, taking a fold of the
skin (which had been previously washed and purified with
carbolic acid, or some such antiseptic reagent), just above
the crest of the ilium, a part very easily exposed from the
arrangement of the attire of both males and females, inserted
the point of the needle, injected about half a syringe full,
withdrew the needle, diffused the fluid as far as possible by
pressure, and the patient passed on. The syringe was
handed back to the assistant, who carefully sterilized it by
plunging it into hot water. The whole operation seemed to
take only a few seconds. A continual stream kept passing
from one room to another, each patient, as he went through,
undergoing the same process of inoculation. Everything
was done in the most orderly fashion, and one could not
but feel that Pasteur, who was standing in the room whilst
this was going on, had every right to feel proud, not only of
the splendid Institute in which he was working, but of the
excellent work that was being carried on, both in the field
of investigation and in the application for the benefit of
suffering humanity of the results of the investigations.

In France they manage some things better than we do,

HYDROPHOBIA, 329

and, although France should not get all the credit of work
towards which most European nations have contributed,
through representatives of all classes, the grants from the
Imperial treasury formed a basis on which to found the now
celebrated Pasteur Institute, and to France and Pasteur
much of the honour is due. The Pasteur Institute is a
building set apart for the study of micro-biology, the most
perfect of its kind in the world, and a short account of it
cannot fail to prove interesting. For many of the details
I have referred to the January number of “ Annales de
l'Institut Pasteur,” 1889, as unfortunately, when I visited
the laboratories, the place was not in full working order,
but I saw enough to convince me that the spirit which
animates the founder of the Institute pervades also those
of his disciples who carry on the work he has begun. It
covers an area of eleven thousand square metres in the
Rue Dutot, and consists of two large blocks running parallel,
one behind the other, united by a long corridor which runs
from the main entrance in the front, connecting the two
buildings, and through to the back, with a number of
separate outbuildings scattered over the gardens behind.
In the large block facing the street on the right are M.
Pasteur’s apartments ; on the left his laboratory occupies the
basement. In this laboratory he continues his researches,
but during the time that patients bitten by mad dogs are
being treated he may frequently be found watching with
great interest the treatment, and chatting with those who
have come to see the place and the method of work. In this
block also is collected everything connected with the pre-
paration of, and despatch of, the various vaccines—rouget de
porc (swine fever), anthrax vaccine, etc. On the first floor
is a large hall, which serves the double purpose of library
and council room. This room is well lighted by four large
windows, and is provided with the current scientific litera-
ture, both periodicals and books, all of which may be
consulted by the workers in the laboratories, who, however,
are not allowed to remove them from the room. The storey
above this is occupied by the servants and attendants of the
institution. The large corridors, four metres and a half
broad (nearly five yards), well lighted, run between the
corresponding storeys from this block of buildings to the
larger one behind, which is entirely devoted to laboratories.

330 BACTERIA.

This block is divided into two wings, each about twenty-
five metres long and fifteen metres from back to front; in
the right wing, on the ground floor, are the rooms set apart
for the examination of patients who have been bitten by
mad dogs, and for their treatment by the preventive
inoculation method. The patients who enter the grounds
at the right are directed by an intelligent porter through
a door at the right of the building to a waiting-room
heated and well lighted, surrounded with benches on
which they may rest while waiting for their turn to
come ; they then pass into a room where their names,
addresses, and particulars of their cases are carefully
recorded. After being duly registered, they are passed on
to the inoculation apartment, where they are treated in the
manner already described. Should any patients show
symptoms of faintness, they are assisted into a small room
in which is a sofa, on which they may recline ; the others pass
out into a central passage, on the opposite side of which
is a room where severe bites and lacerations are attended to
surgically. Here also are an operation room, archive room,
and others. Next to the room in which we left the faint
patients, is the laboratory in which the preparation of the
cords that are used for the manufacture of the virus is
carried on. It is maintained at a constant temperature
of 23° C. by means of a special regulator. Around
this is a regular array of bottles, arranged on shelves fixed
to the wall, and grouped according to the time that they
have been exposed to the dry air. A screen within the
door prevents draughts, and helps to maintain a constant
temperature, even when the door is frequently opened.

In the left wing, on the ground floor, is a lecture-room for
biological chemistry, which’ will: accommodate about fifty
auditors ; it is separated from a laboratory by a wide door-
way, which, when open, permits of those in the lecture-room
seeing what is going on in the laboratory. This opening may
be closed altogether by a large black screen, or by a ground-
glass plate, on which lantern projections may be thrown. M.
Duclaux, professor of biological chemistry in the Sorbonne,
now delivers his course here, and M. Roux lectures on Prac-
tical Micro-biology. From time to time those who are working
in the laboratory, or others, also give demonstrations and ex-
planations of their work and of any recent discoveries in this

HYDROPHOBIA, 331

theatre. Close to the lecture-theatre is a photographic depart-
ment, specially designed and fitted up by M. Roux, for the re-
production of bacteria and other microscopic objects. At the
end of the block, at the right and left of the central corridor, are
tworooms with well-fitted aquaria, which are specially fitted up
for carrying on researches, under the supervision of M. Metsch-
nikoff, on aquatic animals. The two rooms on the ground
floor, specially set apart for experiments upon large animals,
are provided with a good wide door opening to the outside.
The concrete pavement, sloping towards a green, permits of
this being readily cleansed. The remainder of this floor is oc-
cupied bya kind of store-room and a laboratory set apart for
general use, in which are stored and prepared the bouillon and
other nutrient media. Here also is carried on the glass-blow-
ing ; a skilled artizan supplying workers with flasks, pipettes,
tubes, and other vessels they may require. A large staircase
situated at the end of the central gallery puts the labora-
tories of the basement in easy communication with those of
the storeys above. The first storey is divided into duplicate
halves, one on the right, the other on the left of a passage.
That on the left is set apart for M. Duclaux's department of
general micro-biology ; that on the right to practical biology,
over which M. Roux presides. The central corridor leads to
a large concreted workroom, nearly twelve metres square,
which is well lighted with nine large windows. Around the
room are seven work tables, each of which is covered with a
thick sheet of volcanic lava, the surface of which is enamelled
so that it has the appearance of an immense sheet of porce-
lain. Each is fitted for two workers. Every worker has
before him a window, from which light is obtained for the
microscope on his right or left side, according to his position.
At the table is a gas connection, from which gas may be
conveyed to any desired point ; here also is water, which is
received into a sink, also of enamelled lava. A small sliding
board, which may be drawn out and used as a desk, is placed
on the other side of the table, away from the central pro-
jection which carries the sink ; this allows the worker to
make for himself a little retreat, in which he sits surrounded
by all that he requires. When work is over for the day
everything except the microscope is put away in a couple of
small cupboards which are placed at the disposal of the worker.
This is absolutely necessary in order that the room and the

332 BACTERIA.

tables may be kept clean. Two tables placed parallel to one
another in the centre of the room are used for chemical
operations which cannot be carried on at the smaller special
tables. Then there are large evaporating chambers, in which
are placed the sterilizing and incubating apparatus. In
addition to the incubating apparatus used in the common
laboratory, there is a large common room, which is really
made up of three rooms :—An entrance chamber, in which
the temperature varies somewhat from time to time ; it con-
tains the heating apparatus, and is specially designed as an. air
cushion insulator. The second chamber—nine feet ten inches
long, eight feet broad, and six feet high—is heated by means
of hot water, by which it is kept at a constant temperature.
Above this, and heated in connection with it, is a third
chamber, in which the temperature is intermediate between
those of the two preceding ones. This group of incubating
chambers has very little cooling surface, and is separated
from the outer wall by a room in which is collected and
washed all the dirty glass used in the laboratory. In these
constant-temperature chambers are shelves and tables, on
which may be seen all kinds of flasks, test tubes, and sterilized
moist chambers, in which, growing on various media, and
under different conditions, are all kinds of micro-organisms
that are being experimented with in the various departments
of the Institute. The laboratory “ preparateur’’ has a room
next to the laboratory, with which it communicates directly,
so allowing ready access thereto and constant superinten-
dence. From this small laboratory is the only entrance to
the museum, for the care of which the preparateur is directly
responsible. A special laboratory for the performance of
experiments in chemical biology, and a lavatory, are also
added to the suite on this floor. The laboratory and the
room of the director of the department are placed in each
wing. These are entered from the passage which’ leads to
the common laboratory. In the storey above this there are
no teaching laboratories; we have simply two series of
rooms, divided by a passage, each room designed to become
a research laboratory, and fitted up to meet the require-
ments of the savant who occupies it. Investigators have
every facility-for carrying on their work, and may, if
they desire it, have the advice and guidance of any of the
directors of departments, each of whom is responsible for the

HYDROPHOBIA. 333

management and work of the whole wing that is placed
under his supervision. In the left wing M. Chamberland
superintends the department of applied or practical bacteri-
ology; in the right wing M. Gamaleia presides over the
subject of comparative micro-biology or bacteriology. In each
of these departments, in addition to the special incubating
chambers required by the workers, is a general incubating
chamber, constructed like that in the first storey, but on a
larger scale, and also a general laboratory, in which may be
carried on all the sterilizing, preparation of gelatine, broths,
&c., for which special apparatus is required.

This is the extent of the laboratory proper, but. in the
garden which surrounds the main building are found scat-
tered other structures, apparently less important, but all
of them required for the carrying on of the work of this
immense establishment.

First comes a building, parallel to the main block, in which
is accommodation for animals, which are kept in elevated
cages constructed of iron bars on every side; they are so
raised from the sloping asphalt floor, that they can readily
be flushed. At the two ends of the building three small
rooms have been set apart for operation on small animals that
come to be treatedin the Institute. In a special house, which
is very well arranged for the reception of dogs, each animal
has its special cage, where it can be properly looked after,
watched, and fed, without it being necessary for the atten-
dant or observer to come actually in contact with it.
Near this is another series of animal houses, also with
asphalt floors, which are used for stock, or for animals that
being experimented upon require special isolation. The
rabbits that are inoculated for the preparation of the specific
fluid, during the incubation period of their attack of hydro-
phobia occupy a special house, which is also kept at a con-
stant temperature in order that the periods of incubation
may be kept as uniform as possible. A special arrangement
of the cages in which these animals are enclosed allows of
their bedding being changed and of their being kept clean
without its being necessary to open the cage. A gutter, with
walls of glass placed below the cages, which can be well
flushed by a stream of water, serves to carry off the urine
which makes its way- through the floors of the cages. Then
come arun for hens, and smaller hen-coops, an aviary, and

334 BACTERIA.

stabling for large animals, all of which are so constructed
that they can be kept not only ina state of perfect cleanli-
ness, but can be thoroughly washed out with boiling water.
They present no porous surfaces, and there is no material
used into which germs can penetrate and become inaccessible
to the usual microbicidal agents. Lastly, in one corner
of the grounds are two cremating furnaces, in one of which
gas is used, and in the other ordinary fuel. In these every
particle of infected matter and all diseased tissues not kept for
microscopic examination, are carefully calcined in order that
nothing of an infective nature may be carried beyond the
walls of the establishment. The arrangements generally are
such that absolute cleanliness may be maintained with the
slightest possible amount of labour, so that not only the
workers, but also the people residing in the neighbourhood,
may be perfectly at ease as regards any danger of infection.
Even in the old laboratory, situated in the gardens and in
contact with the Normal School in the Rue Vauquelin in
the immediate vicinity of hotels, experiments with the most
virulently infective diseases have for ten years been carried on
by Pasteur and his pupils without the least accident having
happened. The new Institute will accommodate about
fifty workers—about fourteen in the laboratories on the
ground floor, and the remainder in the research laboratories.
The staff of directors and assistants consists of about fifteen
persons, all of whom are studying some branch or other of
micro-biology. The only passport required for working
in the laboratory is a capacity for doing good work. If
there is a place at liberty a good worker is always sure
of it, and Frenchmen and foreigners alike are admitted to
the use of these tables in one or other of the principal
departments, of which there are six, associated with the
names of such men as Straus, Grancher, Duclaux, Gamaleia,
Chantemesse, Widal, Charrin, and others. These depart-
ments are named according to the special kind of work car-
ried on. Thus, one is devoted to the study and treatment
of hydrophobia, another to general micro-biology, a third to
practical bacteriology, a fourth to micro-biology applied to
hygiene, a fifth to morphological and comparative micro-
biology, a sixth to biological chemistry. ost extensive
and elaborate arrangements are made for the instruction of
pupils in both general and special methods of bacteriological

HYDROPHOBIA. 335

investigation, and the pupil, after being instructed in the
first, will, in the laboratory of general micro-biology, follow
out special methods, each one having his own subject and
pursuing his research by appropriate methods. M. Roux
takes them first in small classes, and in five or six
weeks he gives all the general principles of work, and
indicates all the details of technique necessary to ensure the
competence of these pupils in ordinary matters relating to
the study of micro-biology. In the course is included regular
practical laboratory work ; but the main work is the carry-
ing on of original investigation, and the results of the
researches made by the distinguished staff and their pupils
are now known throughout the world, whilst there still
comes a stream of brilliant papers, such as may well fill us
with admiring envy, and, let us hope, provoke in us a
generous rivalry at some not far distant date.

LITERATURE.

The following works may be consulted :
Bases.—Annal. de l'Institut Pasteur, t. 1., No. 7, p. 394,
1888 ; Virch. Arch., Bd. cx., Heft. 3, p. 562, 1887.
Bases Et Lepp.—Annal. de l'Institut Pasteur, t. m1., No. 7,
p- 384, 1889. ;

Cornit ET BaBes.—Les Bactéries, p. 550, 1890.

Dotan.—Hydrophobia ; M. Pasteur and his Methods. 1886.

DowveEswELL.—Lancet, vol. 1, p. 1112, 1886 ; Journ. Roy.
Micros. Soc., vol. vi., p. 669, 1886.

For.—Comptes rendus, t. cl, p. 1276, 1885.

GALTIER.—Comptes rendus, t. CVII., p. 798, 1888.

GAMALEIA.—Annal. de l'Institut Pasteur. 1887.

GipieR.—Comptes rendus, t. XCVI., p. 1701, 1883, and t.
XCVIIL, pp. 55 and 531, 1884.

Hime.— Lancet, vol. 1, p. 184, 1886.

Nocarp ET Rovux.—Annal. de l'Institut Pasteur, t. 1, No.
7, P- 341, 1888. :

Pasteur.—Annal. de l'Institut Pasteur, 1887-1891.

PasTEuR, CHAMBERLAND, Roux ET THUILLIER.—Comptes
rendus, t. xXcv., p. 1187, 1882; t. CIL, p. 531, and t.
Cll, p. 777, 1886.

Rovx ET CHAMBERLAND.—Annales de l'Institut Pasteur,
t. u., No. 8, p. 405, 1888.

336 BACTERIA,

VESTEA E ZAGARI.—Giorn. Internat. d. sc. Med., t. m.,
1887.
WooDHEAD AND Woop.—Comptes rendus, t. crx., 1889.
See also Report from the Select Committee of the House
of Lords on Rabies in dogs, Blue Book, 1887 ; and numerous
papers and reports in the Annales de ‘Institut Pasteur.

‘CHAPTER XIX.
BACTERIA OF THE Movutu.

The Mouth a Good Forcing Ground for Bacteria—Food Material—Kinds
of Bacteria found in the Mouth by Miller and Vignal—Bacteria in the
Teeth in Caries, in Milk Teeth, and in Abscesses—Combination of
Lactic Acid with Lime Salts—Organisms Attack Decalcified Base
Substance—Artificial Decay—Mode of Invasion of Teeth by Bacteria
—Poisonous Saliva—Micrococcus of Sputum Septiceemia—Pneumo-
coccus—Other Organisms found in the Mouth—Septiczemia following
Slight Operations in the Mouth.

It will be remembered that many of Leeuwenhoek’s obser-
vations as to the morphological appearances of the minute
structures which he examined with his-wonderful lenses,
were made on bacteria which were found adhering to the
teeth, and certainly some of his most interesting observations
were on bacteria derived from this source. From what we
now know of the biology of these organisms we can readily
understand that the mouth should form a kind of hothouse
or forcing-ground for their cultivation. Here is a moist
cavity kept at a comparatively high temperature, covered
with an epithelium which is constantly being partially or
completely shed, to which there is ready access from the out-
side air, and through which food material is constantly
being passed, particles of which, despite the exercise of the
greatest care and the utmost cleanliness, always remain in
small crevices between the teeth, or perhaps, more important
still, between the gums and the teeth. It is also said that the
fact that starch is constantly being converted into sugar by
the ptyalin is in favour of the growth of bacteria. Then we
have the dead epithelium, which is readily attacked by
organisms of various kinds, supplying proteid or other
nitrogenous materials; as a matter of fact, there are often
found in these dead epithelial cells a number of micrococci,
which appear to be gradually disintegrating their ‘substance
and utilizing the materials of which they are composed. In

23

338 BACTERIA.

a similar way the various constituents of the teeth are made
to serve as nutrient elements for these bacteria if once the
protecting enamel is removed.

One author, writing of the micro-organisms that may be
found in the mouth, points out that almost every organism
that has been described as occurring in any position has also
been described as growing in this cavity; but it is now

Photo micrograph of scraping from the teeth in which different kinds of micro-organisms
are seen, X 1000, Spirilla very imperfectly stained.
generally accepted that about eight or ten are almost con-
stantly met with, and that six may be said to be invariably
present. Of these we give a short description, as they may
be examined by any one who wishes to see what these
organisms really are, though we warn any who may
attempt to cultivate them that they will almost inevitably
fail, as these special mouth organisms seem to require

BACTERIA OF THE MOUTH. 339

special conditions, as yet not at all understood, for their
existence, and although one or other of them may be found
in enormous numbers in one case, and may be comparatively
few in another, there is usually some explanation to be
offered for such preponderance. 4

(1) The Leptothrix innominata (Miller) is found, usually, on the soft
white material that is deposited on teeth, it may probably be but a generic
term for more than one form, as the constituent elements of which it is said
to be made up vary very much both in length and in thickness; they are
usually .5 to .8u thick, and though there may be shorter cells or rods,
the organisms usually consist of tortuous non-motile threads. These rods
and threads as a rule contain no spores.

(2) The second form, Bacillus dbuccalis maximus, occurs as isolated
bacilli or threads, generally arranged in bundles, which may interweave
with one another. These bundles are sometimes of very’ considerable
length, but each thread is divided into short rods, from 1 to 1.2” broad,
and from 2 to 1o# long. This bacillus is, as a rule, not found within the
dentine tubules. In addition to this bacillary form there is a leptothrix
form, in which the filaments are considerably longer, but otherwise the
organism is much like that already described, although it is scarcely to be
looked upon as related to the leptothrix innominata,

(3) The next organism is one called by Miller the Jodococcus vaginatus,
which is found especially in mouths that are not properly cleansed ; it appears
to be made up of cocci arranged as diplococci or tetrads, which are usually
arranged and held together-ina kind of sheath, though now and again a
single coccus or diplococcus may be seen, around which no sheath can be
demonstrated. hen this organism is stained with iodine a marked
reaction is obtained, the sheath being stained yellow, whilst the cocci are
stained dark blue.

One of the most interesting forms met with in the mouth is: (4) the Spzrz/-
lum sputigenum (Miller), which always occurs in certain numbers, but is found
in especially large quantities in those cases in which the presence of tartar has
set up slight inflammation of the gums around the teeth ; it usually occurs
in the edematous gum just at its junction with the teeth. This organism
has more than usual interest from the fact that it was for long supposed, by
Lewis and Klein, to be identical with the cholera bacillus. Morpho-
logically it certainly resembles this latter organism in many respects. The
simplest form is that of a small curved bacillus ; sometimes two or three
of these may be joined together to form an S-shaped organism, whilst in
other cases it may be found as long spiral threads. This organism, as we
have said, is almost invariably met with in the mouth, but it is curious
that it is very seldom found between the teeth, or in carious cavities ; it
thrives apparently upon the exudation from the slightly inflamed gums, and
so necessary is this food for its existence that it has never yet, so far as is

known, been cultivated in any of the artificial nutrient media outside the, , , .

body, although (5) a somewhat thicker, curved, comma-shaped bacillus,
identical in almost every respect (except as regards size), can be cultivated
in gelatine quite readily at the ordinary temperature of the room. This
differs from the first-mentioned in that it has the true comma-shape, tapering
towards one end, :

340 BACTERIA,

(6) -A third curved or spiral organism is also described—somewhat longer.

than the others; it, occurs in longer or shorter, straight or spiral filaments,
Which may break up into rods and even into chains of cocci; this organism’
when cultivated in gelatine does not give rise to any liquefaction, differing
in this respect from either the Koch’s cholera or comma bacillus or'the
Finckler- Prior bacillus, the latter of which has also been described by Miller,
as occurring in the mouth. Miller maintains indeed that some of these
curved organisms may pass into the lower parts of the alimentary canal,
but that even when he took them from this position he never succeeded in
making pure cultures of them; they will not grow on any of the nutrient
media that have been as yet devised.
_ (7) Then, of these more important forms, is the Spzrochete dentium or
Spirochate denticula, which is usually found associated with the (8) Spirillum
sputigenum, and under similar conditions ; it consists of a long spiral thread.
from 8 to 25z in length, of which the spirals are irregular and of unequal
thickness. Very little is known of this organism, and it is quite possible
that the thicker spirals may represent some stage of development of. the
preceding organjsm, especially as in this case it is also impossible to obtain
any artificial cultures. As already stated, other organisms are very fre-
quently, though not invariably, met with.

Vignal describes a number of bacteria and-bacilli as occur-
ring in the mouth and amongst these certain common forms
that are generally recognized, such as Bacterium termo (?)
Bacillus ulna, the potato bacillus, Bacillus aly, and a number
of others, whilst amongst the cocci are found the Staphylo-
coccus pyogenes albus, and S. pyogenes aureus, &c. Hueppe
describes organisms that give rise to the lactic acid fermenta-
tion, whilst others, especially those giving rise to pigment
and pathogenic. bacteria of various kinds, most of them well
recognized, have a peptonizing action, and a few appearing
to’ secrete a true diastatic ferment may also be met with
in the mouth. a aoe
.' Miller gives some interesting statistics as to the action of
the bacteria found in the mouth upon carbohydrates ; he
finds that of twenty-two kinds of .fnouth bacteria which he
mentions, ‘sixteen brought about’ an acid reaction when
cultivated in beef extract peptone solution, four produced an
alkaline reaction under the same conditions, whilst in the
case of two only'the reaction remained neutral.” Many of
these organisms also exert an enzyme function in a some-
what marked degree; most of .them have the power of
peptonizing coagulated albumens, during which: they give
rise 'to’the usual ultimate products—sulphuretted hydrogen,
ammonia, carbonic acid gas or combinations of these, peptones
of course’also being formed.

BACTERIA. OF THE MOUTH. 341

- In thin sections of decalcified teeth, stained with fuchsin
and vesuvin, it is seen that bacteria are scattered, though
somewhat -irregularly, throughout the dentine (the hatd
substance of which the tooth is principally composed) that
is undergoing decay: or softening: This. is‘ always most
marked near the surface, but it must be noticed that as the
bacteria travel along the dentine tubules (little canals: that
run vertically through the:dentine), there is usually a small
-part of the softened area in which micro-organisms are not
‘seen, as it seems that these cannot make their! way readily
along the fine transverse branches that connect the vertical
tubules, and, as Miller puts it, although the organisms “ keep
up with the softening inthe direction toward .the pulp, they
fall considerably behind in the. lateral: directions, so.that the
invasion, particularly in the lateral direction, is usually much
less exténsive than the softening ;".and the tubules near the
-sutface always -contain ,.more: bacteria than those’ deéper
‘down ‘in the-softened. area. . It is. important to. remember
that bacteria may be found in apparently ‘normal dentine
eanals, whilst a similar invasion. seems to go on in the roots
of, “milk”. teeth, and also'where abscesses have occurred
in the roots of permanent” teeth. . .It is ‘now held by most
of those who have given attention to the subject, that. the
bacteria which are found in decaying teeth are only playing a
secondary part; though a very important one, in the process
of caries.: In the first. instance there appears to be a soften-
ing of the various parts of the tooth by acids; commencing
with the enamel; in the case of people who take perchloride
of iron or nitro-hydrochloric acid this softening may go on
exceedingly rapidly. . As: we have already'seen, lactic acid
is constantly present in the mouth, though often‘in very
small quantities; if left to act on the-lime-it may give
rise. eventually: to softening at the margins of the gums
and: to caries, acids.preparing: the way for the invasion of
various bacteria, by combining with the lime and softening
the tooth. Where once the lime salts have. been removed,
pacteria.can attack the basic substance most easily ; they are
now in a position to make their. way along the dentine
tubules, and by the intertubular spaces,-and once in this
position they attack the surrounding tissue with very great
vigour ; they use, it.as a food material, absorbing and
digesting it until they have made their way into the greater

342 BACTERIA.

part of the softened area. It was at one time stated that
the “leptothrix buccalis,” as it was called, was the organism
that was always found in these cases, but this is now known
not to be the case : micrococci, leptothrix threads, bacilli, and
spiral forms may all be met with even in the same decayed
tooth, and in tubules lying close together ; the softening and
absorption going on indiscriminately, whichever of the organ-
isms may be attacking the basic substance. It is owing to the
peptonizing power of these organisms that they are able to
carry on the disintegrating process, and we thus see that
although the bacilli may not be actually present in all parts
of a decaying area, their products, such as lactic acid and the
peptonizing enzyme, are really carrying on the work that
ends in the decay of the tooth, perhaps cgnsiderably in
advance of the bacteria themselves. In experiments carried
on with the object of proving that decay might take place
in teeth removed from the mouth, Miller placed a number
of perfectly sound teeth in a mixture of saliva and bread ;
this mixture was renewed from time to time, whenever the
slightest trace of alkalinity appeared, and the pulpy mass in
which the teeth were embedded was kept at a temperature
of 37° C. during a period. of three months, with the result
that the dentine became softened, and there was what he
describes as a condition of “white decay”; he found that
where the enamel was perfect, even acids had no power to
attack the dentine beneath, but in those cases where the
enamel was soft or imperfectly developed the dentine had
become softened by any: acid that was present, and the
canaliculi were filled with bacteria; this gave rise to
irregular erosion of these canals which thus appeared to be
unequally distended. Near the surface of the tooth the
organisms are not strictly confined to the tubules, but they
invade the basic substance from the surface, softening it as
they advance, but filling up the microscopic cavities as they
are formed.

The organisms described by Galippe and Vignal that were cultivated on
gelatine were six in number, all occurring in decaying teeth.

1. A short thick bacillus 1.5 in length, and nearly as broad as long ; it
grows somewhat rapidly on gelatine, giving rise to an opaque white growth
along the track of the needle ; it liquefies the gelatine about the third or
fourth day, rendering it somewhat opaque, from which we should gather
that the organism is motile ; on plates it forms colonies which are usually

BACTERIA OF THE MOUTH. 343

2 or 3mm. in diameter before they begin to liquefy ; after th they extend
and liquefy very rapidly.

2. A bacillus slightly constricted in the centre about 3u in length, and
about one-half as thick as long; in cultures it is similar to No. 1, except
that it has a larger surface growth before it gives rise to any liquefaction.

3. A bacillus very similar to No. 2, but with no constriction in the
centre ; it has square ends and frequently grows in long chains, especially in
liquid media ; it causes only slight softening of the gelatine.

4. Is asmall thin bacillus so short that it might even be mistaken for
a micrococcus; it gives rise to a white growth along the needle track in
gelatine which it turns yellow, and then causes to liquefy.

5. Was not found in all cases (in eight out of eighteen decayed teeth).
It is a bacillus with rounded ends, which grows almost exactly ‘ike No. 1.

6. Found five times only, is a large coccus; it was found only in
advanced stages of decay, where the canals had been opened up by other
organisms as it was so large that it could not make its way along the
ordinary dentine canals; it forms a white line along the track of the needle
in gelatine, of which it causes no liquefaction.

The very favourable incubating chamber of the mouth is,
however, not monopolized by the organisms that so far have
been mentioned, and during epidemics, or when people come
in contact with persons suffering from various diseases, the
organisms associated with such diseases are, as might be
anticipated, frequently taken into the mouth, where they
accumulate, multiply, and eventually may set up any of the
various diseases with which they are associated in the hitherto
healthy person,

In the very act of developing in the mouth they are sup-
posed to give rise to ptomaines and other poisonous products
which may render the human saliva toxic when introduced
by a bite or a wound into the individual himself, or into
another individual ; in fact, septic poisoning from injection
of the saliva from mad dogs was one of the great difficulties
with which Pasteur had to contend in his early experiments
on hydrophobia. Quite recently Sternberg, Fraenkel,
Klein, and others have shown that, though the pure saliva
as it runs into the mouth is non-pathogenic, it acquires toxic
properties as soon as it becomes mixed with the organisms
which are usually found in the mouth, this being especially
the case in patients suffering from certain infective diseases.
One observer has found that his own saliva is permanently
so toxic that it invariably causes the death of small animals into
which it is inoculated ; it is almost as fatal as hydrophobic
saliva. Some of these pathogenic organisms, like those we
have already mentioned, cannot be cultivated artificially, for it

344 BACTERIA,

‘is sometimes found that sputum inoculated into mice or
:tabbits causes their death in a comparatively short period,
‘bacteria being found in their blood ; this blood inoculated into
another animal .produces a similar disease, such as acute or
chronic abscess formation, which may be carried on from
‘generation to generation by simply inoculating a healthy
-animal with. the contents of the abscess. In these cases,
chowever, the organisms that are cultivated from the blood or
the pus fail to produce any symptoms at all, and it must be
‘toncluded (until further evidence is obtained) that the patho-
genic organisms cannot live on the artificial culture media,’
sthose that survive on these media being non-pathogenic:*
: There are, however, certain pathogenic organisms found in
‘the mouth which can be readily enough cultivated, the first,
and one of the most important of which is the micrococcus
of sputum septiczemia, which may be grown on blood serum
“or agar-agar at the temperature of the body ; it grows as a
“transparent greyish-white gelatinous coating on the surface of
-the nutrient medium, and looks almost like a dewdrop. It
‘is encapsuléd like the pneumococcus described by Friedlander,
‘and usually occurs in sputum in the form of single or paired
cocci ;. it is found almost invariably in patients suffering
‘from pneumonia, but it also occurs frequently in the mouths
of healthy persons. When injected into animals, either
‘in the sputum or asa pure cultivation, death usually occurs in
‘from twenty-four to thirty-six hours; numerous capsuled
‘cocci are found in the blood, the spleen is enlarged and con-
‘tains a number of organisms ; the symptoms, in fact, are
those of an acute septicemia, It has been observed,
*however, that pigeons and dogs are unaffected by this dis-
ease, whilst rabbits and mice are almost invariably killed
-by its inoculation. It would appear that when it makes
‘its way from the mouth to the healthy lung this organism
“has little or no power of attacking the tissues, but that
4f there be slight congestion or. inflammation, just as in
*the case of inflammation of the gums around the teeth, this
-organism, finding its way from the mouth (where it may have
‘existed for some time without giving any evidence of its
presence) into the air vesicles, is enabled to grow on the
-exuded fluid constituents of the blood, and to setup at once

‘_ ¥ Another explanation. of this will be found in the chapter on
- Leprosy.

BACTERIA OF THE MOUTH. 345,

those intense inflammatory changes characteristic of croupous
pneumonia, or acute inflammation of the lungs, and in some
cases, where the organisms appear to be more virulent, septic
pneumonia and gangrene of the lung. It may be, however,
that this gangrenous pneumonia ‘is the result of the invasion
and action of another organism. It has already been men-
tioned that the Streptococcus aureus and S. albus sometimes
occur in the mouth, but it would appear that these forms
are more frequently met with in the posterior nares and
in the cavities of the nose. To their action ~is. supposed
to be due the suppuration or festering that almost invariably
follows small operations of the’ mucous membrane of the
nostrils, unless the mucous surface is previously prepared. by
careful antiseptic washing out of the cavities and by frequent
application of antiseptics after the operation has been per-
formed. The’ micrococcus tetragonus is also found in the
mouth, whence, in cases of tuberculosis, it makes its way into
the lungs, and is there found, especially in suppurating cavi-
ties ; this organism, which is fatal to white mice and guinea-
pigs, usually occurs in little packets of four, each coccus
being about I in diameter. On gelatine, according to
Eisenberg, it grows as small white colonies, which when mag-
nified appéar to have a peculiar ground glass appearance ;
it does not give rise to any liquefaction of the gelatine.
Various other. septic forms have been isolated from sputum.
Tt will thus be ‘seen that in certain cases injury of the
mouth, of the periosteum of the jaw, or the soft tissues of
the pharynx, may lead to infections of very different, kinds,
but it may be laid. down as a general rule that .séptic’infec-
tion is frequently the result of invasion from these’ regions,
and very numerous are the cases recorded in which death has
resulted even from the most trifling operations in the mouth
and naso-pharynx. I have seen several cases where death
has ensued, with all the symptoms of most acute septicaemia,
or with symptoms of more chronic poisoning, as in pyaemia,
from the extraction of a tooth or the Jancing of the gums in
patients with imperfectly cleansed mouths, or in persons who
have-been engaged in attendance on patients suffering from
certain infectious diseases ; the organisms in such cases find-
ing their way from positions in which they were compara-
tively harmless, into the wounds that were unavoidably
made, whence they invaded the lymphatics or passed

346 BACTERIA.

directly into the blood stream and set up septic or other
mischief. -

It is scarcely necessary here to enter. into the different
forms of septic tooth disease or to consider the points at
which the different kinds of poison may enter, but it should
be mentioned in the interests both of antiseptic purity and
suffering humanity that a good stout tooth brush, plenty of
water and some antiseptic dentrifice applied morning and
night afford a greater safeguard against many diseases than
most people are aware.

LITERATURE,

The following works may be consulted :

AppoT.—Dental Cosmos. 1879.

Bronpt.—Breslauer drztliche Zeitschr, No. 18, Sept. 1887.

Davip.—Les Microbes de Ja Bouche. Paris, 1890.

FRrAENKEL.—Verhandl. d. 3. Congr. f. inner Med. 1884.

GALIPPE ET VIGNAL.—L’'Odontologie. March, 1889.

Hueppr.—Mitth. a. d. k. Gesundheitsamt, Bd. 11, p. 309,
1884., ;

Kirk.—Dental Cosmos, 1887.

K.ein.—Centralbl. f. Med. Wissensch, No. 30, p. 529, 1884.

LEEUWENHOEK.—Opera omnia sive arcana nature ope micros-
copiorum exactissimorum detecta. 1722.

*MILLER.—Micro-organisms of the Human Mouth. Phila-
delphia, 1890.

STERNBERG.—Bull. of the Nat. Board of Health. April 30,
1881.

VicNnaL.—Arch. de Physiol. norm. et. patholog., No. 8, 1886.

* Very full lists of literature given.

CHAPTER XX.

Tue BAcTEerRIA OF COLOUR AND PHOSPHORESCENCE.

Colour- Forming Bacteria—Micrococcus Prodigiosus—Magenta Micrococcus
—Beggiotoa Roseopersicina—Bacillus of Blue Milk—Sulphur Pigments
—lIron Pigments—Bacillus Fluorescens Putidus—Phosphorescent Bac-
teria—Six Species—Method of Cultivation—Conditions under which
they produce Light. :

Irv an organic medium, such as boiled potato, bread
soaked in broth, or nutrient gelatine, be exposed for some
time to the air of an ordinary room, it will be found that at
the end of a few days, in addition to the moulds of various
colours that develop on it, there appear minute yellow,
pink, or brown points, which on examination are found to
consist of yeasts, of sarcina, of micrococci, and of bacteria,
of which there are numerous species that give rise to these
coloured masses present in soil, air, and water. It is a well-
recognised fact that most putrefactive and decomposition
changes are associated with changes in colour of the putre-
fying media ; these colours being due to the activity of some
of the above colour-forming organisms which, in the exercise
of their full-assimilating and colour-forming powers, decom-
pose nitrogenous substances, the elements of which are con-
verted into the protoplasm of the organism, into coloured
material, and into the other excretory products to which
these organisms give rise. In some cases the colour is
actually contained within the substance of the organisms ;
usually, however, it is accumulated in the sheath, as in
the case of the Micrococcus prodigiosus, the organism to
which the bright red colour of “bleeding bread” and
“bloody sweat” is due. This pigment is quite insoluble in
water, but by means of alcohol it may be extracted and ob-
tained in solution ; this and other pigment-forming organisms,
such as the magenta micrococcus, mentioned by Greenfield as

348 BACTERIA.

occurring in-the water of the Tweed, contain a colouring
matter which has been described as resembling in a most
remarkable manner the aniline dyes; in fact, in the case of
the pigment of the magenta micrococcus, the resemblance is
carried so far that in old cultures even the peculiar metallic
lustre of the aniline dyes is reproduced. Other pigments
are soluble in water, but not in alcohol, whilst the bacterio-
purpurin formed by Beéggiatoa ‘Roseopersicina is like chloro-
phyll, insoluble in both alcohol and water. The name
bacterio-purpurin, however, has been ‘given’ by Engelmann
to the pigment produced or possessed by a whole group of
organisms. He concludes that it has, in thesd Tower organistis,
the function of the chlorophyll of the higher plants. If
exposed on the microscope:stage.to the light of. a sub-stage
spectroscope apparatus, these bacterio-purpurin . bacteria
invariably tend to.collect on that part of the slide that is
over thé ultra-red bands of the’spectruin, the portion of the
spectrum ‘where the absorption -of light by .the’ bacterio-
purpurin occurred. ‘He found that the analogy with plants
and. ‘chlorophyll ‘became ‘still closer: from’ the fact’ that
wheréver this: occurred, oxygen ‘was set - free, and” that
light in fact’ was necéssaty ‘for the continued éxistence.
of these bacteria and for the- development : of.” their
characteristic” colour - producing ‘power. In the casé “df
this‘organism; accérding to Ray Lankester, the coldur ‘is
actually contained’ within the protoplasm of ‘the organism,
where it appears in some cases to be‘ combined ‘with sulphur,
to form dark granules.. In some cases the colour, in’placé of
remaining ‘within the organism, becomes diffused into ‘the
surrounding media. As an example of this‘may be cited
the bacillus of “ blue milk,” which, growing along the track
of the needle in a gelatine ‘culture, sends’ out: a, peculiar
iridescent green coloration ‘into the surrounding gelatine’;
as time goes’ on this“ gréen is. replaced by a smoky, brown
colour. Similarly the Bacillus fluorescens putidus’ imparts
to the gelatine, or ‘any other material on ‘which: it is grow-
ing, a peculiar fluorescent green, and- at the same time gives
off an odour of herring brine. ~

That.the decomposition, of the sulphates in the presence of iron and
organic matter plays a most important part in the production of these pig-
ments has now been fully recognized. _If it be borne in mind that sulphides
of the'various metals appear as beautiful precipitates when thrown down from

THE BACTERIA OF COLOUR .AND PHOSPHORESCENCE. 349

solution it is quite evident that very minute traces of iron, say, acted. upon.
by the sulphuretted hydrogen set free by the decomposition of the sulphates,
can easily account for the. production of certain pigments., Cladothrix
dichotoma, for example, growing in water, appears able. to: separate iron
from the surrounding substances ; this becomes accummulated in the form
of. an oxide in the sheath. In the Beggiatoa the power of reducing the
sulphates is especially well marked, sulphur, which appears in regular
granules in the substance of the organisms, being stored up in the proto-
plasm to be utilized as it may be required. The sulphuretted hydrogen that
is formed, acts on the iron and gives rise to the formation of sulphide of iron.
In this way may be explained the presence of the pigment that is formed in

quds where these putrefactive organisms are present, as, for example, in
ihe mud of a tidal river. It should be remembered, however, that iron is
not the only metal that may be present, and that there is always a tendency
for the sulphuretted hydrogen to be set free from the sulphide and to give
way to the formation of oxides, especially in the presence of air and mois-
ture. : at gig Fes :

~ Only. by,the application of some such explanation as this
is it possible to account for certain of the beautiful brown
colorations that make their appearance in gelatine; for
example, that which surrounds the track of a needle inocu-
lation of some of those organisms, which, though colourless
themselves, give rise to most beautiful coloration of the
surrounding gelatine. So important is the presence of iron
in these cases, that Miller holds that to the action of organ-
isms on it is due most of the discoloration that occurs in
decaying teeth.

He points out that the colours characteristic of decaying
dentine only make their appearance some time after the
process ‘has commenced, the depth of the colour being in
direct proportion to the length of time that the decay has
been going on, and he. considers that this coloration is
due to the formation of sulphide of iron in the decomposing
enamel, dentine, and pulp. He performed an experiment
which may be-repeated ‘by any individual who is unfortunate
enough to be compelled to have a tooth of his’own drawn
for. decay, or who is fortunate enough to obtain one from
some other source. He says :—

™ eet = % te
“ A’tooth was cracked in a porcelain mortar, so as to thoroughly expose
the pulp, and then placed in a mixture of dilute hydrochloric acid, to which
was added a small proportion of a ten per cent. solution of ferrocyanide of
potassium. The hydrochloric acid, as well as the water used for diluting it,
rust be free from iron; neither must any iron implement be brought in
contact with the freshly broken surfaces of the tooth. Those parts of the

350 BACTERIA.

tooth containing iron, even in minute quantities, will, after an exposure from
one to sixty minutes, assume a blue colour—Prussian blue being formed.
One source of error is introduced in the necessary use of an iron instrument
in extracting the teeth, but this will only affect those points on the external
surface of the tooth with which the forceps come in contact, and therefore
may be easily eliminated.” He found by these experiments that there were
minute traces of iron in Nasmyth’s membrane, in the dental pulp (though
not constantly), in carious dentine and in enamel, and he considers that it
is quite possible that the sulphide of iron that may be formed during putre-
faction of the pulp—a process that is set up by micro-organisms—may have
something to do with the discoloration, though it is quite possible that
much of the discoloration is due to the iron that is taken into the mouth
along with the food, the putrefactive processes set up in the mouth liberating
the sulphuretted hydrogen, which, combining with the iron brought from
outside, gives the discoloration already referred to.

In addition to this coloration, the result of the formation
of inorganic salts, we have those violet, magenta, green and
yellow colours that appear to be distinctly organic in
character, and to be the result of the metamorphoses of
albuminoid substances. As above stated, these may be
related to the aniline colours, though this has certainly not
yet been proved to be the case. It may be well to bear in
mind, however, in this connection what takes place in the
process of colour formation set up by the Bacillus fluorescens
putidus, in which we have not only a colour resembling an
aniline colour, but we have a distinct odour of trimethyla-
mine, a substance nearly allied to the cyanogen compounds
from which, as we know, the most beautiful red, blue, and
yellow products are readily obtained when combining with
iron in certain definite ways. Of course this is only given
as an example of what might take place, and not as repre-
senting any accurate work that has been done, for it appears
that up to the present very little definite kndwledge has been
obtained as to the nature of the pigments contained within the
protoplasm of micro-organisms or of those diffused from it
into the surrounding tissues. What we do know is, that a
large number of the saprophytic decomposition-producing
bacteria give rise, when grown under certain conditions, to
most exquisite colour products, that by altering the con-
ditions, asin the case of the Micrococcus prodigiosus—subject-
ing it toa higher temperature, for example—the power of form-
ing these colours may remain in abeyance, the energy of the
protoplasm being diverted into the formation of some other
substance—in this instance, lactic acid. It has, however,

THE BACTERIA OF COLOUR AND PHOSPHORESCENCE. 351

been objected that such lactic acid formation can only take
place in the presence of sugar.”

In place of colour a certain amount of the energy of the
organism may be diverted to the production of light.
Although phosphorescent micro-organisms have for some
little time been known to exist, and special organisms have
been described as giving rise to phosphorescence in different
regions, they have not been very carefully studied until com-
paratively recently, when Forster, Tilanus, B. Fischer, Kunz,
Beyerinck, Lehmann, and Tollhausen have added very con-
siderably to our knowledge, not only of the morphology, but
of the biology of these special bacteria. In certain seas, and
especially on clear dark nights at the mouths of rivers,
any one who has rowed over them or steamed through them
may have observed a beautiful phosphorescence or fluorescent
glow at the bows or at the stern of the boat. As the oars
dip into and leave the water, they seem to shine with a pale
phosphorescent light. All kinds of explanations have been
given of this beautiful phenomenon, but it is now known
to be due in part or entirely to the presence of certain low
forms of life amongst which the bacteria take an impor-
tant place. First of all there was described she phos-
phorescent bacillus, then another was added, after this a
third, and now there are described no fewer than six of these
light-producing bacteria, arranged in three groups of two each.
The biological characters of these groups have been very
carefully studied by Beyerinck, who gave the result of his
observations in a most admirable paper presented to the
Royal Academy of Sciences, Amsterdam, 1890.

We may mention briefly some of the characteristic forms
and features of these light bacilli. P%oto-bacterium phos-
phorescens, which is 1.3 to 1.9n long, and 1.5 to 1.74 broad
is motile and is surrounded by a gelatinous membrane ; it is
readily cultivated on fish broth containing a small quantity of
peptone, or in sea-water ; it also grows (though slowly) on ordi-
nary nutrient gelatine or on nutrient gelatine to which herring
brine or 8 per cent. common salt has been added ; it brings
about the fermentation of glucose and maltose, its power of
producing light being apparently closely associated with these
fermentations, as when oxygen is cut off both the light and

* A number of the more important colour-producing organisms will be
found described in the Appendix,

352 BACTERIA,

the ,ferment-forming . powers of the organism: are at once:
interfered with, although growth and multiplication appear to.
go’,on, much as usual. The process of light production’
is evidently somewhat of the nature of an oxidation of the
food elements within the protoplasm under certain definite
conditions, the most important of these conditions being the
presence of oxygen and a temperature. ranging between
3° and 35° C. The Photo-bactertum phosphorescens grows
entirely as a surface colony, and although a. ferment action
is set up, there is no peptonizing power exerted, the gelatine
remaining quite solid. ‘

In a tube culture the organism grows down below the surface along the
track of the needle, but the phosphorescence is’ developed only on the
surface, where the organism can obtain a, plentiful supply of oxygen. In the
neighbourhood of the colonies, after a time, the gelatine takes cn a yellow-
ish-brown tinge. On all surface growths, whether on agar, gelatine or

potato, the grewth increases in thickness rather than in surface area. It
grows best at from 15° to 25° C, ‘ : ‘

A rather pretty’ story is related in connection with this
power of the organism to develop light. A lady, Madame
Salomonsen, the wife of Professor Salomonsen of Copen-
hagen, was able to obtain photographs of the light bacillus
made on gelatine plates and so cultivated them as to form
the letters of a complimentary message to M. Pasteur
(“Hommage a M. Pasteur”) ; the photographs came out very
distinctly, and conveyed in a most delicate and striking
manner the message which the lady wished to send.

Photo-bactertum Fluggerd is the most phosphorescent of
all the light bacilli; it grows in nutrient gelatine as longer
and thinner threads than the preceding form. It differs
from it also in that, although it exercises its characteristic
light function when supplied with peptone and glucose,
maltose cannot take the place of glucose. Both organisms
in setting up their fermentation processes bring about the
evolution of CO, and hydrogen. :

If a number of fresh cod or herring, the surfaces of which have not been
allowed to dry, be placed between a couple of plates and kept at a tem-
perature of about 15° C. or upwards, there may be made out at the end of
about twenty-four hours a number of small p' i ey points, and at
the end of a couple of days the whole of the fishes are coveréd with a
phosphorescent glow; but as putrefaction sets in this glow is gradually
lost, There may be separated from these patches an organism 1.5

-

THE BACTERIA OF COLOUR AND PHOSPHORESCENCE. 353

to 1.94 long and 1.3 to 1.74 broad; these rods have rounded ends
and appear to divide exceedingly rapidly, in consequence of which
the cells are usually almost round, and are then very like large micro-
cocci, in fact they are sometimes compared to the Bacillus prodigiosus
which was for long spoken of as a micrococcus. Sometimes a few organisms
may be held together in a short chain ; the bacterium is motionless, and no
spores have as yet been observed. On plates prepared with peptone gelatine,
to which a small quantity of glucose, and from two to three per cent. of
common salt have been added, the organism develops luxuriantly, giving
rise to small white mother-of-pearl-like colonies, about the size of a pin’s
head, with no surrounding zone of liquefied gelatine. Under the microscope
these are seen as small, round, yellowish-white, granular drops, with sharp
but irregular margins.

Another organism, the Photo-bactertum Fischert, found in
the waters of the Baltic, peptonizes gelatine, causing it to
liquefy very rapidly. It can exist in a medium to which a
small quantity of raw sugar has been added, this addition of
sugar increasing in a most remarkable manner the intensity
of the light given off, although a large quantity of the same
material (three to five per cent.) interferes with, or altogether
stops, the phosphorescent activity of the organism. This
organism is motile ; it occurs in-short chains and grows on
gelatine and agar, the former of which is liquefied by its
action. Grown on plates, the colonies after making their
appearance emit a kind of bluish-white light, and the
organisms themselves as they lie at the bottom of the fluid
gelatine have also a somewhat bluish tinge. It grows best
at a low temperature, from 15° down to o° C., or even lower.

Photo-bacterium Balticum also liquefies gelatine, but more
slowly than the above. It is not, however, dependent upon
glycerine for its growth, and is not nearly so sensitive to the
presence of a considerable quanity of sugar, as it can live in
a medium containing from three to five per cent. of that
substance. These four forms are all what may be called pep-
tone carbon bacteria, as they cannot develop their functions
to their highest point without the presence of some sub-
stance from which carbon may be readily obtained such as
sugar, glycerine, glucose, &c., as well as peptone, but the
Photo-bacterium Fischeri and Photo-bacterium Balticum
do not set up any ferment action as do the first two
mentioned. ;

Beyerinck states that all four are best cultivated in fish
broth made with sea-water, to which are added one per cent.

24

354 BACTERIA.

of glycerine, one-quarter per cent. of asparagin, and eight
per cent. of gelatine.

. The Photo-bacteriun Indicum of the West Indies and
the Photo-bactertum luminosum of the North Sea, both
liquefy gelatine very rapidly, and appear biologically to be
much more like ordinary putrefactive micro-organisms,
though the most favourable conditions as regards tempera-
ture for the performance of their functions differ somewhat ;
that from the West Indies giving off most light at from 30°
to 35° C., that from the North Sea being most active in
this respect at about 15° C. Both of them may multiply
and give off light in peptone geiatine without requiring
the presence of any sugar; in fact, they are both extremely
sensitive to the presence of this substance, one per cent.
preventing the phosphorescence, and three to five per
cent. interfering with the liquefaction of the gelatine and
eventually killing the bacteria, though, if a small amount of
asparagin also be added to the medium in which the West
Indian phosphorescent bacillus is growing, light may continue
to be given off for some time. . The Photo-bacterium Indicum
isa motile rod of medium size which grows very readily on all
the ordinary nutrient media ; the light is best seen in this
case by taking a fish that has been boiled and inoculating
with a fragment of the artificial culture ; there then appears
in a very short time a soft glistening point, which is
found to be covered with bacteria. On darkening the
room a beautiful bluish-white light can be seen at the point
of inoculation. The light of Photo-bacterium phosphorescens
is yellowish.

Beyerinck gives a number of experiments to show that the
formation of light bears no direct relation either to the
respiration or to the growth of the organisms, but he finds
that certain food substances are necessary for this light to
make its appearance, although the growth of the colonies
may go on perfectly well without oxygen, even if the forma-
tion of light be completely stopped. As soon as the
organism is grown anerobically, certain food substances
also become necessary.

THE BACTERIA OF COLOUR AND PHOSPHORESCENCE. 355

LITERATURE.

The following works may be consulted :

BEYERINCK.—Trans. of the Dutch Acad. of Scien. Amster-
dam, 1890.

ENGELMANN.—Bot. Zeit., 42-46, 1888 ; Pfluger’s Arch., Bd.
XXX., p. 95, 1883, and Bd. x1iz., p. 183, 1888.

FiscHer.—Centralbl. f. Bakt. u Parasitenk., Bd. ut., Nos.
4-5, 1888; and Bd. iv., No. 3, 1888.

Forsrer.—Centralbl. f. Bakt. u Parasitenk, Bd. 1., No. 12,
1887. :

GREENFIELD.—Scottish Fishery, Bd., Reps., vols. 11., 1v., and

VI.

Katz.—Centralbl. f. Bakt. u. Parasitenk., Bd. 1x., Nos. 5-8,
1891.

LeHMaANN.—Centralbl. f. Bakt. u. Parasitenk., Bd. v., No. 24,
1889.

MILLeR.—Micro-Organisms of the Human Mouth. Phila-
delphia, 1890.

Ray LANnKESTER.— Quart. Journ. Med. Scz., vol. X11, p. 408,
1873.

Wrnocrapsky.—Bot. Zeit., Nos. 31-37, 1887.

“CHAPTER XXI.
Potsonous ALKALOIDS AND ALBUMINOIDS.

Early Observatigns—Burrows, Kerner, Panum, &c.—Ptomaines—Leuco-
maines—Brieger’s Work—The Alkaloids, Poisonousand Non-Poisonous
—General and Local Reactions—Structure and Composition—Sketch
of Chemistry—Cholera Poisons and other Members of this Group—
Mussel Poisons: two Classes, viz., those Without Oxygen and those
Containing Oxygen—Ptomaines the Result of the Activity of Micro-
Organisms — Loffler’s Experiments on Diphtheria Poison, an
Enzyme—Roux and Yersin’s Diastase -Hankin, Albumose—Brieger
and Fraenkel, Toxalbumen—Method of Preparation of Albumoses
and of Toxalbumens—Near Relationship of Proteid Poisons and the
Ptomaines—Martin’s Observations.

It has long been known that the products of putrefaction,
especially those formed during the putrefaction of fish, are
extremely poisonous. In 1814 Burrows, in this country,

described such a poisonous substance in putrefying fish; ~~

in ‘1820 Kerner described a poisonous alkaloid which
resulted apparently from the decomposition of albumen ;
it resembled in its physiological action a substance that he
had found in poisonous sausages, and which he compared with
atropine as regards its toxic reaction. In 1856 Panum was
able to obtain a substance from decomposing animal matter
which appeared to him to be derived from albuminoid
substances through the action upon them of micro-organ-
isms. He looked upon this as a purely chemical material.
He was able to isolate it by dissolving it in water or
alcohol ; in quantities of about six centigrammes it proved
fatal to dogs, and this substance, to which the name sepsin
was given, came to be looked upon as the cause of putrid
infection or intoxication. Twelve years later Bergmann
and Schmiedeberg obtained what they took to be a similar
substance ; it contained nitrogen, could be crystallized out
and separated, and was also-evidently the result of a putre-
factive process.

POISONOUS ALKALOIDS AND ALBUMINOIDS. 357

The first mention that is made of the probable chemical
composition of such organic poisonous substances is found
in a paper by Zuelzer and Sonnenschein, who describe
as an alkaloid a substance that they were able to obtain
from decomposing animal matter, which they said closely
resembled atropine in its physiological actions; it caused
dilatation of the pupil, paralysis of the inhibitory fibres of
the vagus, so allowing the heart's action to become accelerated,
and paralysed the non-striped muscular fibres of the intestine.
This alkaloid was supposed to resemble the vegetable alkaloids
of which a considerable number had then been described: more
recent observations have shown that these alkaloids are very
nearly related, from the fact that they all appear to have as a
common basis or ground structure a substance named pyri-
dine, of which more immediately. In 1872 Armand Gautier
described as the products of albuminous decomposition a
number of alkaloids; and Selmi, between 1871 and 1880,
described what he called cadaveric alkaloids or ptomaines,
and he was able to obtain two new alkaloids from pure
albumen that had undergone putrefactive changes. Pouchet
in 1880 described an alkaloid in urine, and in 1882 Bouchard
also described alkaloids in the human urine, which, he con-
sidered, were the result of the decomposition of proteid
matters in the alimentary canal, and which were excreted
from the body by the intestines and by the urine, through
the kidneys. He concluded that these alkaloids are usually
found in health in certain definite quantities, whilst in cer-
tain diseases—typhoid fever, for instance—they may be
enormously increased in quantity, and can then be separated
from the urine in very considerable quantities.

The first ptomaine separated pure was obtained by Nencki,
then Brieger obtained several of these alkaloids from pure
cultivations of micro-organisms, but as early as 1880, Pasteur,
after failing with the sterilized products of Anthrax, was
successful in producing the symptoms of Fowl Cholera with
the sterilized products of that organism, z.e., with the
poisonous alkaloids or proteids. Of those in the pathogenic
group Brieger first described the substances that he was able
to obtain from pure cultures of the typhoid bacillus and of
the tetanus bacillus. From the former he obtained typho-
toxine ; from tetanus cultivations tetanine, which produces
characteristic tetanic symptoms in animals, tetanotoxin

358 BACTERIA,

which also produces some of the symptoms of that condition ;
and two other alkaloids both of which give rise to certain
definite symptoms, acting physiologically somewhat like
strychnine. The importance of the presence of these
substances in pure cultivations of pathogenic organisms
can scarcely be overestimated. In the vegetable kingdom
there are recognized whole series of substances that have
an alkaline reaction, combine with acids to form salts,
are evidently formed by the protoplasm of the plants in
which they are found, and which, injected into the tissues
of an animal or taken by the stomach, exert a most
energetic poisonous action either upon the end organs in
muscles or upon the muscles themselves. Of these we may
take such well-known examples as strychnine, atropine,
nicotine, cinchonine, thebaine, morphine, brucine, and others.
They are all of them built up by vegetable cells and all exert
a specific action on animals. Similarly it is found that bacteria
—minute vegetable organisms—can build up substances, as
they grow in dead or living animal tissues in which they are
living as saprophytes or parasites, which substances exert
a most deadly influence on the nerve centres or the parts
above mentioned of animals in which they are formed or
into which they may be injected, but in addition have an
extremely injurious local “ caustic ” influence, giving rise in
many cases to the death of the tissues with which they may
come in contact at the points where the poison is formed, or
at the seat of inoculation. Thus tetanine is a substance that
appears to act through the nervous system much as does the
alkaloid strychnine, whilst in sepsine, material formed by
those bacteria that are found in a local abscess, we have a
powerful acrid substance which by its caustic action causes the
death of the cells with which it is allowed to come imme-
diately in contact. Some of the most deadly of the poisons
formed by micro-organisms, however, are not of the nature
of alkaloids, but are said to belong rather to the classes of
globulins and albumoses. A number of them, however,
give some of the reactions of the alkaloids, but they must
not on that account be looked upon as belonging to that
group. Brieger includes under the term ptomaine all
nitrogenous bases that are formed by the action of bacteria,
such of those as are poisonous being spoken of as toxines.
It thus happens that certain ptomaines that are formed

POISONOUS ALKALOIDS AND ALBUMINOIDS. 359

during putrefactive processes are non-poisonous, whilst others
formed during the same process may be extremely toxic; a
considerable number of the non-poisonous forms especially
have been manufactured synthetically or by analysis ; tri-
methylamine and dimethylamine, for example, both of which
as well as Pentamethyline diamine (Cadaverine) may be
obtained as putrefactive products and may also be prepared
synthetically by the chemist, the artificial Cadaverine in
sufficiently large doses giving rise to all the symptoms
and post-mortem appearances of an attack of cholera.
Of the poisonous kinds a substance known as betaine,
which is closely related to nicotine and glycocol, has been
found in both vegetable alkaloids, and in the human urine,
in the latter being apparently the result of decomposition
changes going’ on in the alimentary canal under the action of
bacteria. It is one of the substances formed during the
processes of decomposition of albuminoid bodies ; it also has
been prepared in the laboratory.

The formule of a number of these substances is exceedingly compli-
cated, but they all appear to be allied to or even to be derived from what
is known as the pyridine base, a non-saturatéd alkaloid, derived from
the products of dry distillation of bone or wood, from the ammoniacal
liquor of coal distillates, and H
from the action of heat or H Cc
strong alkalies on the vegetable
alkaloids. Its composition will
be best understood by reference
to the diagram given by Pictet
(Fig. 1). It is very nearly re- HC——_ ¢
lated to benzol ; the only dif- HC——____ Gyy
ference being that one of the
CH groups is replaced by N.
Thus benzol has a formula
(Fig. 2). Neither of these
substances has all the carbon HC__! cH
bonds satisfied, so that each HC_—_L— CH <

C and the N having a bond
free (those drawn within the

hexagon) to combine with
other atoms, or groups of cS
atoms, there may be enormous - N H
numbers of derivative or addi- Fic 1 Fic. 2.

tion and substitution products formed ; for instance, all the bonds free in
pyridine may be saturated by the addition of a single atom of hydrogen to
each, when we have what is known as the piperidine alkaloid (Fig. 3), and
by adding one, two or three ethyl or methyl groups to the free bonds in
place of one, two, or three of the H’s we may obtain methyl piperidine,

.

360 BACTERIA.

dimethyl piperidine, and so on throughout the whole group. In fact, where
H, there are so many bonds to be acted upon the number
Cc of organic compounds is almost endless, and it can
be readily seen how substances varying much in
chemical composition can still be arranged in groups
under a common head; the members of each series
having very much in common not only as regards
HC CH: their chemical character, but, as has recently been
pointed out, as regards their physiological action. As
a considerable number of the benzol series have been
manufactured by the chemist, it comes quite within
the range of possibility that a number of the ptomaines
(especially those of the pyridine series) may also be
ELC CH built up synthetically.

The most important of the methods used for the sepa-
ration of the ptomaines is that used by Brieger, who is
perhaps the greatest authority on this subject. He
goes to work with the salts of the heavy metals, and

with picric acid. When he wishes to separate an
H alkaloid from any putrefying mass, this mass is first

Fic. 3. boiled with water, and then filtered ; the filtrate is then
treated with sub-acetate of lead; from this the lead is precipitated by
sulphuretted hydrogen which is passed through the filtrate, and the fluid is
again filtered to keep back the lead sulphide. This second filtrate is
evaporated to about one-third of its original bulk, and is mixed with amyl-
alcohol, it is then thoroughly washed with water, and again reduced in bulk
by evaporation, and sulphuric acid and ether are added; the ether is
evaporated, after which the remaining liquid is concentrated by careful
evaporation to one-third of, or one-fourth of, its bulk; the evaporation
driving off most of the volatile fatty acids present, after which the fluid
neutralized by the addition of baryta, is again filtered, carbonic acid gas
is passed through it, by which baryta carbonate is thrown down which
is separated by filtration. After careful heating over a water bath, the
fluid is cooled, and bichloride of mercury is added, when a somewhat
dense precipitate is formed. This precipitate is carefully washed and
decomposed by sulphuretted hydrogen, when sulphide of mercury is
thrown down; the fluid is again filtered and the filtrate is evaporated
to obtain as great concentration as possible. From the liquid so ob-
tained all inorganic substances crystallize out first; these are removed,
and then in the fluid that remains ‘‘organic” acicular crystals are
thrown down, These may be dissolved in water, but they are insoluble in
absolute alcohol, ether, benzine, or chloroform. It is found that the sub-
stances so given, the ptomaines, may be precipitated by the salts (especially
the chlorides) of the heavy metals. These precipitates or crystals
differ, however, very considerably as to their solubility ; hydrochloride of
putrescine obtained by the above method separates out in acicular crystals,
and on the addition of chloride of gold gives very insoluble crystals of an
octahedral form, whilst on the addition of chloride of platinum, octahedral
crystals which are much more soluble, are also formed. Phospho-
molybdic and phospho-wolframic acid added to this substance give respec-
tively a yellow and a white crystalline precipitate. Iodide of mercury
dissolved in iodide of potassium also gives rise to the formation of prisms ;

POISONOUS ALKALOIDS AND ALBUMINOIDS. 361

with ferrocyanide of potassium there is ‘a yellowish amorphous precipitate;
with picric acid a yellow precipitate composed of delicate needle-shaped
crystals ; and with a watery solution of bichloride of mercury an exceedingly
insoluble acicular crystalline precipitate is thrown down. This substance
and the reactions obtained with it may be taken as typical of the whole
group, although there are certain differences; for instance, cadaverine
treated with chloride of gold gives a very soluble substance, whilst with
chloride of platinum there are thrown down well-formed very insoluble
crystals. Mydaleine is exceedingly soluble in most of its combinations,
and it is at present almost impossible to separate it from the mother liquid ;
in fact, its salts have not yet been separated, and in consequence it has
been found impossible to determine its exact chemical nature. These,
along with saprine, were obtained by Brieger from flesh that was being
decomposed by the action of putrefactive micro-organisms.

Cadaverine and putrescine are both poisonous, giving rise
to local death of the tissues along the course of the intestine ;
they have, however, not nearly such marked general toxic
activity as have some of the other alkaloids. They are all
somewhat volatile, and cadaverine as we have already seen
is very readily formed where the cholera bacillus is allowed to.
grow and act on proteid matter such as egg albumen. Of a
more poisonous nature is neurine, a substance that is also
formed in connection with the putrefaction of flesh ; it has
been separated by Brieger and is readily obtained in crystalline
form by the addition of chloride of platinum. Choline,
which was supposed at one time to be identical with neurine,
and is by Brieger said to be the same substance, is really
hydrated neurine. These two substances are exceedingly
toxic and appear to have an action very similar to the vege-
table poison, curare. A very similar substance, muscarin,
has been described by Schmiedeberg and his pupils, as
occurring in a kind of poisonous mushroom, and was found
to have a composition very like that of choline and neurine.
It is an extremely toxic substance and is specially interesting
from the fact that we have it formed as a vegetable alkaloid
in the mushroom, whilst it has also been found in putrid
fish, thus giving us another link between those so-called
animal alkaloids found in decomposing albuminoid matter
and the vegetable alkaloids. This substance is a most
powerful muscle poison, its action being somewhat like
that of eserine.

It will be remembered that not long ago there was a sad
case of poisoning in Dublin, in which the wife and family
of a well-known journalist died after partaking of mussels,

362 BACTERIA.

The poison in this case was probably an alkaloid substance
that was separated from decomposing mussels by Brieger,
who gave to it the name of mytilotoxine. Dr. Vaughan in
America separated from cheese that had undergone putre-
factive changes a substance that he called tyrotoxine, and
‘he was able to separate from some ice cream that gave rise
to most acute poisoning a very similar substance.

Jacquemart, giving an account of these ptomaines, divides them into two
groups—those that are fluid and are volatile, that havea peculiar characteristic
smell, and that contain no oxygen, being in the first group; those in the
second are solid, non-volatile and contain oxygen. Those of the first group
are soluble in ether and also slightly in amyl-alcohol and chloroform. The
members of the second group are usually crystalline, are soluble in water,
but are insoluble in alcohol, benzine, and chloroform. Although they are
extremely unstable they unite with acids, in excess of which, however, they
are soluble, when we have first a red colour and then a brown deposit of
acicular crystals. An excess of chloride of platinum or strong light usually
causes their disintegration. It should be remembered that corrosive
sublimate does not precipitate some of these alkaloids, although there is
undoubtedly a salt formed which can be obtained in white crystals by
evaporating from watery solutions. The only substance that gives invariable
reactions with all these ptomaines is phosphomolybdic acid..

The following ptomaines contzin no oxygen: Parvoline, described in
1881 by Gautier and Etard, who obtained this substance from putrefying
mackerel and horse: flesh ; its formula is C,HisN (also given as CyH,,N).
It is a light yellow substance readily soluble in water, alcohol, ether, and
chloroform, it turns brown on contact with the air ; with chloride of platinum
it forms a somewhat insoluble crystalline flesh-coloured substance, which
rapidly becomes rose-coloured on exposure to the air. These authors also

. described a substance Hydrocollidine (formula CsH,;N, sometimes given
as having two atoms less ate eae It is a colourless, oily fluid, becoming
brownish on exposure to the air; when treated with carbonic acid gas it
becomes sticky. It forms a double salt with chloride of platinum, a pale
yellow crystalline insoluble substance, though it dissolves on heating
without undergoing any disintegration. Collidine, with the formula
CeH,.N, was first obtained from decomposing pancreas and gelatine. It is
a yellowish, mobile fluid with an extremely offensive odour, very soluble in
water, but much more soluble in methyl and ethyl alcohol and in ether.
Neuridine, C;H.,N2, Cadaverine, C,HieN2 (sometimes given as isormeric
with neuridine). Putrescine, C,H:2N., Saprine, C;H,,N., and Mydaleine,
all belonging to this group, have been already mentioned. The
ptomaines that contain oxygen hold a_ kind of intermediate position
between the above group and the Leucomaines or physiological alkaloids.

Neurine, C;H:,N (OH), is a strong base exceedingly soluble in water.
Choline, C,H;,;NO., Muscarine, C;H,,;NO, (or C;Hi;NO., or Choline from
which H, has been removed by nitric acid), and two other ptomaines
described by Pouchet, and having the formulze C,H:;N.O0¢ and C,HzN.O,,
make up the solid ptomaines of the second group; whilst Gadinine
C;H,,NO, is not solid, although in other repects it resembles the members

POISONOUS ALKALOIDS AND ALBUMINOIDS. 363

of this second group. To this group also belong Mytilotoxine, CsH,,NOz,
Typhotoxine, C,H.,NO., and Tetanine C,3;H22N20,, which appears to be
really a double pyridine molecule, and is therefore probably a mixture, asare
also a number of the others above mentioned,

Most of these substances are found to be associated with
the decomposition of dead material by micro-organisms, but
it has long been known that substances similar in many
respects (some of them of an exceedingly poisonous nature)
are formed in the body of the living subject, resulting from
the purely physiological nutritive changes in the protoplasm
of the various organs and tissues in the body; they are
in fact excretory products which must be got rid of, and
which if retained interfere, in some cases very materially,
with the vitality of the protoplasm. These were named
leucomaines by Gautier to distinguish them from the
ptomaines ; they are fully described in physiological text-
books with the uric acid and creatinine groups of sub-
stances, to one or other of which they belong as far, at all
events, as their chemical composition is concerned. Some
of these leucomaines are, as we have said, exceedingly
poisonous, and when retained may give rise to very serious
toxic symptoms. Brieger and others, however, deny that
any such bodies are formed or at any rate have yet been
found in the tissues of the living body or that they owe their
existence to the tissues. They consider that they are simply
absorbed from the intestinal canal where they are formed by
bacteria.

In 1887 Léffler, when examining the products of a pure
culture of the diphtheria bacillus that he had obtained, found
that the fluid from which all the organisms had been
removed by filtration through a porous porcelain cylinder,
when injected into a guinea-pig gave both the local reaction
and the paralytic symptoms that were obtained when the
organism itself was introduced into the subcutaneous tissue.
In order more readily to determine the nature of this material,
he added to.a pure culture of the organism a quantity of
glycerine ; this when filtered and dropped into absolute
alcohol gave a floculent precipitate which could be freely
washed with alcohol without passing into solution, but on the
addition of water it was again dissolved. It could be again
precipitated by alcohol, and after the passing of carbonic
acid gas through the precipitate, it retained a certain toxic

364 BACTERIA.

property,—still setting up distinct local reactions. Léffler
eventually concluded that he was dealing with an enzyme.

I have already mentioned Roux and Yersin’s experi-
ments, from which they also concluded that they had ob-
tained a substance similar in many respects both to a disastase
and to an enzyme. Hankin, working from the fact that a
poisonous albumose had been discovered in snake poison,
set about the task of isolating an albumose from anthrax
cultures. He madea cultivation of anthrax bacilli, then pre-
cipitated it by large quantities of absolute alcohol and washed
the precipitate thoroughly to dissolve out any ptomaines
that might be present; this precipitate was filtered and
dried, then re-dissolved in water and the solution passed
through a Chamberland filter. This albumose is very
similar in its characters to the albumoses ordinarily described,
which really consist of albumen that has been altered by
hydration either by super-heating by steam under pressure,
or during the process of natural digestion, in which albumens
are converted stage by stage into peptones. These albu-
moses are intermediate non-coagulable hydrated albumens.
Some forms are soluble in water, others are insoluble. That
described by Hankin as formed by the anthrax bacillus is a
soluble form. Brieger and Fraenkel obtained what were
apparently similar substances from pure cultivations of cholera
bacillus, typhoid bacillus, tetanus bacillus, from staphylo-
coccus aureus, and diphtheria bacillus, with all of which they
were able to produce toxic effects, some at least of which were
similar to those met with as the result of inoculation with
the bacteria themselves. These observers, however, did not
separate from the albumoses that were formed any enzymes
that might be present, consequently they were working
with a mixture of substances. The products that they
obtained gave most of the ‘reactions of albumoses ; they were
certainly toxic, but they probably contained both enzymes
and albumoses.

As these albumoses, described though not recognized under
that name by Wooldridge, are destined apparently to play a
most important part in the production of immunity against
disease, it may be well here to give ashort description of the
methods adopted by Hankin to obtain his albumose from the
anthrax cultures, and by Brieger and Fraenkel and Babes from
cultures of other organisms (Sidney Martin has been able to

POISONOUS ALKALOIDS AND ALBUMINOIDS. 365

separate from anthrax cultures, in addition to a poisonous
alkaloid, two albumoses, which apparently represent slightly
different stages in the transformation of albumen into pep-
tone) : it may be well also to give the characteristic reactions
that are obtained with these albumoses.

Hankin’s method is as follows : A I per 1,000 pure solution of Liebig’s
extract of meat is carefully sterilized by being heated in a small sterilizer for
two or three hours on two or three successive days ; to the fluid so sterilized
a quantity of pure fibrin is added and. the whole is again sterilized ‘by
repeated heating to boiling point for a short time only on each occasion ” 3
if this is heated for a longer period a considerable quantity of the fibrin is
digested and converted into peptone, a substance that would interfere very
considerably with the after-examination, in addition to which the anthrax
bacillus would have little material left on which to exert its ‘‘ peptonizing ”
function. This is inoculated with blood from an animal that has died from
anthrax and is ‘‘kept at the ordinary temperature.” The cultivation is
allowed to go on for a week, at the end of which time the albumose is
extracted. Ifthe flask be kept at the temperature of the body, 37°C, the
transformation of the albumose into the peptone goes on much more rapidly.
To separate the albumose the culture fluid is first acidulated with acetic acid,
and then thoroughly saturated with ammonium sulphate, when there is thrown
down a bulky precipitate of albumose. In order to concentrate the solution,
instead of using Brieger’s method of evaporation 2 vacuo or under pressure at
a low temperature he resorted to the method of diffusion or dialysis. A quan-
tity of thymol, to prevent putrefaction, is added to a watery solution of the
albumose, and the whole is placed in a parchment sausage skin which is im-
mersed in a foot glass full of methylated spirit. The spirit can be changed
after some hours if it is necessary to prolong the process, but this is not
usually necessary. ‘‘ In this way,” says Hankin, ‘‘I have been able to bring
400 cubic centimetres of albumose solution down to 100 c.cm. in the course
of a single night, at the ordinary temperature without risk to the albumose
or trouble to myself. The concentrated solution is then poured into
absolute alcohol, which precipitates the albumose and removes any im-
purities that might be derived from the methylated spirit. This prolonged
treatment with alcohol will tend to remove any free ptomaines or other
substances soluble in alcohol.” In order to remove any ferments that are_
capable of acting along with the albumose, Hankin, following Roux and
Yersin, adds a quantity of lime water to his solution, so that, on the addi-
tion of a solution of phosphoric acid, a.gelatinous precipitate of calcium
phosphate is produced, in the formation of which ferments are usually
entangled and carried down, and on filtration a purer solution of albumose
is obtained.

Brieger and Fraenkel have adopted Hankin’s method in the
preparation of their toxalbumens, but instead of dialyzing,
they evaporate down zz vacuo at a low temperature until
the liquid has been reduced to less than a fourth of its
original quantity. They again wash in alcohol and filter.

366 BACTERIA.

Brieger and Fraenkel found Millon’s reagent gave a
white precipitate, which on heating became brick red in
colour this indicating its proteid nature ; it is precipitated
by magnesium sulphate in saturated solution ; it is there-
fore not an ordinary albumen ; whilst on the addition of a
drop of dilute sulphate of copper solution and a slight
excess of potash solution (the so-called biuret reaction) a
rose red and not a violet colour is given, indicating that
this material belongs to the albumose rather than to the
globulin group. ‘There are a number of other tests which
it is not here necessary to describe.

It must be remembered, however, that these proteid
poisons and the ptomaines are very closely bound up
with one another. Martin indeed holds, that as some of
the albumoses are less toxic than the alkaloids with which
they occur and as they also have a marked alkaline reaction,
the alkaloid may really be bound up in a nascent condition
in the albumose molecule. This may undoubtedly be the
case with certain vegetable alkaloids and albumoses, but in the
cases of diphtheria and tetanus, it would appear that some of
the so-called poisonous alkaloids owe their specific properties
to the presence of minute traces of an enzyme, or proteid
poison that is present along with them. It has even been
suggested that the alkaloid may be the poisonous agent
formed by bacteria, whilst the albumose is to be looked upon
as the protective agent. This, however, can scarcely be
maintained except in certain cases, as in the case of tetanus
the poisonous agent is certainly not of an alkaloidal nature.
Still, these points should be borne in mind by any one going
to work at the question. It seems to be undoubtedly the
case, that in accordance with the well-known fact that
certain products of micro-organisms, such as those of acid or
alcoholic fermentation, act deleteriously on the bacteria that
produce them, especially as they accumulate in large quan-
tities. On the other hand, it may be that these, or similar
products in a more or less dilute form, may be necessary

* Millon’s reagent is prepared by adding one part of mercury to two
parts of strong nitric acid, gently warming until the mercury is thoroughly
dissolved: to one part of this mixture two parts of water are added; a
precipitate is formed ; the supernatent fluid only is used. A few drops
of this solution give the above characteristic reaction with all proteid
materials held in solution except in the presence of common salt.

POISONOUS ALKALOIDS AND ALBUMINOIDS. 367

in order to allow of the growth of bacteria as parasitic
organisms in animal tissues.

LITERATURE.

The following works may be consulted :
Authors already referred to. Babes, Brieger and Fraenkel,

Hankin, Roux and Yersin.

BERGMANN UND SCHMIEDEBERG.—Med. Centralbl., No. 32,
q- 497, 1868.

Ser mens rendus de la Soc. de Biol., p. 604,
1882.

BriecerR.—Untersuchungen uber Ptomaines, Three parts.
Berlin, 1885-6. :

GAUTIER.—Sur les Alcaloids, &c., Paris, 1886; Bull. de
l’Acad. de Med., 2™ Série t. xv.-xv1., 1886.

maar Et Erarp.—Comptes rendus, t. xciv., p. 1601,
1881.

GrirFiTHs.—Micro-Organisms. London, 1891.

HAtiipurton.—Text Book of Physiology and Pathology.
1891.

Hankin.—Proc. Roy. Soc. May 22, 1890.

JACQUEMART.—Journ. de Med. de Chir. et de Pharmacol.
Bruxelles, No. 18, 1890.

LOFFLER.—Mitth. a. d. k. Gesundheitsamte, Bd. 1, p. 421,
1884 ; Die Geschichtliche Entwickelung der Lehre von
den Bacterien, Leipzig, 1887 ; Deutsche. Med. Woch.,
5 and 6, 1890.

Nenck1.—Ueber d. Zersetz. d. Gel u. d. Eiweisses b. d. Faul-
niss Mit Pankreas. Bern., 1876; Berecht d. deutsch
Chem. Gesellsch., p. 2605, 1884; Journ. f. Prakt. Chemie,
Bd. xxv, pp. 268, 273, 1882.

PanuM.—Bibliotek for Laeger, pp. 253-285, April, 1856 ;
Virch. Arch., Bd. Lx., p. 301, 1874.

PAsTEUR.—Comptes rendus, t. Xc., p. 1030, 1890.

SELM1.—Ber. d, deutsch. Chem. Gesellsch., 1873-1880 ;
Alcaloidi Venefici esostanza amiloide dall’albumina in
putrefazione. Rome, 1879.

SONNENSCHEIN AND ZUELZER.—Berl. Klin. Woch., p. 123,
1869.

Sypney Martin.—Proc. Roy. Soc. May 22, 1890.

CHAPTER XXII.
VACCINATION.

Natural Immunity—Ingrafting of Small-pox—Jenner’s Discovery—Klebs
—Pasteur—Chauveau—Grawitz’s Theory—Buchner’s Theory—Met-
schnikoff s Work—Greenfield’s Observations on Anthrax—Toussaint’s
Vaccinal Fluid—Pasteur’s Classical Experiments-—-Other Methods of
Diminishing Virulence—Chauveau’s Soluble Poison Experiments—
Protection’ by Alkaloids, Albumoses and Toxalbumens—Saprophytic
* Anthrax ”’—Hueppe and Wood—Antagonism by Blue-pus Products
Parone not the same as Antagonism—Summative Action of

acteria.

Ir has long been recognized that in a number of infective
diseases, one attack confers a certain immunity against
that special disease in the future. This protective influ-
ence may last for a very considerable period, or there may
be merely a temporary immunity. It was early noticed,
however, that the severity of the primary attack of the disease
appeared to bear no very definite relation to the degree of
immunity acquired by the patients, although immunity of
an animal usually bears a very direct relation to the strength
of the vaccine which it has withstood. In fact every degree
of immunity may be produced, though this does not always
correspond with the severity of the symptoms. Immunity
appears to be governed by no very regular laws, for it
is found that even in the same species, individuals may
differ very considerably as regards susceptibility to first
or subsequent attacks of some of the infective diseases. It
is known, for example, that whilst certain people have
never more than a single attack of measles, others may
have several, and that similarly, certain individuals are
specially liable to the recurrence of scarlatina, suffering
no fewer than three, four, or even more attacks. When
differences of species are taken into account these differences
of susceptibility are even more marked ; thus sheep and
cattle are protected against subsequent attacks of anthrax by

VACCINATION. 369

a first, whilst in the human being and in the horse, anthrax
may occur again and again, though in all probability a
temporary immunity is conferred by an attack even in
these extreme cases.

Cholera and typhoid fever both belong to the group of
diseases of which one attack usually confer a certain degree of
immunity against a second, the period during which this may
last, however, varying in different cases ; in some it extends
over a few years only, whilst in others it appears to be almost
permanent. The acute exanthemata, of which we may take
small-pox as an example, belongs to this latter class.

Amongst the Turks the ingrafting or inoculating of small-
pox matter was very early resorted to as a routine practice in
order to produce a milder but protective attack of the disease.
We have a record of this in one of Lady Mary Wortley Mon-
tague’s letters, dated 23rd March, 1718, and written from Bel-
grade, in which she describes the inoculation of her little
boy three years old. She says : ‘‘ The boy was ingrafted last
Tuesday, and isat this time singing and playing, veryimpatient
for his supper ; I pray God that my next may give as good an
account of him. I cannot ingraft the girl ; her nurse has
not had the small-pox.” The after progress of the patient
in this case was satisfactory, and, as is well known, Lady
Mary was afterwards instrumental in drawing attention to the
matter in this country.

Then came Jenner’s remarkable discovery that similar
immunity against small-pox might be conferred by the
inoculation of lymph from animals that were suffering from
what was known as cow-pox ; an attack of the one disease
in this case conferring an immunity against an attack of the
other.

There has for long been the utmost anxiety on the
part of physicians and others to obtain some explanation
of these remarkable facts. Klebs and Pasteur explained
them on the assumption that during the course of a first
attack of a disease some material that was essential for the
nutrition of the pathogenic organisms that had by this time
been found to be associated with some of these diseases,
had been used up, and the supply being cut off, the organ-
isms were no longer able to exist in the body and exhibit
the characteristic evidence of their presence. This substance
must have been present in exceedingly small amount, as no

25

370 BACTERIA.

alterations in the composition of the blood or other fluids of
the body could be determined by any methods of chemical
analysis that could then be applied. Then came the theory
advanced by Chauveau and others : that just as micro-organ-
isms when growing in artificial media produced excretory
products, the presence of which was inconsistent with the
continued life of the organism; so in the body, bacteria
during the course of the disease.gave rise to some material
which might act deleteriously on their own protoplasm, and
which, remaining in the body for a considerable length of
time, interfered with the growth of any similar organisms that
might in future be introduced. Here, again, these special
chemical products could not be detected in the blood, and
must have been present in such infinitesimally small quan-
tities that it is difficult to see how they could exert any very
marked influence on the activity of the bacteria ; whilst, as
Fligge points out, our knowledge of the action of the tissues
on foreign bodies of various kinds would lead us to the conclu-
sion that any such material would be very rapidly eliminated.
Then Grawitz suggested that in any battle between the cells
and the bacilli that may occur in the body during the course
of a disease, if the cells can but manage to obtain the upper
hand and to destroy the bacteria, they should become
hardier, as it were, through the training of the contest, their
vital energy and assimilating power should be increased,
and they should thus become able to deal in a more summary
manner with any organisms with which they might afterwards
be brought into contact. Then came Buchner’s theory of
the inflammatory cause of immunity, which offered another
explanation, or modification of Grawitz’s explanation. He
argued that bacteria made their way into the body at certain
special points, these points or seats of election differing in dif-
ferent diseases, and that in consequence of the development of
the bacteria, there was a reactionary alteration, inflammatory
in its nature, in the tissues, which fitted them for the future to
resist the special organism that had previously made the
attack ; this minute alteration in the function of the special
cells at the seat of invasion enabling them to resist the further
action and invasion of the same organism even at a con-
siderably later period. Again based on the same principles
as Grawitz’s theory came the now celebrated Metschnikoff
theory. Metschnikoff holds that the protection against the

VACCINATION. 371

attacks of micro-organisms on the body is entirely due to the
action of the amceboid cells of the body, that these cells are
living pieces of protoplasm, that they are constantly taking
into.their own substance all foreign particles which find their
way into the body, that wherever there is an extra demand on
their energies, a large number are attracted to the point at
which the work is to be done, and that these cells acting on
the micro-organisms just as they do on foreign bodies, take
them up into their substance, digest and convert them
partly to their own uses, and gradually throw into the circu-
lating fluids of the body small quantities of effete substances
which are removed by the ordinary physiological channels.
Some observers, however, hold that the process is not so
simple as it would appear ; certain bacteria secrete substances
which appear to exert a paralysing effect on the cells, and
may so alter them that they are unable to perform their
proper functions ; whilst, on the-other hand, the cells secrete
in the performance of their work a material which has an
unfavourable influence on the activity of the bacteria. This
at first sight is an extremely feasible explanation, but when
we come to consider more carefully the conditions under
which immunity against diseases is conferred, we find that,
although in certain cases an attack of one disease protects
against an attack of a more serious and deadly malady, this
occurs only within certain definite and well-defined groups
of diseases ; there appears, therefore, to be something more
than a mere general protective influence generated within
the body. We must have specific powers of resistance
developed in or by the cells in order that they may be able
to resist specific bacterial activities, and the effects of specific
bacterial poisonous products. I have in previous chapters
spoken of the effects of the bacteria and of their products in
protecting against the various diseases to which they them-
selves give rise ; it may now be well to give a concrete
example of the theories that have been advanced as to the
nature of this protective inoculation ; let us take the develop-
ment of the methods that have been devised for protecting
animals against anthrax.

It was found that on devitalizing the anthrax organism
by one of several methods it might be introduced into the
subcutaneous tissue of a sheep without giving rise to any
very serious symptoms. The first note that the virulence

372 BACTERIA.

of the anthrax could be modified was communicated in this
country by Greenfield, on June 17, 1880, to the Royal Society
of London. This observer found that by cultivating anthrax
bacilli through several successive generations, in fluid taken
from the front part of the eye of the ox, he was able to
obtain a virus so modified that when injected into animals
susceptible of being affected by ordinary anthrax, the
animals did not die, whilst in animals injected with aqueous
humour cultures of the twelfth generation no symptoms
whatever were developed ; earlier cultures giving rise to a
modified form of anthrax only. :

In the following month Toussaint intimated to the French
Academy that he had been able to obtain a protective
anthrax vaccinal fluid, zie., one that might be inoculated
into an animal without causing death, and which conferred
on animals so inoculated protection against a second attack
of the disease. .

His original method of procedure was as follows:—The blood of an
animal dead of anthrax was carefully defibrinated by whipping and straining
through linen and then through ten or twelve thicknesses of blotting-paper.
Some animals were undoubtedly protected by the inoculation of this pre-
pared blood, but the method was very uncertain; and in some cases
the vaccine itself caused the death of the animal. Instead, therefore, of
filtering the defibrinated blood, he heated it for ten minutes to a temperature
of 55° C. With material so prepared he was able, by inoculation, to render
an animal immune to the action of the more virulent anthrax bacillus.

Pasteur, who concluded that the vaccination depended not
on the bacterial products but on an alteration in the viru-
lence of the bacillus which might be the result of the altered
temperature, subjected the bacilli to a temperature of from
42° to 43° C. ; these were found to have lost all their vitality at
the end of about six weeks, this loss of vitality during the six
weeks going on progressively in proportion to the rise of
temperature. It is stated that at the .outset the pure
culture had all the virulence of anthrax blood; whilst
only half of the sheep inoculated with the culture, that
had been heated for twelve days succumbed to anthrax.
On the twenty-fourth day of heating, the culture, when
inoculated, although giving rise to mild febrile disturbance,
did not cause the death of a single animal. It was found,
too, that if now, twelve days after the first inoculation, these
surviving animals were inoculated with a culture from the

VACCINATION. 373

twelfth day, which before had killed half the animals, there
was still onlya slight febrile disturbance, and noneof the inocu-
lated animals died. Virulent anthrax blood, might, after a
further interval of twelve days, be introduced into animals
that had been subjected to the double inoculation, without
giving rise to anything more than a slight febrile condition
similar to that noticed as resulting from the inoculation of
the modified virus. If, however, virulent anthrax blood was
introduced into animals in which only the first protective
inoculation had been made (s.e., with material that had been
cultivated for twenty-four days), a large proportion of the
animals died. It was evident, therefore, that it was abso-
lutely necessary to use both a first and a second vaccine if
the protection was to be complete. This attenuation was
not confined to the generations of bacilli that were directly
acted upon. If the temperature were lowered to about 35°
C., vegetative activity was immediately set up, rods in
enormous numbers were formed, and eventually spores might
be observed in these rods. Now comes the interesting fact :
the attenuated properties of the original bacilli were handed
on to the spores ; these spores might be kept in a latent
condition for a considerable length of time, and on being
introduced into media suitable for their growth they sprouted
out, notinto virulent anthrax bactll:, butinto modified anthrax
bacilli, so that the conservation of the vaccine (on silk
threads) became a comparatively simple matter.

Pasteur attributed the diminution of the virulence of the anthrax bacillus
to the action of heat in the presence of oxygen, but Chauveau, working on
Toussaint’s plan, found that heat alone continued for a very short period was
quite sufficient to modify the virulence of the bacillus. Blood is taken from
a guinea-pig about thirty-six or forty-eight hours after inoculation with an
active virus ; it is carefully defibrinated and is run into small glass pipettes of
about 1 mm. in diameter. ~ One end is carefully sealed by heat so far from
the blood that the heat cannot injure the organisms ; the sealed end contain-
ing the blood is plunged into water at a temperature of 50° C., and is kept
in this for about a quarter of an hour; in this way is prepared what is
known as the primary vaccine, corresponding to that obtained by Pasteur,
by heating for twenty-four days. The second vaccine is heated for only
nine or ten minutes, and corresponds to that obtained by heat at the lower
temperature for twelve days. These vaccines are injected in the same way
as Pasteur’s at intervals of from ten to fifteen days and they are found to
protect very fully against the most active virus. Where large quantities
of the vaccine have to be made by this rapid method, Chauveau used
sterilized broth which is inoculated with anthrax blood from a newly killed
animal; the flasks are then kept at a temperature of 43° C. for about

374 BACTERIA.

twenty hours and the temperature is then raised to 47°C. for a period of
three hours. This is the second vaccine. The first vaccine is a culture
made from one that has been heated for three hours at 47° C.; this is
incubated for from five to seven days at 35° to 37° C., and then for one
hour at 80°. Of these vaccines two drops are used for inoculating a sheep
and four for cattle, the animals being injected, cattle on the outer aspect of
the ear, sheep inside the thigh. The great drawback associated with the
use of vaccine so prepared is that it cannot be preserved for any length of
time, as under cultivation the original virulence is regained at once.

Pasteur’s classical experiments made in May 1881 gave
abundant evidence of the utility of this method of treat-
ment. On the 5th of May, twenty-four sheep, one goat,
and six cows were inoculated with a protective vaccine ;
twelve days afterwards they were again inoculated with a
somewhat stronger vaccine than that at first used, and on
the 31st of May these animals that had already been vac-
cinated, and twenty-four sheep, one goat, and four cattle
that had not previously been inoculated with the protective
virus, were injected with material from a virulent anthrax
culture.

On the 2nd of June all the animals that had been
protected were found in apparent health; of the others,
twenty-one sheep and the goat were dead, two other sheep
were dying, and the other was attacked later in the day.
The non-vaccinated cows were not dead, but they had all
marked local symptoms. Next day one of the vaccinated
sheep died, but its death was said not to be due to anthrax.
The experiment was repeated in a modified form by injecting
a quantity of blood and spleen pulp from a sheep that had
died of anthrax into sixteen non-vaccinated animals, and
into nineteen protected animals, with the result that on the
third day all the unprotected animals but one had succumbed,
whilst the others remained apparently healthy.

Equally good results were not always obtained by other experimenters,
but in some cases, at any rate, the experiments appear to have failed
through want of attention to detail rather than from any defect in the
method itself, and from the failure to recognize that the initial virus is not
always of the same strength, that different animals have very different
degrees of susceptibility and natural immunity, and that the quantity of
the virus injected very materially alters the conditions of the experiments.
No tissues can be expected to cope equally well with large and with
Small doses.

A number of other methods of preparing a less virulent (or vaccine)
material have been described by different observers. Thus Toussaint

VACCINATION. 375

found that if anthrax were treated with a one half per cent. solution of
carbolic acid, it became distinctly attenuated. Chamberland and Roux
found that fresh cultures started from one that had been subjected for
twelve days to .16 per cent. solution of carbolic acid were fatal to
guinea-pigs and rabbits, whilst if the time during which the bacillus was
exposed to this solution of the acid was extended to twenty-nine days and
a cultivation then made, ‘such cultivation was no longer capable of killing
a rabbit. They were thus able to produce a virus of any degree of attenua-
tion and to preserve it for some time, as the cultures made from their
attenuated bacilli inherited the same degree of attenuation that had been
developed by the bacilli that had actually been treated with the acid. They
found that bichromate of potash and other antiseptics exerted‘ a similar
attenuating influence. It has also been shown that the passage of the
anthrax bacillus through a series of animals of a certain species will
render the anthrax bacillus more or less virulent, according to the
species that is used. Thus Klein found that blood taken from a white
mouse which had died of anthrax was a protective vaccine for sheep,
whilst Sanderson and Duguid observed that the virus obtained from a
guinea-pig dead of anthrax was modified so far as cattle were concerned.
Roy made a series of similar observations. It must be borne in mind,
however, that cattle very frequently recover from anthrax under ordinary
treatment, so that these latter observations can, as yet, scarcely be accepted
as fully proved.

Up to this time it had not been recognized that the
immunity was really conferred by the action of the soluble
products of the organism. Pasteur had indeed shown that
the general symptoms of fowl cholera could be induced by
the inoculation of the sterilized products of the fowl
cholera germ, and Chauveau had suggested that an
acquired immunity was due to the action of the soluble
products of the,microbe. He argued from an observation
that, although in pregnant sheep, anthrax bacilli with
which they had been inoculated were unable to pass into the
foetus, the lambs exhibited an extraordinary immunity
against attacks of anthrax, this immunity, he considered,
being necessarily the result of the action of the soluble
products that had been able to pass over from the maternal
to the foetal circulation. We have now a whole series of
diseases from which immunity may be conferred by the inocu-
lation or introduction into the tissues of an animal of the
soluble products of pure cultures of micro-organisms.
In America, hog cholera has been vaccinated against, the
vaccinator using the sterilized cultures of the hog cholera
organism as his protective virus. Wooldridge, who was the
first to adopt this principle in connection with anthrax, was
followed by Pasteur and Perdrix, and by Hankin, whose

376 BACTERIA,

researches on the albumoses formed by the anthrax organism
have opened up a new field for the chemistry of bacteriology.
Fowl cholera, certain forms of septicemia, and a number of
other diseases, amongst which may be mentioned hydro-
phobia, in which, however, the facts do not belong to quite
the same order, all were brought within the same zone,
when it was found that the introduction of the sterilized
products of a specific organism, first in minimal doses and
then in gradually increasing doses, could confer a protection
against the subsequent action of even the most virulent
organism that under ordinary circumstances gives rise to the
same products as those injected. Gradual “ acclimatization”
is the ideal method though in most cases the results were
obtained by a single injection. It was for long supposed
that the products through which this immunity was con-
ferred were of an alkaloidal nature, and there can be little
doubt that in some cases, at any rate, these alkaloids may, if
given for a sufficiently long period of time, and in gradually
increasing doses, have some effect in “acclimatizing ” the
tissues to the action of the poison. As pointed out by
Sewall, who carefully studied the substances contained in
snake poison, the albumose contained in such poison, given
in very minute doses to pigeons, confers upon them the
power of withstanding seven times the ordinary deadly dose
of snake poison, even three months after the inoculation
has been made. A single dose of the ordinary hemi-
albumose of proteid digestion confers a similar immunity
against the action of this same albumose for a period of twelve
hours. Hankin, working on this analogy, concluded that
the albumose that he was able to separate from anthrax
bacilli was really the substance that conferred the immunity
against attacks of the bacillus itself, and he found that, -
although he could kill rabbits with doses of the five
millionth of the body weight, a dose of one-ten millionth
of the body weight rendered the animal immune to the
action of virulent anthrax. He found that he had obtained
an instrument of such delicacy, although of so great power,
that he was able to protect even mice against anthrax, which
had only been done once before—by Hueppe and Wood. It
would appear, however, that it was necessary to allow a certain
interval to come between the inoculation with the albumose
and the injection of the virulent anthrax organism. This is

VACCINATION. 377

an exceedingly interesting fact, for from it we gather that the
protective material acts in much the same way as does the
poison of the anthrax bacillus itself, showing that the
poisonous and the protective agents may be one and the
same, in certain diseases at any rate. We have, in fact, if we
introduce it at the same time as the living organism, a
cumulative action during the earlier stages, the albumose
helping the anthrax bacillus (by additional albumose being
formed) to do its work. Where, however, there is an
interval allowed between the introduction of the albumose
and the inoculation of the virulent material, there is time
allowed for the tissues of the body to become acclimatized,
as it were, to the action of this special material, so that when
the stronger poison is introduced the cells are more ready to
deal with it. A very interesting fact in this connection is, as
pointed out by Hueppe and Wood, that a certain putre-
factive organism, the earth bacillus, which in all morpho-
logical characters resembles the anthrax bacillus, and differs
only in the fact that it does not give rise to any fatal disease,
even in mice, was able when inoculated into mice and rabbits,
to afford protection against anthrax that otherwise proved
fatal to these animals, These observers concluded from this
fact that the saprophytic organism must be closely related
to the anthrax organism, and that it formed much smaller
quantities of the same specific poison as the disease organisms,
so that by its previous introduction these cells were prepared
for the attack of the specific anthrax poison, and the disease
was checked or modified. They indicate that the relation of
the saprophyte to the parasite is merely a quantitative one,
having an analogy in the relation that one of our cultivated
flowers bears to its wild progenitor. On the other hand Wood
and I have observed in a series of experiments that we
carried on with the products of the blue pus bacillus that
we had a kind of antagonistic influence exerted by the
blue pus products on the action of the anthrax bacillus.
That the favourable influence exerted by the blue pus pro-
ducts in the course of an attack of anthrax was not due merely
to an antiseptic action was proved by the fact that the anthrax
bacillus could actually grow in the blue pus products, al-
though, under these conditions, it was undoubtedly somewhat
weakened ; and we came to the conclusion ‘that we had to
deal with a kind of biological antagonism acting indirectly

378 BACTERIA.

through the cells. We observed that animals treated by
inoculation of the blue pus products and then with anthrax
bacillus not only passed through the disease at the time, but
they were protected against anthrax, even of a virulent
order, when inoculated later. This we looked upon as most
interesting, as in other experiments that had been carried on
with the blue pus bacillus itself as a protective agent against
anthrax, it was found that although an attack of anthrax was
cut short in the presence of the blue pus bacillus in a
rabbit, it could still be so inoculated at a later date that the
animal died with symptoms of true anthrax. The inference
we drew was, that the action of the products of the blue
pus bacillus and the action of the anthrax albumoses on the
cells are essentially different but that the one may interfere
with the action of the other. Thus, an animal that has been
rendered immune to blue pus is none the less susceptible to
anthrax. In our experiments the products of the blue pus |
bacillus were injected only at intervals, and during part of
the time between these intervals there was little of the
substance in the fluids of the body ; during these intervals
the albumoses of the anthrax had the opportunity of acting
on the tissue cells and of so acclimatizing them to its
presence that immunity was conferred. Where, however,
the blue pus bacillus was in the body, forming its products
continuously and acting antagonistically to the anthrax
bacillus, the tissues had never any opportunity of becoming
acclimatized to the action of the albumoses and no immunity
was conferred. Immunity produced by the attack of a
specific disease must then be looked upon as an acquired
tolerance or adaptation of the cells of our body to the specific
poison of the special bacterium of that disease, and the pro-
cess of recoveryfrom an attack of anthrax, for example, is really
the development of such immunity, which gradually passes
into the more perfect form during the course of the disease
and remains after the patient has recovered. The antago-
nism of the products of one organism on another which occurs
in mixed infection can never in all probability act directly
in the body, but through the agency of the cells in the
body such action may come to play a most important part
in holding in check the active poisons until an immunity can
be acquired. On the other hand we may look forward to a
period when it will be possible to obtain, by the action of

VACCINATION, 379

one organism in the body neutralizing the action of another
or by means of antagonizing drugs, a method of treating
and ameliorating disease, and at the same time allowing of
an acquisition of a condition of immunity by the patient.
It is a well-known fact that certain substances which
are in themselves non-poisonous or are only slightly “ de-
pressant” in their action may so restrain the powers of the
tissue cells that organisms that are otherwise incapable of
growing in the body are enabled to give rise to septic and
putrefactive changes even during the life of the animal.
Thus papain and the soluble poison of the Jequirity bean
when introduced into the circulation, both so alter the
conditions in the body that ordinary putrefactive organisms
can make their appearance in enormous numbers in the
blood of the animal injected.

It appears probable that both the antagonistic action and
this summative action are due to the bringing into play, or
the depressing, of certain specific functions of the proto-
plasms of the cells by the products of different micro-
organisms. It is not necessary that these functions should
always be manifesting themselves ; after being once evoked
and exercised they may remain latent for a considerable
period and only be again called into action under the regular
specific stimulus. It is a case of writing on the looking-
glass with ink and with French chalk ; the ink is always in
evidence, and we might say that it corresponds to the enzyme
or the peptonizing functions exerted by certain cells, animal
and vegetable, whilst the French chalk, though always there,
is only brought out when the glass is breathed upon. This
may be said to exemplify very roughly the specific power
that the cell has acquired of resisting the anthrax or other
special poison. It only comes into play when the specific
poison is present ; it is actually present though in a latent
condition the whole time.

“i

LITERATURE.

The following works may be referred to :
Authors already quoted. *Crookshank, Fliigge, Hankin,
Sewall.
Bucuner.—Eine Neue Theorie i. Erzielung von Immunitat
Gegen Infectionskrankheiten. Munich, 1883.

380 BACTERIA.

CHAMBERLAND ET Rovux.—Comptes rendus, t. XCVI., pp.
1088 and 1410. 1885.

CHARRIN ET GUIGNARD.—Comptes rendus, t. CVIIL, p. 764,
1889.

CHauveau.—Comptes rendus, t. xcIv. 1882, and t. XCvIL,
1883.

CREIGHTON.—The Natural History of Cow-Pox and Vaccinal

_ Syphilis. London, 1887.

CrooxsHank. — History and Pathology of Vaccination.
London, 1889.

GREENFIELD.—Proc. Roy. Soc. London, 1880.

Huepre anp Woon.—JLancet, Dec. 7, 1889; Berl. Klin.
‘Woch., No. 16, 1889.

JENNER.—Inquiry. into the Causes and Effects of Variole
Vaccinz,.a Disease known by the name of Cow-Pox.
London, 1800. >

KvEmn.—Micro-Organisms and Disease; Report of Med.
Officer of Loc. Gov. Bd., p. 166, 1882. ;

Kirt.—Wert un Unwert der Schutzimpfungen Gegen
Thierseuchen. Berlin, 1886.

Kocu.—Ueber die Milzbrandimpfung. 1882.

METSCHNIKOFF.—Fortschritt. der Med., Bd. ., p. 558, 1884,
Bd. v., pp. 541, 583, 732, 1887; Annales de 1’Institut
Pasteur, 1887-1890 ; Virch. Arch., Bd. xcvit., p. 502,
1884, and Bd. cix., p. 176, 1887.

PastEuR.—Comptes rendus, t. XcIL, p. 1378, 1881, t. Xcv.,
p- 1250, 1882, t. XCVI., p. 979, 1882; Revue Scientifique,
m1 series, t. v., p. 82.

PasTEuR ET PeERDRIx.—Comptes rendus, t. CVvi., p. 322,
1888.

Rurrer.— Quart. Journ. Micro, Sct, Vol. XXXIL, p. 99.
1891.

Sinouonsey unp Curistmus DirckIncK-HoLMFELD.—Fort=
schritt der Med., Bd. m., p. 78, 1884.

*WooDHEAD AND Woop.—Comptes rendus, t. crx., No. 26,
1889 ; Lancet, Feb. 22, 1890; Hain. Med. Journ.,
1890.

Woo.pripGEe.—Proc. Roy. Soc., London, p. 312, 1887.

* Full list of references given.

CHAPTER XXIII.
BacTErRiA IN Arr, EaRTH, AND WATER.

Spores in the Air in Hospitals—Effects of Currents and Altitude—Direction
of Wind—Nature of Country over which it passes—Frankland’s, Car-
nelley’s, Haldane’s and Petri’s Experiments—Few Bacteria in the Air of
Sewers—Tyndall’s Glycerine Chamber—Examination of Air—Koch’s
Method—Miquel’s Method—Hesse’s Apparatus—Modified Hesse’s
Apparatus—Miquel’s Sugar Method—Bacteria in Water—Effect of
Standing—Sluggish Movement—Oxidation of Organic Matter in Water
—Organisms carried by Sewage—Number of Organisms in Water—
Method of Procedure in Analysing Water—Pfuhl’s Flasks—Petri’s
Dishes—Petruschky’s Flask—Plate Cultivations—Cooling Apparatus—
Von Esmarch’s Tube Cultures—Effect of Rains, Frosts and Thaws—
Relation of Bacteria to Ammonia—Basis on which to determine
whether water is fit for Drinking or not—Filtration—Method of
Examination of Soil.

As will have already been gathered from what has been
stated in connection with the distribution of the cholera
bacillus, tubercle bacillus, anthrax bacillus, and similar
organisms, there are marked differences in the facility
with which organisms may be carried even by currents of
air. Those organisms that do not produce spores, and that
are easily killed by drying, are very seldom contained in
the air, at any rate in a condition capable of growing, whilst
it is found that those which resist ‘drying, and especially
those which form resting spores, may be carried about from -
place to place, either alone or adhering to dust or other
particles.

In connection with this question of micro-organisms in
air, there are certain general rules that should always be
borne in mind. Their presence must, we are afraid, be
looked upon as inevitable in those hospital wards in which
patients suffering from infective diseases are gathered to-
gether for treatment ; tubercle bacilli, for example, are found
in the air of wards where phthisical patients are collected,

382 BACTERIA.

whilst the special bacteria associated with other diseases
have also been demonstrated in the air of wards specially
set apart for the treatment of these diseases. Then,
again, it must be remembered that there are always
more micro-organisms in the air in those regions where
decomposing organic matter of any kind is allowed to ac-
cumulate, this being especialfy the case in positions in
which the air cannot be continuously renewed by currents
setting in from other and purer regions. Consequently,
it is found that in valleys and in low-lying country generally,
especially where there is any accumulation of decayed vege-
table matter, or where people are massed together in towns
or villages, many bacteria are usually present in the air.
Some of the organic growths obtained from such air
belong to the mould fungi, the spores of which, as is
well known, have considerable resisting power, and may
be readily carried from point to point by gentle currents
of air. As the low-lying lands are left, and the hill
country is reached, micro-organisms are comparatively
few in number, and at certain elevations, especially where
the temperature is low, the number of these germs of
moulds and bacteria may sink almost to zero. Again,
if the air brought by breezes coming from the sea on
the one hand and from the land on the other, be examined,
it will be found that in the former case it is im-
possible to demonstrate the presence of organic life of any
kind ; whilst in the latter, collected it may be at the same
point, an enormous number of micro-organisms of different
kinds may be met with. It is, however, impossible to give
any general rule as to the number of organisms that should
be found under these various conditions, though we may
take it as a result of Aitken’s experiments on the presence
of solid particles in the air and their relation to fogs, that
the more solid particles there are in the air, thé more micro-
organisms of various kinds are to be found. Some idea of
the number of organisms present at different seasons of the
year may be gathered from P. F. Frankland’s investiga-
tions, the results of which were presented to the Royal
Society in 1886. He determined the number of colonies
found in two gallons of air (examined by Hesse’s method),
collected on the roofs of the Science Schools at South
Kensington. He found fewest present during the month of

BACTERIA IN AIR, EARTH, AND WATER. 383

January, on an average of 4 per two gallons (ten litres) ; the
numbers gradually rose until August, when there was an
average of 105, and then gradually fell, though he records
no observations made during November and December.
He had previously confirmed the general results of Miquel,
Hare, and others, that as we leave the ground the number
of micro-organisms in the air rapidly diminishes. On Nor-
wich cathedral spire, at a height of about 300 feet he found
in ten litres of air only seven micro-organisms on one occasion,
and on the tower, at a height of 180 feet, he found nine,
whilst at the base of the cathedral (in the close) eighteen
were found. In another series of experiments made at St.
Paul’s cathedral a similar volume of air taken from the golden
gallery yielded eleven, that from the stone gallery thirty-four,
and that in the churchyard seventy micro-organisms. He gives
a number of other most interesting experiments, for which,
however, the reader must refer to the original paper. Car-
nelley, Haldane, and Petri have been able to show that the
number of micro-organisms in any air depends to a very great
extent on the moisture or dryness of the atmosphere, for they
found that in the air of sewers, which is necessarily very
damp, the number of micro-organisms present is extremely
small, unless rapid fermentation is going on, or there is
splashing from irregularities in the course of the drain, or from
falling in of sewage from a height, the bacteria always
tending to settle and to adhere to the moist walls of the drain,
so that unless a considerable number of the organisms are
carried into the air by the escape of bubbles and gas, or by
other agencies, the tendency of these organisms to gravitate
allows of their removal from the air. Here, however, it
appears to be the moisture on the walls that prevents the
escape into the air, rather than any moisture in the air itself.
Tyndall demonstrated that exactly the same thing occurred
in his chamber coated with glycerine ; bacteria, or other solid
particles to which these bacteria were adherent, fell-to the
floor, or were carried on to the walls, where they were held
fast by the glycerine, the air in the chamber thus becoming
practically sterile, or deprived of its micro-organisms ; there
might still be an enormous number of organisms attached
to the floor and walls, but the air itself remained absolutely
free, and flasks opened in this chamber would remain sterile
for a very considerable, or even an indefinite, period. Griffiths,

384 , BACTERIA.

gives the result of a number of similar observations, in which
the above statements are in the main confirmed.

Of the methods of examination of the air for micro-organisms the
simplest and most convenient, but perhaps the least reliable, is that of
allowing the germs to settle on plates of sterilized gelatine or potatoes, that
are left uncovered for a definite length of time. Currents of air may
completely destroy the accuracy of these results, but in rooms that
have been left undisturbed for some time, in which the doors are
closed, and in which no currents are set up by heat coming through the
windows or from fireplaces, moderately accurate average ‘‘ countings”’
may be obtained. In place of these glass plates covered with gelatine,
shallow glass trays with glass lids soon came to be used to contain the
gelatine. Those used by Koch for this purpose are shallow glass capsules
about half an inch deep and a couple of inches in diameter, in which the
sterilized gelatine is placed. These are placed inside tall glass cylinders
about five or six inches high, the mouths of which are closed with large
cotton wadding plugs. The glass capsule is lowered into the cylinder and
again removed from it by means of a piece of soft metal bent.at right
angles. After the whole has been sterilized the cotton wadding plug is
removed, the gelatine is left exposed, say, for ten minutes, the plug is
re-inserted, and organisms that have settled on it are allowed to develop at
the temperature of the room. These soon make their appearance as small,
white, yellow, or pink points, according to the nature of the germs that are
present in the air. In addition to these, however, a number of fluffy
white, green, or black, forms make their appearance. The former consist of
bacteria, sarcina, or yeasts ; the latter of penicillia, mucors, and aspergilli.

_ Another method of examining the dust and micro-organisms contained
in the air is one in which an apparatus somewhat like a chemical ‘* wash
bottle” with the bottom knocked out is used. In the neck of the bottle
is an india-rubber cork with two holes ; through one of these holes the long
limb of a tube bent into a U shape, with a long limb and a short limb is
passed ; the long limb, which is drawn out into a pretty fine point, projects
about two-thirds down into the bottle ; in the other hole of the stopper is a
short glass tube, to which is attached a piece of india-rubber tubing; the
bottom of the bottle carefully ground, is luted with vaseline on to a
glass plate on which has been placed a microscope slide, so supported as
to rest with its upper surface immediately under the drawn out opening of
the longer tube. This slide has previously been coated (as recommended.
by Miquel) with a mixture of oné part of grape sugar and two parts of
glycerine. A given quantity of air is then drawn into the bottle by means
of an aspirating apparatus, and all particles of dust, spores, or moulds,
and organic and inorganic fragments of all descriptions are made to
impinge on the sticky surface, where they are retained and may afterwards
be examined under the microscope, or, if necessary, the glycerine and
sugar mixture may be washed into a nutrient fluid such as peptonized meat
gelatine, and a regular biological examination of the organisms may be
made. A modification of this zeroscope used by Miquel is a flask con-
taining a small quantity of fluid, into which the air isdrawn, Thisapparatus
is somewhat complicated ; it is like a Pasteur flask, with three openings,
through one of which the air enters, the neck of the flask being continued
as a kind of tube down into the fluid in which the organisms are to be

BACTERIA IN AIR, EARTH, AND WATER. 385

cultivated. Hesse’s apparatus, for the estimation of the number and nature
of bacteria, &c., in the air, consists of a glass tube of about 18 to 25 inches
long, and 14 inches in diameter, slightly funnel-shaped at each end } in one
end an indiarubber bung is fixed. This has a central opening through
which passes a short glass tube about 5 or 6 inches in length, and one
third of an inch in diameter. In thisshort tube is inserted a plug of cotton
wadding. At the other end of the larger tube are two membranes of india-
rubber tied on separately ; the inner one has a perforation of about one-third
of an inch in diameter, the outer one fits over this and so closes the opening.
The tube, bung, and indiarubber membranes are then sterilized with a one
per cent. solution of bichloride of mercury and rinsed with boiled distilled
water. The inner cap is firmly tied in position with good stout thread,
the tube is half filled with water, and with a pair“of scissors a hole is
clipped in the centre of the indiarubber membrane. The second cap
is then firmly tied on, water is again poured into the tube, the bung is
replaced, and the whole apparatus is thoroughly boiled for a quarter of an
hour in the steam sterilizer. After allowing time for the glass to cool, the
bung is removed, the water is carefully poured out and liquid nutrient
gelatine is poured in to replace it ; the bung is again fixed in position and
the apparatus is boiled for ten minutes at 100° C, after which the tube is
put in a cool place in a horizontal position, so that the gelatine may con-
solidate in a thin layer along the whole length of the tube. Just as the
gelatine is beginning to ‘‘ set,” the tube may be gently rocked from side to
side so as to obtain a rather larger cultivating gelatine surface. This tube,
so prepared, is placed in a V-shaped support resting on a tripod, where it is
held in position by a couple of elastic bands. To this tripod an aspirating
apparatus is fixed. This consists of two bottles, each fitted with corks
and tubes like those of a wash bottle. These bottles are placed at different
levels and are connected by an indiarubber band, in the middle of which
is a pinch cock. The indiarubber tube connects the longer tubes in the
flasks. A litre of water is then measured into the upper flask, and, by
applying suction to the orifice of the lower flask that is now left free,
water commences to flow from the upper into the lower flask. If now the
second tube in the upper flask be attached to the little tube projecting from
the bung of the tube containing the gelatine, the aspirating apparatus will
communicate with the chamber in which the gelatine is contained; the
outer layer of indiarubber membrane which covers the orifice in the inner
membrane is then removed, and by setting the syphon system in operation
water is slowly and regularly run from the one flask into the other, and a
corresponding volume of air is drawn in at the small opening in the india-
rubber membrane. As soon as one litre of air has been drawn through,
all that is necessary is to close the pinch cock, reverse the positions of the
bottles, and repeat the whole process. This may be done until a volume of
from 1 to 5 litres of air has been drawn into and through the tube containing
the gelatine, the micro-organisms, drawn in along with this air, settling on
the nutrient medium in the tube. If the estimation is to be made in the
open air, it may be necessary to draw through the tube 20 or 30 litres.
It is, however, usually necessary to expose a number of tubes, drawing
different quantities of air through each; then by taking an average of
those in which the organisms are most readily counted, a fairly accurate
result is obtained. The flow of water, and consequently the rate of flow of
air, may be regulated by the introduction of pieces of glass tubing of known
20

386 BACTERIA.

calibre into the indiarubber tube that connects the two flasks. When the
operation is complete the tube with the contained organisms is disconnected,
the imperforate indiarubber membrane is again tied in position, and the
whole is set aside until the organisms begin to develop; they may then be
counted 2% sdt#, or with a long platinum needle special points may be
removes for microscopic examination, or for the purpose of making pure
cultures.
In place of Hesse’s tubes I have, with Mr. Coghill’s assistance, devised
a flat glass bottle with a large central opening at the top and two side
openings just at the level of the gelatine surface, These orifices are placed
at the opposite sides of the jar. They are used just as are Hesse’s tubes,
but possess several very obvious advantages over them.
Another method used in estimating the number of bacteria in the air
was that of filtering the air through tubes containing sterilized glass
- wool, asbestos, or sand; these are now seldom used, as they all interfere
more or less with the transparency of the gelatine. Indeed, it is often a
very difficult matter to distinguish grains of sand from young colonies of
organisms. In place of these substances Miquel recommends that sterilized
cane sugar should be used. Here the method of procedure is as follows :
Loaf sugar is carefully ground in a mortar and passed through a couple of
metal sieves, the latter of which allows grains of not more than half a milli-
metre in diameter to pass; this sifted sugar is packed into a glass tube about
eight inches long and the sixth of an inch in diameter. Near one end of
this tube is a slight constriction, on each side of which is placed a little
sterilized cotton wadding or glass wool, one of which serves to prevent the
sugar from escaping at the lower end, the other acting as a sterilized plug.
At the other end of the tube is a glass or indiarubber cap (see description
of Pasteur-Chamberland flask in Appendix) carefully sterilized and plugged
with cotton wadding. The tube is sterilized for an hour at 150°C., and
allowed to cool; the cap is then removed and the sugar, which has been
previously well dried, is poured into the tube, which should be filled to a
depth of about 4 to 44 inches. The whole is again sterilized at a tempera-
ture of 150°C, When the filter is to be used the sugar is packed against
the plug by tapping the tube gently, and the tube is held vertically whilst
the air is being drawn through the filter; this may be done slowly for
twenty-four hours, or very rapid aspiration may be carried on where it is
required to ascertain the number of micro-organisms present in the.air at
any definite period of the twenty-four hours. When the process is com-
pleted, the plug nearer the aspirator is carefully removed with forceps and
placed in a sterile glass box, which should be got ready beforehand, the
inner plug is then removed and is put into a test tube containing a small
quantity of liquefied nutrient gelatine ; this test tube is carefully plugged and
laid aside, the outer plug is immediately replaced, the cap is removed and
the sugar is poured out into a flask filled with sterilized water. A short
indiarubber tube is then slipped over the plugged end of the glass, the
upper end, from which the sugar has been emptied, being placed in a test
tube containing sterilized water; this water is alternately sucked up into the
filter tube and then expelled, until the whole of the sugar with its contained
germs has been dissolved and driven into the test tube. This water is added
to that of the flask and well shaken until the whole of the sugar is dissolved.
The number and character of germs so obtained from the air are determined
by making plate cultures. One portion should be utilized for the demon-

BACTERIA IN AIR, EARTH, AND WATER. 387

stration of the different zerobic organisms present, another for the anzrobic
species, and others may be used to determine the mere numbers of these
bacteria that are present, there being sufficient material for a dozen or even
twenty analyses. If a large number of organisms be present it is necessary
to dissolve the sugar in a large quantity of water so as to obtain a sufficiently
dilute ‘‘ solution,” whilst if a small number only are expected to be present
a smaller quantity of water should be used.

Water is one of the most convenient vehicles for the
distribution of micro-organisms. It has been noted that a
shower of rain diminishes the number of bacteria suspended
in the atmosphere in a most remarkable manner ; these
organisms being afterwards found in the puddles in the
road, in the pools in the sidewalks. In stagnant water and in
surface water of all kinds bacteria may be found in enormous
numbers, though the’ numbers and varieties in which they
are present may be very considerably modified by the amount
of organic material contained in the soil through which such
surface’ water comes, by the depth at which the water is
taken from the surface, and by the facilities for oxidation or
aeration that may be present. In the sluggish streams of
valleys, where there is a constant drainage from the surface
land, and where the water is so little disturbed that the air
of the atmosphere cannot obtain free access to the organic
matter suspended in the water, the number of micro-organisms
will, as a rule, be very considerable, whilst the rapidly-flowing
shallow streams that make their way over shingle or gravelly
beds will be found to lose their micro-organisms very rapidly
indeed ; if the organic matter has not already been left
behind in the sluggish reaches of the stream it is very
rapidly converted by oxidation into the ultimate products
of decomposition, and ordinary putrefactive micro-organisms
at any rate are no longer able to obtain any subsistence,
although, as Bolton maintains, the ‘water’ bacteria
can still flourish. They can even grow in distilled
water. In the water that comes from springs there may
be scarcely a single bacterium as the water rises to the
surface, but if such spring water be allowed to stand exposed
to dust and contamination of various kinds it very quickly
swarms with micro-organisms just as do the waters from
other sources. A very small quantity of sewage, which
really consists of water in which is suspended an enormous
quantity of organic matter, finding its way into a water
supply may contaminate it for a whole neighbourhood, such

388 BACTERIA.

contamination being only too plainly indicated by some of the
epidemics of typhoid fever and cholera that have from time
to time devastated our badly drained villages both at home
and in new countries. In view of all these facts, the
biological analysis of water has come to be a subject of im-
portance of the first rank, and too much stress can scarcely be
laid on the necessity for such analysis in order to determine
the suitability of any water supply for the purposes to which it
is to be put ; fortunately such examination is comparatively
easily carried out. In addition to this, however, regular
examination is absolutely necessary both to provide an
indication that no contamination is creeping in during the
process of distribution and to test how filter beds where such
are used are performing their work. Water should always
be examined for bacteria immediately after the sample
is drawn from its source, for although the amount of nutri-
ment for micro-organisms may be comparatively small, it is
all in solution, and in such a form that it can be easily
utilized by the organisms which im the fluid make their
way with the utmost rapidity from one point toanother. In
consequence of these extremely favourable conditions their
rate of multiplication is most remarkable. If a sample of
even the purest water (containing, say, 200 germs per cc.)
be left to stand in a room, in which the temperature is
comparatively high and therefore suited for rapid growth of.
these organisms, it may be found that in place of 200 germs
per cc, there may be present on the second day 5,000, on the
third day 20,000, whilst on the fourth, as pointed out by
Carl Fraenkel, they are almost innumerable. This multi-
plication may go on for some time, but at length there
comes a period at which the small quantity of food contained
in the water is used up, the bacteria begin to die, and the
number of living cultivatable organisms gradually falls until
eventually it may become extremely smail.

Water for analysis is collected in a sterilized Ehrlenmeyer flask, well
plugged with sterilized cotton wadding ; in place of this a wide-mouthed
stoppered bottle may be used. In either case the stopper or the wadding
should be covered with an indiarubber cap previously carefully steri-
lized in corrosive sublimate solution and boiled distilled water. If the
water is to be collected from a tap it should be allowed to run for several
minutes before any is taken ; water from the surface of a pond or a river
should be collected by means of large sterilized pipettes ; whereas if itis to
be taken from a depth or from a well a stoppered and weighted flask is

BACTERIA IN AIR, EARTH, AND WATER. 389

lowered, then, when it has reached the required depth, the stopper is
removed, the bottle is allowed to fill, after which it is then drawn to the
surface, the water so obtained being at once transferred to « number of
smaller bottles prepared as above.

Quite recently an exceedingly convenient apparatus for the collection of
water has been described by Dr. Pfuhl. It consists of a tall glass vessel with
a flat bottom 2.5 centimetres in diameter and 10 centimetres long, with a
glass tube 6 or 8 centimetres long, which can be easily closed, leading from
this vessel. To prepare this it is only necessary to sterilize by heating in
a ‘flame, and while the air is rarefied to seal up the point of the tube by
heat. When this has to be filled it is plunged under water or in the running
stream from a tap or pump, the tip of the tube is broken off with a pair of
sterilized forceps, water rushes in and about half fills the vessel. The outer
surface of the tube is then carefully dried with blotting-paper, gently
warmed to drive away the moisture from the glass near the opening, and
finally sealed as before in a spirit lamp flame. After itis thoroughly cooled .
the flask is well shaken, and should there be any leak thisis made good. For
transport these flasks are packed in zinc cases with cotton wadding and ice.
To remove the water the tube is nicked with a file, broken off, and a steri-
lized pipette is introduced. In all cases, however, there is the difficulty of
transport, and it is a great matter if plate cultivations can be made at the
time that the water is drawn. Petri gets over this difficulty by making
his cultivations in double glass dishes, which are kept in position by india-
rubber bands; and various other pieces of apparatus have been devised,
perhaps the best of these being Petruschky’s flask, which can be used for the
cultivation of either zerobic or anzerobic organisms. This consists of a thin
flask flattened on two sides with an indentation at the neck to prevent the
flowing out of the softened gelatine. It may be used simply with a plug of
cotton wool, The mixture of gelatine and water is poured in and then allowed
to settle on one of the flat sides. For anzerobic cultures, however, a couple
of glass tubes similar to those used in a wash bottle are introduced through
openings in an indiarubber cork. Hydrogen is driven through the bottle to
displace the air, and the ends of these tubes are carefully sealed, the flask
is laid on its side, and the gelatine is allowed to cool. These flasks are so
constructed that a microscopical examination may be made through the thin
glass walls, especially if care be taken to obtain a layer of gelatine or agar
sufficiently thin and of equal thickness throughout. Measure the size of
the drop delivered by a pipette in the following manner. Weigh a filter
paper on a fine balance, then put a gramme weight into the opposite scale,
and drop by drop deliver exactly one gramme of water on to the filter paper,
counting the drops as this is done ; then mark the pipette with the number
of drops that it delivers per gramme, after which it may be used for measur-
ing the water in making gelatine plate cultivations. It is now sterilized in
the hot air chamber at 150°C., or by being thoroughly washed out with bi-
chloride of mercury, then with distilled water that has been boiled and
allowed to cool, and then with absolute alcohol, the last traces of alcohol
being driven out by the heat of a spirit lamp. If a very large number of
organisms are present a.single small drop of the water, corresponding to about
the fiftieth part of a gramme or cc. will be sufficient, whilst-a larger drop,
the twentieth part of a gramme, or even six or eight of these drops, may
have to be used in order to obtain a sufficient number of organisms in a
plate cultivation. Then prepare a number of glass plates in the following

390 BACTERIA,

manner :—Thin plate glass is cut into squares of four and a half inches, the
sharp edges are removed witha file, and the glass is carefully cleaned. It may
then be sterilized in one of the following ways: Wrap each plate separately
in a sheet of hard tissue paper, and place in the hot air chamber, leaving it
for about an hour subject to a temperature of 150° C. It may then be taken
out and kept in a dry place until required for use. If the plate is required
at once, leave it slightly damp after cleaning, then, laying hold of it with a
pair of strong forceps, heat it carefully over a Bunsen burner or spirit lamp
until the whole moisture disappears, great care being taken that one side at
any rate is sterilized by the action of the flame, wrap up in a piece of steri-
lized paper and leave it until the other materials are ready. Or the plate
may be tilted against a clean wooden block or iron upright with the more
carefully heated surface downwards. Bell jars and glass benches have pre-
viously been prepared by being thoroughly washed with soap and water,
and then with a 1 per 1,000 solution of bichloride of mercury; a piece
of absorbent filter paper thoroughly saturated with the bichloride solution
is placed in the bottom of one of the jars; on to this all germs that are
suspended in the air within the jar gradually fall and are destroyed. On
a surface made as level as possible by means of a tripod levelling apparatus
and a small round spirit level, (if these can be obtained), a plate of metal
resting on three metal feet is placed in a mixture of ice, salt, and water; on
this the sterilized plate of glass, with the more carefully prepared side upper-
most, is laid, and the whole is covered with a bell jar that has been previously
sterilized by means of heat or of bichloride of mercury. A test tube, with
a large overhanging cotton wadding stopper, containing gelatine or a
mixture of agar and gelatine is taken, the stopper is removed for a second
or two, and the lip of the glass tube is carefully heated in a flame, the
cotton wadding stopper is also thoroughly singed and then replaced. The
gelatine is melted by placing the tube in water at a temperature of about
35°-39° C. for gelatine, and a much higher one for agar gelatine. As soon
as the medium is thoroughly melted, the quantity of water that is to be used
is dropped from the sterilized pipette into the tube, then with a rolling
motion the water is thoroughly incorporated with the nutrient medium,

the gelatine is poured out so as to form qual and regular lay&:; it is
spread gradually from the centre o: . to near the imargits,“over
which, however, it is not allowed is, then allowed to solidify,
after which the plate is trans : many red for its recep-

zinc benches,
At vell jar; they
F’, and at the
\Vappearance

carefully sterilized, three plates may be placed
are then allowed to incubate at the temperature
end of two or three days colonies of bacteria yy
(each one from a single seed if the mixing has bec
be isolated and described. Where the numbe eae
the method described for the isolation of chix.
Cholera, p. 155) should be utilized. Te is som 85 necessary tof make
a whole series of plate cultures to obtain, a sing] growth, especially
where zoogloea masses are formed. This.smethod “is also used for the
separation of different species of micro-organisms, and it can be easily
understood how micro-organisms may be more or less isolated by being
shaken in a fluid medium, and how when once they are isolated they

anisms (see finder

BACTERIA IN AIR, EARTH, AND WATER. 391

are prevented from running together again, for a time at any rate, by
the solid gelatinized medium. Where it is necessary to isolate and
obtain pure cultures of any special organisms, the method described as
used in separating any colony of organisms should be used (see page 155).
Salomonsen recommends a cooling apparatus made of an ordinary plate
on which a shallow glass dish with a ground rim rests. This is filled up
to the surface, but not to overflowing, with water and lumps of ice, a
plate of ground glass is fitted on to this, and then a sterilized glass cover
or bell jar is used to cover the glass plate; the further procedure is then
much as above. The organisms may be counted as a whole, but it is more
convenient to use a plate of glass marked into squares, which is sup-
ported over the gelatine plate, a low power lens being used to define the
smaller colonies. The colenies in a number of squares in different positions
are counted, then the number of squares that cover the gelatine plates
is determined, and an average is obtained from which the whole number
of organisms may be reckoned. It must be remembered, however, that a
small proportion of the gelatine still remains in each tube, therefore it is
necessary when making the plate cultivation to spread the remaining
quantity of gelatine over the walls of the tube by keeping the tube rotating
in water until the gelatine is fixed in position; the organisms left in the
tube also give rise to colonies. These should be counted and added to
those found on the plates. In place of plates von Esmarch recommends
the use of test tubes. The inoculation is made, exactly as above, into
wide test tubes containing from eight to ten cc. of nutrient gelatine, a
second tube may be inoculated from the first, and a third from the second
by means of the sterilized pipettes. The plug is then thrust well home
after being singed, and a tight-fitting indiarubber cap is placed over the
mouth of the test tube, or melted paraffin is run in to protect the wadding.
The mixture is then effected by rolling the tube rapidly between the
hands, keeping it in a vertical position. When the fluids are sufficiently
mixed the tube is placed into ice-cold water and, in a horizontal position,
is kept rotating on its longitudinal axis until the gelatine is ‘‘set” in a
thin layer all over the walls of the tube; the tubes are then put aside
and kept under observation. Should the presence of a large quantity
of oxygen be necessary, the indiarubber cap may be removed and
the plug may be pierced at one or two points with needles that have been
carefully heated, these needles passing through both paraffin and gelatine.
These tube cultivations may be made at once and on the spot, are readily
carried about, and serve as control experiments even when plate cultivations
are made in the ordinary manner. In place of glass plates and large moist
chambers, glass capsules are sometimes used for making plate cultivations,
and Petri has devised a shallow glass tray with a lid which answers the purpose
admirably. The method of procedure is much the same as in the above
cases, but is much simplified from the fact that the gelatine is allowed to
cool on the floor of the capsule, the space above serving as a moist chamber.
Before sterilization these boxes are wrapped in a sheet of the hard tissue
paper, they are then subjected to dry heat and are ready for use at any time.
In working with gelatine there is the disadvantage that certain organisms
peptonizing it cause it to liquefy exceedingly rapidly ; it has the advantage
that it is exceedingly clear, is readily melted, and solidifies rapidly. Agar-
agar on the other hand is not liquefied by the peptonizing organisms, but it
is not nearly so clear as the gelatine, and requires a much higher tempera-

392 BACTERIA.

ture (at least 90°C.) to melt it, although, as should be borne in mind, once
melted it will remain fluid at 40°C., at which temperature any ordinary
inoculation may be made A mixture of agar and gelatine prepared by
adding 5 per cent. of gelatine and .75 per cent. of agar to the peptone beef
broth, orothernutrient fluid combines the advantage of both gelatine and agar-
agar media. It is almost as clear as gelatine, is readily manipulated, and
melts at a considerably. lower temperature, although it will remain solid
between at 30° and 40° C., and it is not nearly so difficult to prepare as the .
pure agar.. The number of organisms in water is always calculated per
cubic centimetre, but having obtained the number of organisms in the fraction
of acc., it is easy enough to convert the figures to the desired standard.
In every case a sample of water should be allowed to stand in conical
glasses, so that any sediment may be obtained for microscopic and biological
examination. This is especially important where the presence of pathogenic
micro-organisms such as those of typhoid is suspected. These organisms
are usually brought in along with solid particles of sewage matter, and to
these they usually adhere so that they may all be deposited along with
such particles,

The conditions under which micro-organisms are most
numerous in water have been already referred to in general
terms. It is of course found that where great rain storms,
or thaws after the action of frost, break up the earth's
surface, many organisms are set free and are carried away
into the water supplies, the number usually varying to a
certain extent with the amount of solid matter that is
suspended in the water; as a general but by no means in-
variable rule it may be stated that most bacteria are found
where there is most ammonia. It is sometimes said that if
awater does not contain more than one thousand organisms
per cc. that it may be used with safety for drinking purposes,
but it must be borne in mind that this thousand organisms
may contain a larger number of pathogenic organisms,
whilst on the other hand five thousand organisms in the
‘same quantity might not include a single pathogenic germ.
It has been found indeed that no general biological examina-
tion will give us absolutely accurate indications as to the
nature of bacteria in water ; to obtain such information a
rigid examination of every species present must be carried
out. The number of liquefying organisms has indeed
been taken as giving an indication of the quality of water,
and this is undoubtedly a safer plan than to take into
consideration the mere number. A safer rule still, however,
is to take the number of different species of organisms in a
drinking water as indicating its purity or impurity for
drinking purposes, for it follows that if any water contains a

BACTERIA IN AIR, EARTH, AND WATER. 393

considerable number of species, there must be several. centres
from which these must be derived, each additional source, of
course, bringing in an additional element of danger. After ex-
amining 400 springs, wells, and streams, W. Migula concluded
that when there are more than ten species of bacteria in any
sample of water, especially when these are species not ordin-
arily met with, the water should not be used for drinking
purposes. In only 59 out of 400 was such a number of species
obtained, whilst 169 contained more than 1,000 individuals
per cc., 66 of these having over 10,000, and 40 over 50,000.
He found in all 28 species, and observed that the number of
colonies does not by any means correspond with the number
of species, though in some cases it undoubtédly does.
Ordinary putrefactive bacteria are almost invariably absent
from spring water, but they are usually found where the
number of colonies is between 1,000 and 10,000 per cc., but
they also occur where the number of germs is below 50 cc.,
but very seldom where the number is over 10,000. Of
course the only perfect method is to examine each separate
species by itself and to examine carefully any organisms that
bear the slightest resemblance to any of the pathogenic
species. It is a good rule to observe that all water taken
from near the surface, or spring water that has been allowed
to come to the surface and remain there for some time before
use, should be filtered through sand or through porcelain
filters. If in the process of filtration pure air can be mixed
with the water so much the better ; the best of all filters for
this purpose being the Pasteur-Chamberland, which may be
readily applied to every household water supply. In large
water-purification works gravel and sand are by far the best
filters, especially if these are frequently renewed, the old
filtering medium being burned before being again used.

For the examination of soil the first method used was simply to sprinkle a
little of the earth on a plate of nutrient gelatine, and then to examine the
organisms that grew on it. This, of course, was an exceedingly imperfect
method. The next step was to mix a small quantity of the earth with sterilized
nutrient gelatine in a test tube, and then to make a plate cultivation either
on a tube or on a flat surface. Now, however, that it has been found that
there are so many organisms on the surface of the earth, the mass of earth
to be examined has been diluted by adding a considerable quantity of
sterilized distilled water. This is allowed to stand for a considerable time
in order that the two may be thoroughly incorporated into a thin brown
liquid with as little sediment as possible, and from it plate cultivations

394 BACTERIA.

are made as described in the case of water or cholera organisms. Perhaps,
however, the most certain way of obtaining all the organisms that are in
any sample of earth is to break it down in liquid gelatine as above
described, and then make an Esmarch tube cultivation. It is of course an
easy enough matter to take a sample of earth from near the surface, but it
is much more difficult to take samples from the deeper layers, which can
only be done by means of special boring rods, unless access can be gained
to a clean cut surface such as those met with in the making of a drain or
other such cutting. As most organisms, however, such as the bacilli of
anthrax, malignant cedema and tetanus, and the zrobic putrefactive bacteria
are usually found near the surface, and as the deeper layers are so frequently
almost entirely free from micro-organisms, just as is the ground water that
we find in these deeper layers, this is as a rule a matter of comparatively
little importance.

LITERATURE.

The following works may be referred to: .
Authors already mentioned. Fraenkel, Griffiths, Hueppe,
Koch, Salomonsen, Tyndall.

AITKEN.—Trans. Roy. Soc., Edin., vol. xv., Feb. 6, 1888;
Proc. Roy. Soc., Edin., vol. xvi, p. 139, 1886.

Bo.ton.—Zeitschr. f. Hygiene, Bd. 1, p. 76, 1886.

Burpon SANDERSON.—Reports on the Intimate Pathology
of Contagion. 1876.

CARNELLEY, HALDANE AND ANDERSON.—Phil. Trans., vol.
CLXXVIIL., p. 61, 1887.

FRANKLAND.—Phil. Trans. Roy. Soc., vol. CLXXVIUIL., p. I13,
1887 ; Journ. Chem. Soc. p. 373, 1888; Proc. Roy.
Soc., vol. XL., p. 509, 1886.

FRANKLAND AND Hart.—Proc. Roy. Soc., vol. XLL, p. 446,
1887. ‘

eae eer | f. Bakt. u. Parasitenk, Bd. vir, No. 12,

P- 353, 1890. ; ;
Miouet.—Annuaire de l'Observatiore de Montsouris. 1877-
1890.

NicoLatER.—Deutsch. Med. Woch., No. 52, 1884.

Petri.—Zeitschr. f. Hygiene, vol. 11, p. 1, 1887.

Petruscuky.—Centralbl. f. Bakt. u. Parasitenk., Bd. vu,
No. 20, 1890.

Pruni.—Centralbl. f. Bakt. u. Parasitenk., Bd. vimt., No. 21,
1890.

Severe SGeimalbh f. Bakt. u. Parasitenk., Bd. 1v., No. 7,
1888, and Bd. vit., No. 4, 1890.

BACTERIA IN AIR, EARTH, AND WATER, 395

SmirH, ANGuS.—Second Report Med. Officer Local Gov.
Board. 1884.

Soyka.—Fortschritte. der. Med., Bd. 1v., No. 9, 1886.

STrRAus AND Wurtz.—Annales de l'Institut Pasteur, t. 1.,
p- 171, 1888.

Von Esmarcu.—Zeitschr. f. Hygiene, Bd. 1., p. 293, 1886.

WarinGton.—Journ. Chem. Soc., p. 642, 1884 ; p. 727, 1888.

*WOooDHEAD AND Hare.—Pathological Mycology. Edin.,
1885.

APPENDIX.

—+—

THE short outline of bacteria and their products contained in the preced-
ing pages will not have had the effect desired if it does not induce in a
certain number. of readers an ambition to undertake some experiments in
bacteriology, in at least some of its branches. As it is often extremely
difficult to obtain the most elementary knowledge of the technique of a
subject without being compelled to dive into elaborate and erudite articles,
it has been thought advisable to supplement the descriptions of methods
already given by a short réseemé of some of the simpler methods of experi-
mental investigation (the apparatus required for which is extremely simple,
and can be obtained at comparatively trifling cost), and to give a short
description of some of the commoner forms of bacteria that they may be
readily identified.

Hot Air STERILIZING APPARATUS.

C. Salomonsen recommends a very inexpensive form of sterilizing oven in
which glass utensils, metal apparatus, wadding, paper, &c., may be heated
to about 150° C. It consists of an ordinary large-sized biscuit box,
such as may be obtained from any grocer. A round hole is cut in the
middle of the lid, and into this a cork is fitted to carry a thermometer
registering to 200°C. A small hole is punched in each of the four sides near
the top, and similar holes are made close to the bottom. On the bottom
of the box, inside, is fitted a piece of strong sheet iron, with the ends bent
at right angles to raise it about two-thirds of an inch, so that materials to
be sterilized do not touch the metal which is in immediate contact with the
flame. The lid is covered with a piece of felt, in the middle of which isa
hole for the thermometer. Another piece of felt, the breadth of which is
three-quarters the height of the box, and long enough to go round it out-
side, is fastened round the chamber, just below the upper, and above the
lower, air holes, and is kept in position with stout copper wire. The box
so prepared is supported on an ordinary tripod, between which and the box
rests a loose piece of thin sheet iron or tin (this latter prevents the burning
of the thin tin bottom of the box). Such a chamber may be heated by gas
or by a small oil stove. An ordinary cooking oven may also be used as a
hot air sterilizer. A more complicated apparatus is a double-walled sheet
iron box of about the same size as the above, or a little larger, with a hole
in the top to receive a thermometer, a door in front, and a movable bottom,
which can be easily renewed from time to time as it is burned through. All
objects placed in this sterilizing chamber must be protected against dust on
their removal. This is done in the case of flasks and test tubes with their
cotton wadding plugs by covering them with a layer or two of strong, hard,

398 APPENDIX.
thin “ preserve” or ‘ type-writing” paper; other objects, such as watch
glasses, slides, cover glasses, cultivation plates, &c., may be enclosed in
small iron or copper boxes, or they may be carefully wrapped in cotton
wadding, or in a couple of thicknesses of the above paper, before they are put
into the oven to be sterilized. One must always be careful that the paper
or wadding does not come into actual contact with the bottom or sides of
the oven, the hot metal of which may cause singeing. Even hot air at 150°C.
will ‘‘ brown” cotton wadding or paper slightly, so that such a temperature
in the chamber may be at once recognized, even when no thermometer can be
obtained, by the appearance of a slight brown coloration of these materials,
STEAM STERILIZER.

For, sterilizing by means of steam, an ordinary fish kettle or potato
steamer may be used ; either of these placed on a fire will, for private work,
usually serve the purposes of the more complicated Koch steam sterilizer.
In this steaming apparatus the heat penetrates rapidly and does its work
thoroughly, moist heat being much more effective as a sterilizing agent than
dry heat. Ifit is thought necessary to obtain a piece of special apparatus
for the purpose, the best is a tin or zinc cylinder about eighteen inches high
and six inches in diameter, to which a small water gauge is added. The
bottom of this should be made of block tin ; the top is closed by a conical
lid in which is a hole for a thermometer. The lid and sides are usually
covered with felt, though this is not essential, unless economy of heat is
advisable. The felt should not in any case extend quite to the bottom, or
it is readily singed by the heating flame. Inside the cylinder are a couple
of shelves, one about one inch, and the other about ten inches, above the
level of the water ; on each rests a perforated tin plate on which tin vessels
about four or five inches in diameter and with perforated bottoms are sup-
ported. The objects to be sterilized are placed in these vessels, the cylinder
is filled to a depth of three or four inches with water, the pails are put into
their places, and the lid placed in position. After the water has com-
menced to boil briskly, the steaming is continued for about twenty minutes,
at the end of which time most of the pieces of apparatus are thoroughly
sterilized. This sterilizer may be lengthened by the addition of a tin
cylinder with a ring or collar near the base, which fits into the -top of the
sterilizing cylinder, the lid being placed at the top of the additional cylin-
der. This lengthening portion is especially useful for sterilizing Hesse’s air
analysis tubes.

INCUBATING APPARATUS.

Most of the ordinary micro-organisms may be cultivated at the temperature
of the room, but many of the pathogenic organisms, such as the tubercle bacil-
lus, will only grow at a temperature approximating that of the human body,
and for the cultivation of these some sort of incubating apparatus is necessary.
The simplest apparatus, however, if supplied with a good body of water
or a wrapping of felt and cotton wadding, will in most cases serve our pur-
poses. Any one with a little ingenuity will be able to devise an incubator
for his own use, if he has a greenhouse or any warm room at his disposal,
an ordinary oil lamp being sufficient, if properly trimmed and regulated, to
keep a double-walled chamber covered with felt at a fairly constant
temperature, varying only three or four degrees for months together. Where
systematic investigation is to be carried on, however, one of the ordinary

APPENDIX. 399

thermostats with regulator should be obtained, or, failing this, one of the
anal egg hatching machines, of which there are several in the market, may
e used.

STERILIZED VESSELS FOR THE RECEPTION OF VARIOUS MEDIA.

Ordinary test tubes, flasks, and other special glass apparatus, are first
carefully washed with soap and water, then with boiling solution of per-
manganate of potash, to which a few crystals of oxalic acid are added.
They are then rinsed with distilled water, and are allowed to drain ona
rack for some time, after which they are carefully plugged with cotton wool,
care being taken that the wadding inside the neck is perfectly smooth and
firm, the tuft outside being large enough to overlap well the lip of the test
tube. These plugs 7% sét# may be covered with paper, which keeps off
the dust. They are heated for an hour in the hot air chamber at 150°
C. For the reception of fluid media, Salomonsen recommends the use of
a small flask or test tube of which the neck or upper part is so drawn
out that it has a comparatively narrow mouth. The mouth is closed by
a piece of indiarubber tube a couple of inches long. The tube is washed
with bichloride of mercury solution, then with distilled water, is wrapped
in hard parchment paper and sterilized at 100° C. in the steam sterilizer.
It is then filled for half its length with cotton wadding that has been
sterilized at 150° C. in the hot air chamber. The flasks are sterilized as
above. For these stoppers the following advantages are claimed :—(1) In
opening and closing the flasks, the wadding and dust that is collected on
it are not touched. (2) The apparatus is opened and closed at a point
which can always be easily kept free from dust. (3) The opening through
which the inoculation is made is smaller than in the case of the ordinary
test tubes. Chamberland has devised flasks and test tubes which differ
from these only in having glass in place of indiarubber caps.

PREPARATION OF FLUID CULTURE MEDIA.

Beef Broth—Bouillon.

To prepare beef extract for the nutrition’ of micro-organisms, take a
pound of lean beef, mince it fine; add to this a Ktre of pure water, mix
thoroughly, and allow to stand in-a cool place for twenty-four hours; again
mix thoroughly and squeeze through a cloth, passing sufficient additional
water through the meat to again make up the quantity of fluid to a litre;
boil the extract thus obtained for half an hour, render it neutral, or very
slightly alkaline, by adding a saturated solution of mixed sodium hydrate,
sodium carbonate, and sodium phosphate; with a bit of gummed paper
fasten a strip of neutral litmus paper and one of turmeric paper to the end
of a glass rod; as soon as the faintest alkaline reaction is obtained, add no
more of the alkaline solution ; boil for an hour; allow to cool, and remove
the fat ; again filter into a large stock flask or into test tubes that have been
plugged with cotton wadding and sterilized as above. These vessels, with
their contents, should now be boiled in a potato steamer or other steam
sterilizing apparatus for a quarter of an hour on each of two or even three
successive days, the wadding plugs being protected from dust by several
layers of paper tied over them, or by means of thin indiarubber caps that
have been washed in a solution of corrosive sublimate. The meat extract
may be modified by the addition of various materials, such as .§ per cent.

400 APPENDIX.

of common salt, recommended by Miquel, or of 5 per cent. glycerine,
which is added before the nutrient fluid is finally sterilized (first used by -
Roux and Nocard). This glycerine meat extract is, as we have-already
seen, an excellent medium for the growth of the bacillus tuberculosis.
Albumen peptone, cane or grape sugar, acetic acid, mannite, &c., have all
been added in various proportions and for various purposes. Liebig’s extract
of beef, in the proportion of five parts to one thousand, or Cibil’s extract,
twenty grammes to a litre, may also be used. These latter require to be very
carefully sterilized by Tyndall’s method of discontinuous heating, in which
the fully developed organisms are killed off in a very short time on exposure
to a comparatively low temperature. Some of the spores that remain in
these develop intp the vegetative form during the next twenty-four hours.
This crop is again killed by a second heating. The remaining spores, if
any, are again encouraged to develop, and then this crop is also killed off,
usually leaving the fluid sterile, though in some cases the process may have
to be repeated three, four, or even five times. Various infusions and
decoctions of wheat or hay, of different fruits or vegetables, yeast water, .
beer wort (the latter especially for the culture of the mucors and yeasts),
a mixture of beer wort and prune juice (especially useful for the growth of
the various aspergilli) may be used. These should all be sterilized by
discontinuous heating at 100° C. in the steam sterilizing apparatus for
twenty minutes on three or four successive days. Urine, aqueous humour,
or other fluids of the body drawn with antiseptic precautions, may all be
used as cultivation media for certain organisms.

Milk.

Milk may also be used asa culture medium, but although it is a substance
easily obtained, it is a somewhat difficult matter to render it absolutely
sterile. If heated under pressure to 120° C., milk may be sterilized in
from ten to fifteen minutes; but in the steam sterilizing chamber, at 100°
C., it is necessary to heat it for an hour on- the first day, and from twenty
to thirty minutes on each of the two following days.

SoLip CULTURE MEDIA.
Bread Crumb.

One of the simplest of the solid culture media, bread crumb, is prepared
by taking the crumb of a loaf, drying it in small pieces, spreading it out on
a sheet of clean paper in an oven, or on the top of a stove, or even in front
of a warm fire; then rubbing it through a fine sieve, or passing it through
a coffee-mill. A small quantity of this dried crumb, sufficient to cover the
bottom of a flask to about the depth of a quarter of an inch, is put into a
wadding-stoppered small sterilized flask ; distilled water is added until the
bread crumb is thoroughly moistened, no superfluous water, however, being
left unabsorbed. If after allowing the bread crumb to stand for about a
quarter of an hour, it is found to be properly moistened, the flasks con-
taining it are heated in the steam chamber on each of three successive
days for halfanhour, In place of using water, the crumb may be moistened:
with beef extract, sugar solution, dilute glycerine, or any other of the fluid
media already referred to. This medium is used chiefly for mucors and
should be rendered slightly acid by the addition of a small quantity of
Tartaric or other organic acid. i

APPENDIX. 401

Soyka’s Ground Rice Medium.

A medium which can be used instead of bread paste is that described by
Soyka. It is composed of ground rice, 10 grammes ; milk, 15 cc.; neutral
beef bouillon, 5 cc. These ingredients are thoroughly mixed, and put into
small covered glass dishes or small glass flasks, which are sterilized, as is
the bread paste. It forms a beautiful solid white opaque mass, on which
coloured organisms may be even more easily studied than when they are
grown on bread paste.

Hueppe’s Method of Cultivating on Egg Albumen.

Eggs may also be used as culture media. The yolk is broken down and
mixed with the white by means of thorough shaking (or the white only
may be used, in which case the yolk is left unbroken) ; the shell is then
disinfected with bichloride of mercury solution, a hole is chipped at one
end, and the membrane cut through with a pair of sterilized scissors. The
inoculation is made with a pipette or a platinum needle, and then the open-
ing is covered with a piece of sterilized cotton wadding or paper, which is
painted over and sealed with surgical collodion.

Potatoes.

The simplest and most effective way in which potatoes can be used as
solid culture media is by introducing small wedge-shaped strips into sterilized
test tubes. All that is here necessary is to clean the potato thoroughly, then
steam it for five minutes, allow it to cool, and, with a cork-borer or apple-
corer, cut out a cylinder from the longest. diameter, remove the two ends of
the cylinder with a knife, and cut obliquely across from end to end, so that
two wedge-shaped portions are formed ; each of these is put intoa test tube,
preparéd as follows:—Into a plugged test tube, sterilized by dry heat, a
small piece of sterilized cotton wadding, well moistened with distilled water,
is introduced and pushed to the bottom; the potato wedge is then intro-
duced so that the base rests on the surface of the moist wadding. The
whole is boiled in the steam sterilizer for at least three quarters of an hour
(better an hour or an hour and a half), when it is ready for use.

If test tubes are not readily obtainable, a soup-plate and a basin, well
washed and rinsed with a 1 per 1,000 solution of bichloride of mercury may
be used as a sterile chamber, a couple of layers of blotting-paper soaked with
the. bichloride solution being placed in the soup-plate, on to which any
organisms in the air under the basin may fall after the sterilizing process
has been completed and the parts of the apparatus placed in position.
A clean and smooth-skinned potato is thoroughly scrubbed, and the eyes
and any diseased portions are removed with a sharp-pointed knife ; it is
then soaked for fifteen minutes in a I per 1,000 solution of bichloride
of mercury, after which it is washed in water, wrapped in paper, and
steamed for half an hour; at the end of twenty-four hours it may again
be steamed for fifteen minutes, and allowed to cool. (A single steaming
is, however, usually sufficient.) The hands are carefully washed first in
soap and water, then in a I per 1,000 sublimate solution. A knife is
sterilized by heat in a naked flame, or in the hot-air chamber at 150°C. (in
the latter case it should first be wrapped in cotton wadding or in paper),
and then allowed to cool. The potato is taken in the left hand, and, with
the sterilized knife, is cut through the middle, and the two halves are intro.

27

402 APPENDIX.

duced under the basin with their cut surfaces, on which the inoculations
are to be made, uppermost. Various modifications of these methods may
be made by individual workers, but in most cases the potato used in the test
tube is the most convenient.

Koch's Gelatinized Meat Peptone Medium.

To prepare Koch’s-peptone meat jelly or solid gelatine medium, cover
one pound of beef, freed from fat and finely minced, With 1,000 cc. of water,
. to which a drop or two of hydrochloric acid may be added ; allow the mix.
ture to stand in a cool place for twenty-four hours, and squeeze through a
cloth, as described in the preparation of meat extract. To this fluid add 10
grammes of albumen peptone, 5 grammes of common salt, and 100 grammes
(75, or even 50, if the weather be cool) of Coignet’s go/d /aéel gelatine.
Mix in a two- or three-litre flask, boil for half an hour, or until the gelatine is
thoroughly dissolved, neutralize with the alkaline solution (p. 399), and again
boil for nearly anhour. This,length of boiling in some cases appears to be too
prolonged, the gelatine afterwards not becoming properly solidified. Insuch
cases it is necessary to add a little more gelatine, boil, and again neutralize.
The mass is then filtered through a layer of fine white filter-paper, moistened
with hot water in a funnel, which must be kept heated, to prevent solidifica-
tion of the gelatine on the filter. Salomonsen recommends a very ingenious
device, a device that may be used at almost any time, especially where only
small quantities of gelatine are to be filtered. He pours a little of the warm
gelatine into a filter which has been previously sterilized, warmed, and
moistened as follows : a layer of water is poured into a flask to the depth of
half an inch, in the mouth of which rests a funnel with a clean filter-paper.
The top of the funnel is covered with several thicknesses of filter-paper,
over which a sheet .of asbestos or a plate of zinc is laid. By heating
the water to boiling-point for a few minutes, flask, funnel, and filter are
at once sterilized, warmed, and moistened. The hot water is then poured
from the flask, the funnel is replaced, its cover carefully removed, and the
gelatine is poured into it. If the flask be placed on a non-conducting sur-
face, a very considerable quantity of gelatine may be thus filtered. Where
larger quantities have to be filtered, it may be necessary to obtain a double
walled metal funnel, or, better still, an enamelled funnel with the limb
bent at an angle. This funnel is fitted into the top of the steam sterilizing
apparatus, the bent tube coming through the side. This, of course, can be
heated for any length of time, and is useful when large quantities of gela-
tine or agar-agar are dealt with. (This apparatus, which is comparatively
cheap, may be obtained from Frazer, Teviot Place, Edinburgh.) Before
filtering, it is sometimes necessary to clarify the gelatine by a process well
known to cooks. The gelatine is allowed to cool to about 50° C., the
white of an egg is broken into 100 grammes of water; this, along with the
broken-up shell, is added to the gelatine, with which it is thoroughly mixed.
The whole is then boiled until the albumen coagulates and a perfectly clear
liquid appears between the flakes; the mass is then filtered.

Gelatinized Milk Serum.

One litre of fresh milk is warmed to 60° or 70° C. Add from 7o
to 100 grammes of gelatine (according to external temperature) and
dissolve. Boil for « few minutes, until all the casein is precipitated,
and pass through a fine muslin strainer. The fluid is allowed to

APPENDIX. 403

stand for about twenty minutes at the temperature of the body, in order
to allow the fat to come to the surface, after which it is allowed to cool,
and the layer of cream is carefully removed. To the resulting slightly
opalescent fluid, 1 per cent. of albumen peptone is added, and the whole is
neutralized, boiled, filtered, and sterilized and used-as nutrient gelatine.
This material, like agar-agar (which was first used by Frau Hesse), was
introduced into bacteriological work by a lady, Madame Raskina.

Agar-Agar Peptone Meat Jelly.

In place of 30 per cent. gelatine, we may use 1.5 per cent. of agar-
agar. This is prepared in much the same manner as the gelatine jelly,

Photographs. Growths on oblique Agar-Agar surface a

1. White Torula,

2. Yellow Torula,

3. Aspergillus Albus.

4- Scheuerlein's Cancer bacillus,

Numbered from left to right.
except that it requires a more prolonged boiling before it can be properly
filtered. It is prepared in the following manner :—Cut into pieces, about
half an inch long, 15 grammes of agar-agar, place in a porcelain basin, and
soak in a strong solution of common salt for twenty-four hours, pick out
the coarse particles of dirt and wash thoroughly, then drain off the water ; to
the washed agar-agar add one of the following solutions: (1) Water, 1,000
grammes; beef, 1 pound; peptone, 10 grammes; common salt, 5 grammes;
or (2) water, 1,000 grammes; Liebig’s extract of beef, 5 grammes; albumen
peptone, 39 grammes; common salt, 5 grammes.
In (1) the beef is first extracted, as in the preparation of gelatine. The

404, APPENDIX,

preparation after this is the same in both cases. Boil the mixture thoroughly
over a naked flame for about an hour (great care being required at first to
prevent the mixture boiling over in consequence of the large amount of
air contained in the agar-agar, which is, however, gradually boiled out). As
a small quantity of the agar is dissolved, the boiling point is gradually raised
until the temperature of the mixture is about 105° C., instead of 100°C., and
the complete solution of the agar-agar is more readily brought about. As
soon as the whole of the agar is dissolved, which is usually in about an hour,
the mass should be filtered as quickly as possible through a very hot filter.
To Make Glycerine Agar.—After filtration of the agar jelly, add from 5
to 8 per cent. of glycerine, 7.¢., 50 to 80 cc. per 1,000, sterilize and put into
test tubes. After sterilizing, allow to cool in the oblique position in order
to obtain a larger surface. This also applies to the other agar-agar media.

For the preparation of Agar-Agar and Gelatine (see p. 392-)
Koch's Sterilized Blood Serum.

A large stoppered glass jar, well washed with soap and warm water, is
sterilized either by being wrapped up in paper and heated for an hour at a
temperature of 150° C., or by being rinsed with a 1 to 1,000 solution of
bichloride of mercury, and then with absolute alcohol to remove the mercury
salt. The stopper is greased with pure vaseline. When the animal from
which the blood is to be collected has been stabbed the first few drops of
blood that come should be rejected, and that which follows must be col-
lected as carefully as possible. The jar is then placed in cold water until
the blood coagulates, removed to the laboratory and placed in an ice box ;
and the first few drops of coloured serum that soon make their appearance on
the surface of the clot are carefully removed with a pipette. It is then left
for from twenty-four to thirty-six hours, at the end of which time a quantity
of clear serum will be found to have been expressed. In the meantime a
pipette has been prepared by exposing it to the action of boiling water for
ten or fifteen minutes, or by thoroughly cleaning it with hot water and
rinsing with bichloride of mercury and alcohol and then passing it through
a flame for the purpose of driving off the alcohol. This pipette is used to
transfer the clear serum from the space around the clot to the sterilized
plugged test tubes, care being taken that the serum is not allowed to smear
the sides of these tubes. The test tubes, with the contained clear serum, are
placed for an hour in a water bath, which is kept at a temperature of 58°
to 60° C, every day for a week. Each day the serum becomes clearer, and
a small precipitate of grey powdery material collects at the bottom and a
thin film of cholesterin forms on the surface. This water bath may be
constructed very cheaply. It is made of tin, is about twenty-seven inches
high and four inches in diameter, with a collar projecting about half an
inch. Over this collar a piece of iron netting serves both as a lid and as a
support to the thermometer, which is fixed in a cork passing through a hole
in the centre ; or a bulb thermometer may be used. This is supported by a
second piece of wire gauze resting at some little distance—about three-
quarters of an inch—from the bottom of the bath. When the water has
reached a temperature of 58° C. it is easily maintained there by means of a
small spirit flame. In place of this simple apparatus any of the small incu-
bating chambers sold by the chemical apparatus makers may be used for this
discontinuous sterilization at comparatively low temperature. Special serum

APPENDIX. 408

sterilizers and inspissators may also be obtained from the apparatus makers.
Blood serum so sterilized may be used in various ways. It may be rendered
solid with the tube in an upright position, or when a large surface is required
in an oblique position, by placing the tube on the sloping floor of a warm
chamber. This slope should be such that the blood serum does not come
within an inch of the wadding plug. To bring about the solidification the
temperature should be kept between 65° and 68° C., at which temperature
solidification takes place slowly, but the serum remains comparatively clear.
Sheep’s blood coagulates more rapidly than that of the calf, the time required
varying from half an hour ‘to an hour, according to the temperature that is
maintained. In place of test tubes, watch glasses or covered glass dishes may
be used to hold the serum. As the serum cools there collects in the tube a
guantity of condensed vapour ; this, however, may be prevented by adding a
small quantity (I per cent.) of gelatine or 6 to 8 per cent. of glycerine. This
latter prevents the formation of a dry scaly surface, and at the same time
absorbs a considerable proportion of the condensed vapour. When glycerine
is added the temperature required to bring about solidification is over 75° or

78° C,
Lifters Serum Medium.

Loffler added to three parts of blood serum one part of the flesh infusion
already described, 1 per cent. of peptone, I per cent. of grape sugar, and
5 per cent. of common salt. The meat broth, prepared as above described
(p- 399), is allowed to cool down to 50° C., and is then mixed with the serum.

Hueppe’s Agar Serum.

Hueppe uses a mixture of blood serum and agar-agar for plate cultiva-
tions, especially where he wishes to use the plate method for the separation
of the tubercle bacilli. He takes the sterilized serum at a temperature of
37° C., inoculates it, and shakes it thoroughly in order to distribute the
organisms through it ; he then pours it into the fluid agar-agar meat peptone
solution at 42° C. ; the mixture is again well shaken to continue the distri-
bution of the germs equally through the fluid, and then either plate, tube,

-or flask cultivations are made ; the mass is allowed to become solid, after
which it is kept at the temperature of the body (about 37° C.), at which
temperature it remains perfectly solid. :

To Fill Test Tubes or Flasks with Fluid Media.

Before transferring fluid gelatine or agar-agar meat broth or other fluid
media to test tubes or flasks, these should be previously plugged and
sterilized, as mentioned on p. 399. With a sterilized pipette (see p. 389)
the fluid is run into these vessels, the plug is returned, and the vessels with
the contained nutrient medium are sterilized for ten minutes or a quarter
of an hour on each of two successive days. As soon as the vessels are
taken from the steamer the plugs are covered with sheets of sterilized paper,
which are kept in position by string or indiarubber bands.

In place of the sterilized pipette a small carefully-sterilized funnel may be
used. The stem of this is simply inserted into the flask or test tube, and the
amount of the medium required is poured in from small stock flasks, A’burette
with a small funnel may be used in the same way, or better still, the stock
flasks may be fitted with an indiarubber bung in which are bored two
holes, Through one of these holes is introduced a glass tube, at-one end

406 APPENDIX.

of which is a thistle-head funnel filled with sterilized cotton wadding. The
other end dips down zearly to the level of the nutrient medium. Through the
other hole a short glass tube passes just to the other side of the bung ; its
outer end is slightly bent, and is fitted with a piece of indiarubber tubing,
on which a pinch clip fits. Into the other end of the rubber tubing a wash-
bottle nozzle is fitted. The gelatine is first filtered into this flask, and the
whole is sterilized as usual, a piece of cotton wadding or paper tied in position
serving to keep dust away from the nozzle. When tubes or small flasks are to
be filled the gelatine is melted, the flask, inverted, is fitted into a retort stand
ring, the nozzle is allowed to fall into the mouth of the vessel to be filled
and the pinch clip is compressed. If care be taken to cover the nozzle
carefully each time that the flask is used, there is very little danger of con-
tamination from without. Fluid media may also be kept in these flasks,
but Lister’s flasks are perhaps adapted better for this purpose.

Apparatus used for Inoculating Animals or Artificial Nutrient Media.

Inoculating needles are made of pieces of platinum wire mounted in glass
rods ; these are of various shapes—(1) perfectly straight, (2) with a loop for
inoculating liquids, or (3) with a hook or short rectangular limb for
inoculating extensive free surfaces, such as ‘‘ oblique” blood serum, agar-
agar or gelatine, potatoes, &c. These are usually allowed to stand upright
in a wide-mouthed bottle in which a piece of cotton wadding is placed.
Before it is used the wire should be heated to a white heat; the glass rod
is also thoroughly heated ; the needle is then allowed to cool and a small
quantity of the substance to be inoculated is taken on the end of the wire and
introduced as required. Where the inoculations have to be made into
narrow-mouthed flasks or tubes, short fragments of platinum wire are
oftenused. These, held in a pair of forceps, are heated in a flame. When
they are quite cool, one of them is dipped into the inoculating fiuid and then
dropped into the fluid culture medium. Capillary tubes, five or six iriches
long, pipettes, or glass needles, may be used for the same purpose. Where
large quantities of fluid have to be inoculated, the Pasteur pipette, which
consists of a piece of tubing with one end drawn out into a fine capillary
tube, the other being plugged with sterilized cotton wadding, is often used.-
These capillary pipettes are always kept closed at the ends. All that is
necessary before using them is to snip off the end with a pair of sterilized
forceps, pass the glass once or twice through the flame, allow it to cool, and
then draw up the amount of inoculating fluid required and inject into the
media, tissues, or vessels.

Cantani uses a similar pipette, but fits it up as follows :—Over the end
of the pipette that is filled with cottoa wadding he fits a piece of india-
rubber tubing. Into the other end of this latter is placed a piece of glass
tubing, in which is a hole at the side ; then comes another piece of india-
rubber tubing, then a glass mouthpiece, and the apparatus is complete.
‘When the apparatus is to be used, the end of the pipette is broken off after
being carefully heated and allowed to cool ; it is then introduced into the
inoculating fluid, a finger is placed over the orifice in the side of the middle
glass tube, and by suction the required quantity of fluid is drawn into the
pipette; the finger on the opening controlling the pressure or vacuum, To
inject, reverse the process. As soon as the finger is removed the pressure
within the tube becomes equal to that outside, however much suction or
blowing there may be, and the operation stops. It is a valuable apparatus,

APPENDIX, 407

because when it is used ‘there is never any danger of drawing toxic fluids
into the mouth.

Glass needles are especially useful when anrobic organisms are being
dealt with, as the smooth surface of the glass does not allow of oxygen
being carried down with it along the track, which closes up as soon as the
needle is withdrawn. ;

To Inaenlata Solid Culruve Media:

The test tube or flask is held inverted in the left hand, and the plug of
cotton wool is twisted once or twice in the mouth of the test tube to break
down any adhesions between it and the neck of the vessel. If the plug is at
all dusty, it is well to singe the surface by passing it rapidly through a
flame before removing it from its position. Wadding burns very rapidly,
and must be extinguished at once. The plug is removed and held between
two of the unoccupied fingers of the left hand, great care being taken that
no part of the plug that passes into the test tube shall come in contact
with any source of infection other than the air itself. At the same time
this portion of the plug is directed downwards, in order to avoid any falling
germs that may be present in the atmosphere. The platinum or glass
needle with its charge of seed material, is plunged straight into the gelatine
mass, then carefully withdrawn and the plug replaced. Where the seed
material is also in solid gelatine, the two tubes may be held inverted in the
left hand, one between the thumb and finger, the other between the first and
second, the plugs being held between the second and third and third and
fourth fingers. as?

Methods of Cultivating Anerobie Organisms.

When it is necessary to cultivate such an organism as that of malignant
cedema or of tetanus the experimenter has to resort to the methods
used for keeping out the air. To isolate these organisms, Koch first
used the ordinary plate cultures, placing at irregular points on the
surface of the gelatine, thin sheets of mica that had previously been
sterilized (p. 172). Fraenkel, to render the exclusion of oxygen more
perfect, ran a little melted paraffin round the margins of these plates. By
filling a tube with gelatine or agar, Liborius succeeded in obtaining cultures
of anzrobic organisms at the lower part of his culture medium. If
the tube is filled with the solid medium close up to the plug, and the air is
carefully excluded by means of melted paraffin poured over the plug, this
method often succeeds exceedingly well. It is, however, difficult to obtain
pure inoculating material from such growths without removing the contents
of the tube or breaking it up altogether.

One of the simplest methods of isolating anzrobic bacteria in gela-
tinized media is that described by Fraenkel, who uses a test tube fitted up like
a wash-Lottle, with a long and a short glass tube passing through an india-
rubber cork. This, after being carefully sterilized, has about 10 cc. of
nutrient gelatine poured into it. An inoculation is made, and the india-
rubber cork, with its two tubes, is pushed home into the mouth of the test
tube, and the whole is luted down with paraffin, great care having been
taken to sterilize, by heating in a flame, those parts of the tubes that are
to go down into the test tube. A stream of hydrogen, purified by being
washed in an alkaline solution of pyrogallic acid, is passed through this
liquefied gelatine (kept at 35° C.) for four or five minutes, The tubes are then

408 APPENDIX.

sealed in a flame, and a “‘tube plate” cultivation is made by distributing
the gelatine on the walls of the-test tube. In this way excellent anzrobic
cultures may be obtained. The advantage of this method is that fresh inocu-
lations may be readily made from the growths that appear on the thin layer
of gelatine that covers the wall of the tube. Anzrobic cultures may also be
obtained by drawing the media, that have been de-oxygenated by means of
hydrogen or by means of the air pump, into cApillary tubes, which are then
sealed so as to prevent the access of any fresh oxygen. Fluid media, of
course, may be used in exactly the same way. Where an air pump or
water exhaust apparatus is obtainable, an ordinary tube, with thick walls
and a long neck, may be used in which to make the culture; but a com-
bination of these methods, as a rule, gives the best results. To such a tube
may be fitted » ‘‘T” piece with a stop-cock, through which a vacuum
is first created ; then hydrogen is allowed to come in, the process being
repeated several times until all the oxygen is removed. One very simple
method of obtaining a solid medium in which some anzrobic organisms
will grow is to add a small quantity of glucose (1 to 2 per cent.) to agar-
agar or gelatine. For the more complicated methods we refer the reader
to special treatises.

Various methods have been utilized for preserving anzerobic cultures or
of obtaining ‘‘ needle” cultivations of such cultures in gelatine, &c. In
some of the earlier experiments on abiogenesis a layer of warm oil was
used to prevent the access of air to the nutrient medium, and this method
is still sometimes used when inoculating a solid medium with an anzerobic
organism. A puncture cultivation is made with a glass needle, and then
a layer of acouple of inches of boiled and cooled olive oil is carefully poured
over the surface. Roux used two methods—one physical, the other biological
—for the attainment of this object. For the former he draws out one end of
a pipette into a long capillary tube, then a short distance from the other
end a constriction is made; the whole is carefully heated to 150° C. in the
hot air chamber, or by a Bunsen burner, the point of the pipette being
closed by fusion, and the other end plugged with sterilized cotton-wadding.
This pipette is then filled with sterilized nutrient gelatine that has been
brought to the boiling point in order that as much air as possible may
be driven off., To do this the capillary point is broken with sterilized
forceps; the fluid is drawn up to the level of the constricted portion of
the tube by means of suction ; the capillary end is then fused, after which
the tube is also fused at the constricted part at the other end. Such tubes
may be kept for an indefinite period. To inoculate the gelatine in this
tube one end is broken, a fine glass needle on which is the material to be
inoculated, is carefully introduced’ and the end is again sealed. Under
these conditions no air can make its way to the organism, or to the medium
into which it is inoculated.

Roux’s second, or biological, method is to boil a quantity of nutrient
agar in a test tube, and then cool it as quickly as possible in cold
water. It is then inoculated with the anerobic organism on a smooth
glass needle.: A layer of melted nutrient gelatine is poured on the surface,
and when this is cooled a drop of a broth cultivation of ‘‘ Bacillus subtilis ”
is run on to the surface from a capillary pipette. The tube is then
fused, or the cotton-wadding plug is rendered impervious by being luted
with warm paraffin. As the Bacillus subtilis develops and grows it uses
up the oxygen at the surface. The organism below receives none, and

APPENDIX. 409

is thus placed under favourable (anzrobic) conditions for its growth.
To obtain inoculating material from such a tube it is necessary to break it at
the bottom when the growth is easily taken from the lower part of the
medium. Salomonsen uses a modification of this method, placing one
tube within another. A small inner tube containing gelatine or agar
is inoculated with the anzrobic organism, the outer tube, the neck
of which is sealed, or the opening of which is closed with paraffin,
contains bouillon which is inoculated with a strongly zrobic organism,
such as the Bacillus subtilis. In place of the bouillon and Bacillus
subtilis a solution of one part of pyrogallic acid to ten parts of a ten
per cent. solution of caustic potash may be used. Buchner, in making
use of this method, plugged his inner tube in the ordinary fashion with
cotton wadding and supported it on a kind of stage, closing the orifice of
the larger tube with an indiarubber cork. This is a very convenient
plan, as it enables the observer to gain access to the cultures very readily
indeed. All these methods may be used with both solid and fluid media ;
and now that potatoes can be used within test tubes they also may be used
for the cultivation of anzerobic organisms. Here all that is necessary is a
strong test tube with a lateral stem. The inoculation is made through the
open mouth of the tube which is then sealed, after which, by means of an
exhaust apparatus, the air is extracted from the potato and then from the
tube ; the lateral tube is then sealed.

To Separate Bacteria from their Products.

To obtain the products of bacteria apart from the bacteria, and, there-
fore, ina sterile condition, several methods of filtration have been suggested,
all of them depending on the close porous nature of unglazed porcelain and
baked clay. When fluid that originally contains bacteria is aspirated or
forced through such a filter, all the organisms are kept back, and a perfectly
clear sterile fluid comes through on the opposite side. The best form of filter
is a tube of this unglazed porcelain, one end of which is closed, and the
other so constricted that a thick walled indiarubber tube may be affixed.
The Chamberland filter consists of a wide tube, as above described, the
upper piece of which is composed of a glazed funnel-shaped end with a
nipple on which a piece of indiarubber tubing can be fastened. The other
end of this tubing is fitted to the long glass tube of an ordinary strong wash
bottle or flask which acts as a receiver; the second or short tube of the wash
bottle is attached to an aspirating apparatus, either in the form of a siphon
bottle placed at a lower level, or of a Geissler water exhaust pump. The wash
bottle, the filter, and the glass tube, are all carefully plugged, covered with -
paper or cotton-wadding and sterilized for an hour at 150° C. The
indiarubber stopper of the wash bottle and the tubing are sterilized by
being well soaked in a 1 per 1,000 solution of bichloride of mercury,
washed with sterilized distilled water, and then boiled for twenty minutes
in the steaming apparatus. As soon as everything is cool the hands are
thoroughly cleansed, and the apparatus is put together as above described,
a small cotton-wadding plug being left in the short tube of the wash bottle
to prevent the return of unfiltered air when the exhaust apparatus is re-
moved. When all is ready, the filter is lowered into a tall glass jar with
a firm base, which contains the fluid to be filtered. The aspirator or other
exhaust apparatus is set to work (care being taken that the exhaust is not
too great), and the fluid is drawn into the flask which is gradually filled.

410 APPENDIX,

Another flask may be inserted, and so on until the whole of the fluid is
filtered. At the end of the process, such fluid as remains in the filter is
withdrawn by means of a sterilized pipette, and may be used along with
the other, though it is better to use a fresh filter for each flask, as then the
indiarubber communication between the flask and the filter may be clamped
and the flask removed. The fluid within the flasks so removed may
be kept sterile for a considerable length of time. Another very con-
venient filter is Kitasato’s, which consists of a large thistle shaped funnel
attached by an indiarubber connection to a piece of very thick pipe stem
of unglazed porcelain, which is well-plugged with baked porcelain at the
bottom. This pipe stem passes down through the indiarubber cork into a
bottle which serves as areceiver. From the side of the neck of the receiver
a lateral tube is given off, to which the exhaust apparatus may be attached.
The apparatus is thoroughly sterilized as above. The fluid to be filtered is
placed in the funnel, the exhaust is applied and the filtrate passes down
into the bottle. Here also it is well to place a small plug of cotton-wadding
in the lateral tube. In all cases where the water exhaust pump is used it
is well to have a bottle intervening between the receiver and the pump, so
that should there be any backward flow of water it may pass into this flask
and not into the receiver. This filter requires a somewhat stronger exhaust
than the Chamberland pattern, and an ordinary aspirating siphon bottle
is not sufficient to obtain the required suction. Various modifications
of this apparatus will at once suggest themselves to an ingenious worker,
but one that may sometimes be used, especially where the culture
fluids have to be kept at a low temperature, is that recommended by Miquel.
It consists of a flask with a long wide neck; at some distance above the
bulb (about halfway) this neck is considerably constricted, and between
the constriction and the bulb is a long narrow lateral tube which is pointed
somewhat downwards. To prepare this as a filter, the tube above the
constriction is packed with asbestos, above this is poured some thoroughly
dried and sterilized plaster of Paris made into a cream-like paste with
boiled distilled water, this should be nearly an inch in thickness. The
plaster is allowed to “set,” and the filter is ready for use. A small
quantity of water is put into the flask and thoroughly boiled, the steam
escaping from the lateral tube. As soon as the water is boiled away, but
while the flask is still full of steam, the lateral tube is sealed. The whole
apparatus is now thoroughly sterile: an indiarubber bung with a funnel _pass-
ing through the centre is fitted into the tube above the plaster of Paris ;
into this the fluid to be filtered is poured. As the air in the flask cools
(and eventually ice may be packed round the flask) the fluid is drawn
through the plaster of Paris; this flask can seldom be more than about half
filled in this manner. After the indiarubber cork has been removed, the
flask may be kept in ice, the contents remaining sterile for an indefinite
period.
Hanging Drop Cultures.

The best form of moist chamber for making drop cultures is an ordinary
slide into which is cut a deep round groove which surrounds a central pillar
or disc of glass.which has been ground and polished so as to be slightly .
below the level of the remainder of the slide. This, after careful steriliza-
tion, is used as follows: A small drop of the fluid is placed upon the lower
central pillar (a small portion of the fluid may also be allowed to run into

APPENDIX, 4Il

the groove), the cover glass, after being thoroughly heated in the flame, is
allowed to cool; a ring of vaseline is painted on the slide around the
groove ; on to this the cover glass is lowered, where it compresses the drop
of culture fluid, which thus presents a flat surface and allows of the
development of the bacteria being exceedingly well followed. The groove
around the disc forms a kind of air chamber. If it is required that the
oxygen should be absorbed this groove may be partially filled with the
alkaline pyrogallic acid solution.

Various modifications of the hanging drop culture method are described.
Buchner dries the spores upon a sterilized cover glass, then places on this
a drop of his culture fluid and inverts the cover glass ; fragments of cover
glass are then arranged around the drop so as to support the cover glass cn
which it is hanging and the whole is cemented down to form a perfectly
air-tight cell in which the development of the organisms may be readily
watched.

Salomonsen describes a moist chamber constructed of a thin sheet of card-
board which is sterilized by boiling, and is rendered so soft that it fits
accurately to the slide and allows of the cover glass with its hanging drop
of inoculation fluid being pressed down so as to form an air-tight chamber ;
the moisture from the cardboard serving to prevent the evaporation of the
fluid. It is difficult to keep these cardboard supports sufficiently moist and
sterile without the use of somewhat complicated moist sterilized chambers.
The best way of keeping them, however; is under bell jars, the air of which
is saturated with moisture. These hanging drop cultures may be dried, and
the organisms are then stained as in the case of an ordinary cover glass pre-
paration. Watson Cheyne has utilized this method in a most ingenious
manner for obtaining a permanent record of the various phases of develop-
ment of micro-organisms. He makes a series of hanging drop cultures of
any organism that is to be studied, and then dries and stains them,
taking them at stated intervals, say, five, ten, fifteen, twenty minutes, and
so on through a whole series. Jn this manner the whole cycle of develop-
ment may be watched and referred to again and again, especially if different
series of these cultures be grown at different temperatures.

METHODS OF STAINING BACTERIA.
Methylene Blue.

Methylene blue may be kept as a saturated alcoholic solution; a few
drops of this filtered into water will give a very beautiful stain in cover
glass preparations. The sputum, &c., on the cover glass, after being dried
and passed three times rapidly through a spirit lamp or Bunsen flame (see
page 206), is floated on the surface of the methylene blue, it is allowed to
stain for five or ten minutes, washed in water (sometimes first in alcohol),
tilted on edge and allowed to dry. It is then mounted in xylol balsam.

Kiihne’s Methylene Blue Method.

One of the best general stains for bacteriological work is Kiihne’s methylene
blue solution. 1.5 grammes of methyl blue is dissolved in 10 cc. of absolute
alcohol and 100 cc. of a 1 to 20 watery solution of carbolic acid. Specimens
are stained in this for from five minutes to two hours, although sections
may be left in it for a whole day without becoming overstained. They
are then carefully washed in water, then with acidulated water made by
adding a couple of drops of hydrochloric acid to 100 cc. of water. As soon

412 ‘APPENDIX.

as the sections become a pale blue colour they are transferred to a solution
of lithium water made of the strength of about 1 part of lithium carbonate
to 20 of water; they are then thoroughly washed in pure water, de-
hydrated in absolute alcohol in which a little methyl blue may be
dissolved, placed in aniline oil, which may or may not contain a small
portion of methyl blue in solution, and rinsed in pure aniline oil. They are
after this treatment transferred to terebene, where they are left for about a
couple of minutes. They are then washed in two lots of xylol and mounted
in Canada balsam. Almost any organisms may be stained by this method,
even the glanders bacillus coming out fairly distinctly.

Another great stand-by of bacteriologists is fuchsin. With this reagent
almost every bacillus may be brought into prominence. It is especially
useful for the bacilli of tubercle, leprosy, and mouse septicaemia.

Zichl-Neelsen Carbol-Fuchsin Method for Tubercle Bacilli.

The Ziehl-Neelsen method of staining the tubercle bacillus is a modification
of the Weigert-Ehrlich method. The sections or cover glasses are stained in
Neelsen’s solution, made as follows :—Fuchsin 1 part, is dissolved in 10 parts
of absolute alcohol, to this solution are added 100 parts of a 5 per cent. watery
solution of carbolic acid, and the mixture is heated until steam rises pretty
freely. Cover glass preparations are stained in three or four minutes, or
even less; sections are usually sufficiently deeply stained in seven or eight
minutes. In the cold they may be left for twelve or even twenty-four hours.
Thesuperfluous fluid is drained off and the preparations are placed for a second
or two in alcohol (90 per cent.), then in a 25 per cent. solution of sulphuric
acid, when the pink tinge should immediately be replaced by a yellowish
brown. The preparations are then washed in alcohol, and if they are
sufficiently decolorized they are transferred to a solution of lithium
carbonate. They may afterwards be stained with a watery solution of
methylene blue, cleared up with clove oil or with aniline oil, terebene, and
xylol, and mounted in Canada balsam. Exceedingly good results are obtained
by this method, which is preferable in many respects to the aniline oil
method. In place of sulphuric acid nitric or hydrochloric acid may be
used..

The Kiihne-Gram Staining Method.

Kiihne’s modification of Gram’s method is, perhaps, superior to the
original. Instead of usiag Weigert’s saturated alcoholic solution of methyl
violet (or gentian violet) in 100 parts of aniline water and 10 parts of abso-
lute alcohol, he stains with a 2 per cent. solution of gentian voilet in dilute
alcohol, to which has been added one sixth of its bulk of a 1 per cent. watery
solution of ammonium carbonate, or with a similar solution of Victoria blue
without the ammonium carbonate, for about five or ten minutes. The pre-
parations are then rinsed in water, and are placed in Gram’s solution made up
of iodine 1 gramme, iodide of potassium 2 grammes, distilled water 300 cc.
for two or three minutes ; they are again washed in water, dehydrated with
fluoresceine alcohol, which is prepared by dissolving 1 gramme of yellow
fluoresceine in 50 cc. of absolute alcohol, the part undissolved being allowed
to settle at the bottom of the bottle. The section is washed in pure alcohol,
then with aniline ofl, and mounted in xylol balsam.

The Kiihne-Unna Method.
Kiihne describc 2 modified method of making dry preparations adopted

APPENDIX, 413

from Unna’s method. The preparations are stained in methylene blue,
and up to the stage of the washing with lithium carbonate are treated
exactly as above The section is then spread out on a cover glass, and
the moisture is allowed to run off the edge on to blotting-paper, the upper
surface being carefully wiped with a cloth. With a balloon syringe a stream
of air is then directed down on to the section as it lies on the cover glass,
and beginning at the centre of the section gradually driving the water
towards the margins, and then on to the cover glass, whence it may be
removed by scraps of blotting-paper. The sections are then placed ona plate
of glass which is gently warmed over a lamp to the body temperature, the
sections gradually becoming transparent and glassy, the heating is continued
for about five minutes after this occurs, after which they are treated with
terebene, then with xylol, and mounted in balsam in the usual fashion.
For other methods the reader is referred to works specially devoted to the
treatment of this subject.

Zo demonstrate Tubercle bacilli in Milk.

To demonstrate tubercle bacilli in tuberculous milk the best plan is to
pass the milk through a centrifugal apparatus and to take the sediment for
examination, as almost the whole of the bacilli that were originally in the
milk will be found along with the mucus and solid particles in this sediment,
Where it is not possible to obtain the use of such apparatus the milk should
be allowed to stand for from twelve to twenty-four hours in a glass ‘‘ sepa-
rator ” such as is used by chemists or in a conical or funnel-shaped vessel
surrounded by ice, The sediment with the contained bacilli is drawn off
from the separator by the tap placed at its lower part, or the cream and the
upper layers of the milk may be carefully removed by means of a siphon,
then with a pipette a few drops of the milk from the bottom of the funnel
are taken, dried on a cover glass, and examined in the ordinary way. In
place of the separator or other funnel-shaped vessel, I have used, at Mr.
Coghill’s suggestion, a long wide burette in which to place the milk. In
drawing off the sediment from the separator or burette the first few drops
are rejected, the fluid from immediately above the stop-cock, which contains
most of the bacilli, being taken.:

To demonstrate Flagella on Bacilli.

Make a potato broth composed of two parts cooked potato mashed and
boiled in ten parts of distilled water ; carefully sterilize ; on this make a
cultivation of the required organism. A drop of the culture is then diluted
from five to ten times with distilled water. If the organisms will not
grow on this potato broth they may be cultivated in meat bouillon, which
must be diluted forty or fifty times before it is used for microscopic ex-
amination, or on gelatine, which must be diluted about one hundred
times. A drop of the diluted fluid is spread on a cover glass; cn this
a drop of 10 per cent. alcohol is allowed to fall; the whole is dried in
the open air or in a warm room at a temperature of 40° C. ; the bacilli are
then stained in a solution made up as follows :—10 per cent. tannin solution
20 parts, water 80 parts, cold saturated solution of sulphate of iron § parts,
fuchsin or methyl violet 1 part ; to this mixture a drop of hydrochloric acid
in some cases, or of an alkaline solution in others, will bring out flagella
most beautifully. Acetic or sulphuric acid may be used. Cholera bacillus,
vibrio Metschnikoff, spirillam rubrum, spirillum concentricum, and proteus

414 APPENDIX.

vulgaris all stain on the addition of larger or smaller portions of acid. With
alkali the bacillus crystallosus, micrococcus agilis, and the typhoid bacillus all
show flagella. The glanders bacillus, although said to be motile, has
apparently no flagella.

IDENTIFICATION OF SPECIES OF BACTERIA.

The Organism is a Micrococcus.
The i » Bacillus, see p. 421.
The 4 » Spirillum, see p. 438.

Ihe Organism 7s a Micrococcus.

I. The gelatine is not liquefied.

II. The ,, _ is liquefied, see p. 418.
III. No growth on gelatine, see p. 421.
The gelatine is not liquefied.

A. The colonies are white.

B. The ‘ yy yellow, see p. 417.
c. The i » red, see p. 418.

pb. The y black, see p. 418.

‘A. The colonies are white.
a. The colonies are small, but confluent, growing slowly.
4. Colonies confluent, growing luxuriantly, p. 416.

a. The colonies are small, not confluent, growing slowly.

(1) Streptococcus pyogenes.—On plates grow as small punctiform masses
$-mm., in diameter, at first appear white, pale yellow, and then brown,
under low power of microscope ; no tendency to run together in either
plate, puncture, or stroke cultivations, except on blood serum, or agar-agar,
where the mass is thicker in the centre ; terraced towards edges, and then
again discrete as in gelatine cultivations at the extreme margins ; no growth
on potatoes ; Cocci Iu. in diameter arranged in chains or diplococci; not
pathogenic to mice or healthy rabbits ; frequently found in pus in human
subject and in lymphatics, near the spreading margin of a suppurating area.

(2) Streptococcus erysipelatosus.—Very like the above, but differs in that in
stroke cultivations the colonies have a somewhat greater tendency to run
together ; these appear whiter and more opaque, and have at the periphery
numerous outgrowths which consist of projecting chains, which give to the
cultivation the appearance of a fern-leaf; found in the lymphatics of the
spreading zone of an erysipelatous area ; it sets up erysipelatous inflammation
when inoculated into the ear of a rabbit ; sets up typical erysipelas and not
suppuration in man.

(3) Streptococcus pyogenes malignus.—Cultivated by Fliigge (also
described by Krause), from necrotic masses in a leucaemic spleen.
Colonies only visible at end of forty-eight hours ; stroke cultivations, like
those of No. 1, fatal to mice and rabbits in about fourdays. Symptoms at
first like those obtained with 1 and 2, but soon followed by suppuration
and general infection.

APPENDIX, 415

(4) Streptococcus articulorum.—Found in the mucousmembrane and tonsils
of cases of diphtheria and scarlatina; colonies grow slowly; appear as
transparent watery greyish drops with delicate feather-like protrusions at
the margins; chains have here and there larger cocci; slight indications
of transverse division ; often kills rabbits and mice with formation of pus
in joints in which these streptococci are found ; this occurs specially when
cultivations are injected directly into the veins.

(5) Dzplococcus albicans tardissimus.—Grows very slowly on nutrient
jelly, the track being only about 1mm. broad after several weeks ; grows
more rapidly on blood serum at the body temperature, when colonies form
as greyish-white points; these have a peculiar moist appearance and an
irregular outline ; identical in form with the gonococcus (see p. 421), but
individuals are more adherent and form small masses.

(6) Streptococcus septicus.—Colonies grow very slowly indeed ; seen as
fine points on fourth and fifth days in plate and puncture cultivations ; cocci
have a special tendency to form chains or diplococci ; fatal to mice in forty-
eight to seventy-two hours, to rabbits in three or four days, when injected
into veins ; vessels in various organs plugged with organism, this leading
to the formation of purulent or necrotic foci.

(7) Micrococcus or Diplococcus of Trachoma. (Sattler).—An organism
found in the contents of the follicles of the eyelids in cases of acute con-
junctivitis met with in Egypt; in the contracted follicles met with in
trachoma. It grows on plates in the form of whitish clouds; in gelatine
tubes it grows as pearly white tufts, little beads running along the line of
the needle ; later, these become slightly yellow ; on agar-agar, potatoes,
and blood serum we have a similar growth on the surface, which is usually
somewhat viscid ; grows best at the body temperature ; is a diplococcus, but
the line of division is not very distinctly marked ; the only motion that
has been noticed is a rotatory, or oscillatory one ; gives rise to trachoma
when inoculated into the eyelids of the human subject, but does not effect
rabbits,

(8) Adécrococcus of Cattle pneumonia (Micrococcus der Lungenseuche
der Rinder). (Poels and Nolen).—This organism grows on plates as sharply
circumscribed white rounded colonies with a delicate yellow tinge; in
gelatine tubes it grows very much like Friedlander’s pneumonia bacillus,
but in place of being white it has a delicate cream colour ; has a similar
growth on agar-agar; on potatoes it forms a moist yellowish layer; on
blood serum it is at first white but gradually assumes the cream colour
above mentioned ; this organism, which grows best at about 37°C., consists
of cocci of various sizes of an average diameter of .gu ; it is single, or may
be arranged in short chains of from two to six cocci; is usually surrounded
by a somewhat deeply stained capsule; pure cultures introduced into the
trachea of rabbits, guinea-pigs, dogs, and cattle produce pneumonia.

(9) Micrococcus of Mastitis (Micrococcus der Mastitis der Ktihe).—Obtained
by Kitt from the inflamed udder of the cow. | On gelatine plates it grows as
little opalescent white rounded well-defined drops from the size of a pin’s
head to a lentil ; in gelatine tubes it grows as a white opaque fungus-like
mass along the needle track ; on potatoes it occurs as a prominent layer,
whitish or dark yellow in colour, which after several days becomes moist
and glistening looking ; grows in milk at the temperature of the body, and
gives rise to a lactic acid fermentation ; a micrococcus .2p to .5u in diameter,
usually in pairs, masses, or chains.

416 APPENDIX.

6, Colonies confluent, growing luxuriantly.
(a) Cocci arranged irregularly..
(8) Cocci occur as diplococci, or dumb bell shaped organisms.
(y) Cocci arranged as sarcinze.

a. Cocci arranged irregularly.

(1) Micrococcus candicans forms irregular masses, small yellowish white
discs with smooth margins in substance of gelatine ; opalescent or milk
white moist flat colonies 2mm. or more in diameter, with indented and
sinuous margins at the surface; dark brown in the centre when seen by
transmitted light, but transparent near the thin margin; nail-head appear-
ance in puncture cultivations ; micrococci quite round, moderately large.

(2) Micrococcus uree.—Grows as miliary points like mother-of-pearl,
smooth on surface, sharp margins; these grow rapidly, are well formed
in twenty-four hours; project above surface of gelatine, colony gradually
divided by fissures; along the track of the needle in a tube culture there
appear long delicate threads ; there isa large surface growth ; has a peculiar
paste-like odour ; grows best at higher temperatures, (30° C.), coccus 0.8 to
1 in diameter, occurs as diplococci, tetrads, or chains ; along with other
organisms, causes decomposition of urea into ammonium carbonate.

(3) Staphylococcus cereus albus grow moderately rapidly ; white points on
gelatine during the fisst few days ; in stroke cultivations forms a wax-like
layer, with slightly thickened irregular margins along the needle track;
grows on blood serum and potato; found in pus; usually saprophytic
in its action.

B. Cocci arranged as diplococci, or dumb-bell shaped organisms.

(1) Diplococcus lacteus faviformis grows rapidly along track of needle
in small points, which run together to form milk white colonies ; found in
the sputum and certain secretions as isolated diplococci; in cultivations
occurs as parallel bands of diplococci, each organism being about 1.25 in
length and consisting of two hemispheres, between which there is a distinct
but narrow fissure.

(2) Diplococcus albicans amplus.—Very like No. 1, found in the same
positions, but grows in thick white lines along the track of a stroke inocula-
tion, organism is comparatively large, measuring 2.25 in diameter.

_(3) Diplococcus der Pferdepneumonie.—Obtained from the lungs of a horse
affected with acute pneumonia. Has only been cultivated on gelatine and
agar-agar at the temperature of the room ; forms small white, somewhat
transparent rounded colonies in agar-agar ; along the line of the needle
track in a gelatine tube culture there is seen a row of small white granules,
which gradually become larger and coalesce, but there is no special surface
growth; this is an oval micrococcus which sub-divides in its shortest
diameter, two of them usually lying together surrounded by a clear homo-.
geneous capsule ; pathogenic for mice, guinea-pigs, rabbits, and dogs.

y. Cocci arranged as sarcinze.

(1) Micrococcus tetragonus forms small white points in gelatine in from
twenty-four to twenty-eight hours; under lens deep colonies have a faint
yellow tinge ; mulberry-like surface ; is somewhat raised on the surface of
the gelatine along track of needle in puncture inoculation ; first there appear
rounded points, which run together; these grow most readily at surface,

APPENDIX. 417

spreading into cracks and forming a layer of considerable thickness ;
micrococci about 1 to I.5# in diameter, dividing into four, which remain
united by a gelatinous envelope, or there may be a large round cell in
which there are found indications of division ; kills white mice, but not
ordinary mice; produces local abscesses or septicaemia in guinea-pigs ;
ral bis and dogs are unaffected ; unlike sarcina in dividing in two planes
only.
B. The colonies are yellow.

a. The colonies form raised drops.
6. The colonies form flat deposits.

@. The colonies form raised drops.

(1) Staphylococcus cereus flavus.—Forms white points in two days;
colonies spread on surface with irregular margins gradually assuming a dark
citron yellow colour ; in the early stages the growth is almost like micro-
coccus cereus albus ; cocci 1.15 in diameter, single, in groups, or in short
chains, found in pus; set up no pathogenic action.

(2) Micrococcus flavus tardigradus.—Grows very slowly (four tosix days) ;
in gelatine occurs as rounded or oval, dark chrome yellow coloured points ;
on the surface these have smooth wax-like surface, and project slightly,
especially near the centre ; colour is always darker in the deep layers ; along
track of needle in puncture occurs as minute yellow isolated points, which
do not make their appearance for six or seven days ; large coccus, sometimes
with peculiar dark poles.

(3) Diplococcus citreus conglomeratus occurs in certain forms of pus and
in dust ; on gelatine plates forms citron yellow colonies, raised at the
margins, at first moist and slimy, gradually becoming cracked and scaly,
forming tuberculated masses which when crushed and diluted with water
are seen to be made of cocci resembling gonococcus, or tetrads ; average
diameter 1.5y.

(4) Sarcina lutea grows rapidly on gelatine plates ; appears in two days
as yellow points with somewhat irregular and scalloped outlines, yellow in
the centre, grey in the intermediate zone and transparent at the periphery ;
occurs in air ; made up of rounded cells 1.24 in diameter, dividing in three
axes, thus giving rise to the well-known corded packets.

(5) Sarctna aurantiaca (Orange sarcina).—Forms small colonies in plate
cultures with smooth outlines ; along the track of. the needle of a gelatine
tube culture it grows very slowly, but best at the ordinary temperature of
the room, as a whitish growth; at the surface it forms an orange yellow
layer ; cocci which look as if cut in two, arranged in twos or fours, or in
regular packets.

4. The colonies form flat deposits.

(1) Aficrococeus versicolor grows rapidly, forming white points in twenty-
four hours, which twenty-four hours later become yellow ; spherical
growths in the gelatine, outline sharp, substance yellowish green in
colour, and opaque; on the surface growth is irregular or square ; has a
peculiar gelatinous consistence, and a yellowish green iridescent shimmer ;
although the growth is flat it may be slightly raised in the centre ; along
the needle puncture the yellowish colonies are developed separately; small
cocci are arranged in pairs, or in neg a

2

418 APPENDIX,

c. The colonies are red.

(1) Micrococcus cinnabareus forms cinnabar red drops, grows very
slowly, only just visible at the end of four days in the deeper gelatine, and
colonies are very small on the surface; at the end of eight days appear
as small wax-like drops on the surface of the gelatine, these gradually
deepen in colour ; the deeper cultivations along the needle track remain
white ; on plate cultivations superficial colonies when seen magnified bya
lens, are light brown, rounded with somewhat irregular outline, and slightly
nodulated surface ; margins transparent.

(2) Micrococcus roseus, a rose-coloured growth, flourishes luxuriantly on
the surface of gelatine and at the ordinary temperature ; somewhat raised,
especially at the margins ; moist and granular with distinct rosy red colour ;
arranged as diplococci with a broad division between the two halves, 1
to 1.5y in diameter.

_ (3) Pink Torula (not a micrococcus, but frequently met with). A coral
pink mass, growing freely on the surface of gelatine. Small white or
grey points along the needle track. On bread paste grows as a rose-coloured
succulent film. It consists of rounded or slightly oval cells 5 to 8 in
diameter ; these contain pigment of delicate yellow colour, under micro-
scope, pink only in mass.

D. The colonies are black.

(1) Black Torula (not a micrococcus, but sometimes met with in air).
Grows on gelatine as a black heaped-up mass. Along the track of the needle
it forms small black nodules. On potato and bread paste grows asa dull
sooty crust with a dry slightly furrowed surface. In milk it forms a black
crust, with a dusky grey tint on the upper surface. The milk itself becomes
of a muddy colour from an invasion of the deeper layers by colonies of the
organism. Under the microscope is like the Pink Torula, but with a dark
brown pigment. :

II. The gelatine is liquefied.
A. The colonies are white.
B. The colonies are yellow, see p. 419.

: A. The colonies are white.

(1) Staphylococcus pyogenes albus.—Grows rapidly in plate cultivations ;~
colonies seen under lens are dark in the centre, with smooth borders ;
liquefy the gelatine on the second or third day, forming a little clear cup,
with a white mass at the bottom ; liquefying centres gradually run together.
Along track of needle white mass is formed ; liquefaction commences at
surface and extends along the whole track ; at the bottom of the liquefied
gelatine is a greyish or white deposit ; a micrococcus .8 to .gu in diameter ;
occurs as irregular masses, diplococci, tetrads or short chains; is fatal in
large doses to mice, guinea-pigs and rabbits, if injected into the veins or
into the peritoneal cavity, otherwise usually forms abscesses ; appears to be
especially associated with suppuration, pyemia, ulcerative endocarditis,
osteo-myelitis and similar diseases ; it is found in pus, necrotic tissues and
in capillary vessels of internal organs. ae

(2) Micrococcus uree liquefaciens.—In plate cultivations forms small white
points, somewhat opalescent with well-defined margins which appear in two
days; grows more rapidly near the surface ; surface granular, and as the

APPENDIX. 419

gelatine becomes liquid the border becomes wavy ; along the track of a
needle in puncture cultivations there is first a continuous growth, liquefac-
tion takes place along this track commencing at the surface, and in the
later stages the gelatine is liquefied to the depth of the needle track, a
sediment of light yellow deposit being thrown down, the liquefied gelatine is
somewhat turbid but uncoloured ; organism rounded 1.25 to 2u in diameter,
occurs singly or in short chains; this organism is supposed to convert urea
into carbonate of ammonia by a kind of fermentation.

(3) Sarcina alba.—An organism obtained from the air. Grows slowly on
plates as small white colonies along the needle track; in gelatine grows
slowly and forms a white projecting head on the surface of the gelatine,
causing very slight liquefaction near the surface ; on agar it forms a whitish
yellow layer, which surrounds the point of inoculation ; grows like a small
coccus arranged in twos, fours, or packets.

B. The colonies are yellow.

a. Gelatine liquefies slowly and imperfectly.
4. Gelatine becomes completely liquid.

a. Gelatine liquefies slowly and imperfectly.

(1) Micrococcus flavus desidens occurs in the dust of the atmosphere ;
organism grows slowly in the depth of gelatine where the colony has somewhat
irregular outline, grows more rapidly at the surface ; it isthen dull yellow or
brown in colour, is smooth and almost slimy in consistence ; gelatine under-
neath is softened, and there is slow sinking of the surface growth, the soft
jelly becomes opaque ; the organism is a small coccus; may be arranged in
diplococci, in triangles or in short chains ; is non-pathogenic.

(2) Micrococcus erogenes (Miller).—Found in the intestinal tract. On
gelatine plates forms dark coloured round scalloped colonies with smooth
outlines ; under the microscope these may be either opaque or transparent ;
in gelatine tubes the growth occurs along the track of the needle as a
brownish yellow mass, forming on the surface a flat greyish white porridge-
like layer of some thickness; liquefaction takes place at a later stage; the
same yellowish white porridge-like layer is seen on both agar-agar and
potato growths ; it is a large non-motile oval coccus.

4. Gelatine becomes completely liquefied.
a. Colonies remain limited to the centre of the liquefying area.
B. Colonies are found occupying both in the centre and the periphery
of the liquefying area, see p. 420.

a. Colonies remain limited to the centre of the liquefying area.

(1) Staphylococcus pyogenes aureus (Probably identical with Micro-
coccus of Osteomyelitis).—Grows rapidly in plate cultivations ; seen under
microscope as light brown circular masses, darker in the centre and with
smooth borders ; these become yellow and liquefy the gelatine on the second
or third day, forming a little clear cut funnel with an orange yellow mass at
the bottom ; liquefying areas gradually run together ; along puncture track of
needle in gelatine tube a white mass is formed, which only becomes yellow
on the access of air, liquefaction commencing at the surface and extending
the whole length of track ; micrococcus .8 to .gu in diameter; occurs in
irregular masses, diplococci, tetrads or short chains ; is fatal in large doses
to mice, guinea-pigs and rabbits if injected into the veins or into the
peritoneal cavity; inoculated subcutaneously usually gives rise to abscesses,

420 APPENDIX.

but to little other disturbance. Like the Staphylococcus pyogenes albus
appears to be associated with suppurative processes and is found under
similar conditions.

(2) Staphylococcus pyogenes citreus.—Found in the pus of acute abscesses 3
differs from No, 1 only in the fact that instead of being dark orange yellow
it remains bright citron yellow in colour.

(3) Déplococcus subflavus.—Grows rapidly on nutrient jelly and blood
serum, first as whitish points which gradually become yellowish and then
deep yellow; in large quantities produces abscesses; occurs in several
secretions as a diplococcus from 0.5 to 1.5 in diameter; it is made up of
two hemispheres with a central division and resembles the gonococcus
somewhat in appearance, but retains the aniline dyes much more tenaciously
than that organism.

(4) Streptococcus coli gractlis.—Occurs in the intestinal canal and feeces
of the carnivora ; on plates it forms small sharply outlined dark colonies
in the centre of an area of clear liquefied gelatine; these, later, become
somewhat crenated at the margins; in a gelatine tube the medium is
liquefied rapidly along the track of the puncture and after six or eight days
there is precipitated a white finely granular mass; on agar-agar, potatoes
and blood serum there is very little superficial growth even at the body
temperature, which is most favourable to its growth; it is a coccus from
.2 to .44 in diameter; in fresh gelatine cultures it forms curved chains
consisting of from six to twenty cocci.

B The colonies are found occupying both the centre and periphery of
the liquefying area.

(1) Micrococcus coronatus.—Appears on the second day in plate cultivations
as whitish yellow points; deep colonies, under the microscope appear as
opaque sharply-defined plates; superficial growths project slightly, thisis made
more marked by a slight zone of depression surrounding the gelatine ; at
intervals tooth-shaped processes advance beyond the general circular peri-
phery; the older growths are dark in colour, newer growths are yellow or
yellowish brown ; liquefaction takes place around the growth in presence
of air; the coccus, I.I to 1.24 in diameter, occurs singly in short chains or
in irregular masses.

(2) Micrococcus radiatus.—Growths visible in twenty-four hours; 1 mm. in
diameter in two days ; white or yellowish green, sharply defined, granular,
or with outgrowths like the rays of a star fish 5 colonies sink as gelatine be-
comes liquid, and a series of circles of rays formed of delicate threads project
radially, this zone increasing in breadth towards the periphery ; one, two or
three of these circles are seen according to the age of the growth, the
rays of the outer circles always being shorter than those: of the inner ones,
each circle forms in about two days; in a puncture cultivation isolated
points form along the track of the needle; from these, lateral branches
project; a funnel-shaped area of liquefaction is formed very slowly, it
extends for a short distance only into the gelatine; micrococci .8 to .gp in
diameter ; usually grouped in small masses but sometimes in short chains.

(3) Micrococcus flavus liquefactens.—Occurs on gelatine plates as small
yellow circular, oval, or irregular finely-toothed colonies ; superficial colonies
distinctly yellow, cause liquefaction. Smaller colonies are found at the
-border of the liquefying area which has a very sharp outline ; lines of cocci
radially disposed run from the centre to the periphery in the clear liquefying
axea, giving an appearance that is said to resemble the wheel of a waggon ;

APPENDIX, 421

in puncture cultivations yellow points are seen in two days, these become
confluent and rapidly liquefy the jelly, which remains clear with a yellow
deposit below ; it is a comparatively large coccus, occurring in irregular
masses or in twos or threes.

III. There is no growth on gelatine at 22° C.

(1) Micrococcus gonorrhea.—Grows on blood serum at 37° C. as a thin
greyish yellow layer with moist smooth surface ;-organism consists of two
hemispheres slightly concave on the opposed sides with a clear line of division
between them ; it is from 0.8 to 1.6 in length and from 0.6 to 0.8 in
diameter; unlike most other organisms it is contained within the protoplasm
of the tissue cells, and is readily decolorized by Gram’s method.

(2) Diplococcus intracellularis meningitidis.—Found in fresh exudation
of cases of acute cerebral meningitis. Grows on a mixture of agar-agar and
gelatine at the temperature of the body; the growths in the deeper layers
are very small, those on the surface are larger, and are somewhat grey ; -at
first they are round when seen under the microscope, they then become
irregular, are finely granular and yellowish brown, the centre is usually darker
than the periphery ; on the surface of agar this organism grows well but not
along the track of the needle; it forms a grey, viscid growth as the various
colonies run together; it only remains virulent for about six days, affects
mice, guinea-pigs, rabbits and dogs; is probably very closely allied to the
diplococcus of pneumonia ; grows as a coccus sometimes singly but usually
arranged in pairs, fours, or small masses; in single cocci a line of division
may usually be seen; is almost invariably found within the cells contained
in the exudation.

(3) Alicrococcus pyogenes tenuis.—An irregular coccus, larger than the
staphylococci, and not forming masses; found in a certain proportion of
unopened abscesses by Rosenbach; cultivated on agar, chain-like micro-
cocci in Endocarditis ulcerosa; micrococci found in disease of the hands
and fingers of butchers and tanners, but not yet fully studied.

Micrococci have also been described in small-pox pustules and in the
various internal organs in the lymph of vaccinal vesicles, in scarlatina by
Crooke, and in measles, in diphtheria, in inflammation of membranes of
the brain, in influenza (doubtful), in ozcena, in hemophilia neonatorium,
in acute yellow atrophy of the liver, and in many other diseased conditions.

In addition to these may be mentioned Pathogenic micrococci, in the
blood of patients suffering from ‘‘ Clou de Riskra or Bouton d’Alep,” which
excite gangrene when injected subcutaneously into rabbits, or death sixteen
hours after they are injected into the blood.

The Organism 1s a Bacillus.

I. The nutrient gelatine is not liquefied.

II. The nutrient gelatine is liquefied, see p. 429.

JIf. Organisms do not grow on nutrient jelly, and only on
other media at higher temperatures in the presence of air,
See p.434. he

IV. Organisms will only grow under conditions of anzro-
biosis, see p. 436. : .

V. Organism has not yet been artificially cultivated outside

422 APPENDIX.

the body,'ze., it does not grow under ordinary conditions,
see p. 437.
I. The nutrient gelatine is not liquefied.

A. Colonies white, nutrient gelatine near growth not
stained. ‘

'B. Colonies colourless, nutrient substratum near growth.
stained, see p. 427.

c. Colonies cream coloured, see p. 428.

pv. Colonies of a yellow colour, see p. 428.

a. Colonies white, nutrient gelatine near growth not stained.

a. Colonies form minute small translucent drops on
plates, delicate growths in stroke and puncture cultiva-
tions.

4. Colonies form thin films on plates, and on the sur-
face of tube cultures, see p. 423.

c. Colonies form white nail-head projections on plates,
and nail-shaped growths in tube cultures, see p. 425.

@, Colonies are branched, not circumscribed, see p. 426.

a. Colonies form minute small translucent drops on plates,
delicate growths in stroke and puncture cultivations.

(1) Bacillus cholere gatlinarum (Fowl cholera).—Grows on gelatine
as small, round, white, superficial, finely-granular colonies, light yellow in
the centre, a dark zone further out, outlines irregular; on potatoes do
not grow at the ordinary temperature of the room, but at 37°C. grow
slowly as yellowish grey transparent drops. Under the microscope average
1.2 to 1.54 in length, and.are seen as short rods with rounded ends,
which are always more deeply stained with aniline colours at the ends than
in the middle, so that they appear like diplococci. Fatal to fowls in from
24 to 36 hours, also to mice and rabbits (probably identical with Koch’s
bacillus of rabbit septiczemia). Not fatal to guinea-pigs, sheep, and horses,
but causes abscess formation.

(2) Bacillus (Bacterium) der Wildseuche (Described by Kitt and Hueppe).
—Grows on plate cultivationsas white or greyish-white colonies, about the size
of a pin head, which under the microscope appear to be slightly granular ; in
the needle track in puncture cultures we see small isolated colonies which
run together to form a greyish-white line ; on the surface there are small,
white, rounded layers, which grow up from the surface; on agar-agar they
have much the same appearance, but are greyer and more transparent ; on
potatoes forms greyish yellow, slightly prominent layers; on blood serum
it has a peculiar iridescent appearance ; grows best at the temperature of the
body; occurs as short rods two to three times as long as they are broad,
with somewhat rounded ends; sometimes appear as cocci, or may be
ellipsoidal ; Arthrospores are said to be present. Said to be the cause of
certain forms of infective pneumonia. It gives rise to most marked symptoms
in a number of animals, and is classed by Hueppe with the organisms of

APPENDIX, 423

swine erysipelas, rabbit septicaemia, and fowl cholera, all of which give
rise to hemorrhagic septiceemias.

(3) Bacdilus septicus agrigenus.—A bacillus found in cultivated ground.
On plates it grows as rounded, finely-granular colonies, with sharp outlines,
centre of colony light yellow, margin darker. Under the microscope it is
exceedingly like the bacillus of fowl cholera. In the body it adheres to
the red blood corpuscles, and is fatal to mice and guinea-pigs. Colonies
form thin films on plates and on the surface of test tube growths.

6. Colonies colourless, form thin films on plates and on the surface of
tube cultures.
a. Cultivations odourless.
8. Cultivations giving off an odour, see p. 424).

a. Cultivations odourless. '

(t) Bactllus acidt lacticé,—Found by Hueppe in sour milk. It grows on
gelatine plates as small, white points, which gradually become opaque and
moist looking, forming a thick layer of from I to 2mm. in diameter. Under
the microscope these colonies appear to be dark yellow in the middle. The
margins are irregularly indented and toothed. In tubes, growth appears
as small granules along the line of puncture; surface growth thick, moist,
and opaque; grows very slowly, and in milk can only develop at a
temperature above 10° or 12° C., and below 45° C.; occurs as short, plump,
motionless rods, I to 1.7 in length, and .3 to .4u in thickness; usually
arranged in pairs, sometimes, but rarely, in chains of four; well-marked
refractile bodies which are regarded as spores, which are usually placed at
the end of the rods.

(2) Bacillus of typhoid fever (p. 194) and Pseudo typhoid bacilli (p. 202).

(3) Bacterium col commune.—Is found in the intestinal canal of man
and animals, especially at the lower end. It grows on gelatine in the form
of superficial colonies, from 2 to 4mm. in diameter, which are granular, or
may be slightly wrinkled ; in the deeper layers of the gelatine appear as
yellow granular discs ; grow pretty rapidly. Organism occurs as thin rods,
about 2 to 3# in length, and .4u in breadth; sometimes it occurs as short
ovoid, or even rounded forms ; rods are slightly curved, and may be slightly
motile. When injected into the veins of rabbits or guinea-pigs, kills these
animals with symptoms of violent diarrhoea and fever, but guinea-pigs are
not quite so susceptible as rabbits. Does not form spores,

(4) Brieger’s Bacillus or Bacillus Cavicida.—Found in feces and putre-
fying fluids. The growth on plates occurs as colonies 2 to 4mm. in
diameter, composed of white concentric rings, like the scales on the back
of a tortoise; grows rapidly as dirty yellow masses on potatoes; small
rods about twice as long as broad; injected into guinea-pigs, cause death
in about 72 hours, They act like ordinary putrefactive bacilli, produce
propionic and other acids which give characteristic odour ; do not cause
death of rabbits or mice. .

(5) Bacillus diphtheria columbarum.—An organism separated from the
false membrane of the diphtheria of pigeons. The colonies are from 2 to
4mm. in diameter, and occur as white nodules in the deeper layers of
gelatine, but grow on the surface as whitish or brownish yellow films. On
potatoes it is sometimes difficult to distinguish it, as it is almost the colour
of the potato itself, having, however, a slightly greyer colour 3 bacilli are

424 APPENDIX.

longer and thicker than those of fowl cholera, but the ends are somewhat
rounded as in that organism ; are usually grouped together in small masses.
Kills pigeons, sparrows, rabbits, and mice, but does not affect fowls,
guinea-pigs, rats, and dogs. \

(6) Bacillus of Diphtheria of Rabbits (der Darmdiphtherie der Kanin-
chen).—In rabbits, as in pigeons, there has been described an organism
which grows in the “ diphtheritic ” processes of the intestine. On gelatine
plates is seen as small transparent grey colonies, which gradually become
brown ; the surface is finely granular, and has a peculiar pearly shimmer ;
growth in tubes along the track of the needle is comparatively slight, as the
organism requires a considerable amount of oxygen for its growth; but on
the surface it forms a slowly growing whitish layer ; rods 3 to 4p in length,
and 1 to 1.4m thick, rounded at the ends, arranged in pairs or in long
pany 3 in rabbits causes an inflammatory exudation in the alimentary
canal.

& Cultivations give rise to a strong odour.

(t) Bacillus urvee.—Found in ammoniacal urine; grows on gelatine
plates as small, semi-transparent points, which make their appearance on
the second day; on the tenth day they are about the size of a sixpence.
These are described as having the appearance of a ground glass plate that
has been breathed upon; the growth extends in the form of concentric
rings, the, outer one of which has a somewhat zigzag outline ; in gelatine
tubes it grows along the track of the needle very slowly as an exceedingly
delicate grey film ; on the surface it grows rather more rapidly, and in old
cultivations gives rise to a characteristic trimethylamine or herring brine
odour. The organism occurs as plump rods with rounded ends, 2p in
length, and half as broad as long; it converts urea into carbonate of
ammonia.

(2) Bacillus pyogenes fetidus.—First obtained from a phlegmonous
abscess. Occurs in a very short time (24 hours) as white points, which
rapidly spread out as greyish-white films over the surface, and may gradually
become confluent; the margins are usually somewhat more translucent
looking than the centre, which is thicker; along the needle puncture in
gelatine are delicate, greyish-white points of various sizes, whilst on the
surface there is formed a layer similar to those described as occurring on
plates; the gelatine is not liquefied; it becomes slightly opaque in older
cultures ; on potatoes, is seen as glistening light brown growth ; occurs as
short rods with rounded ends, about 1.45 in length, and .6z in breadth;
sometimes in chains of two or more, and is slightly motile ; causes death of
mice and guinea-pigs, when injected into the abdomen, in ahout 24 hours ;
the bacilli are then found in the blood, but not at the point of inoculation ;
spores may be indistinctly made out in these bacilli.

(3) Schottelius’ intestine bacillus (Bacillus Coprogenes fatidus).—Found in
the intestinal canal and liverand spleen of pigs suffering from swine erysipelas.
This organism grows on gelatine as light yellow rounded deep colonies,
or as a fine transparent grey layer on the surface; does not give rise to
liquefaction of the gelatine; on potatoes it forms a dry, clear layer; the
organism is about as thick as the hay bacillus (2), but is only 4 or 5 in
length ; it has rounded ends, and is motile ; the spores appear in distinct
rows along the course of the threads. It does not cause any affection in
pigs, and is only toxic in large doses to rabbits.

“APPENDIX, 425

(4) Bienstock’s putrefactive bacillus (B. Putrificus Coli, “ Drumstick”
bacillus).—Was first separated from feces. On gelatine it has first
a peculiar opalescent appearance, but later it becomes yellowish; on
agar-agar it has much the same appearances; is an extremely motile
organism, which occurs in longer or shorter threads; usually the long
threads break up into shorter rods, about 3 in length, at one end of
which may be seen a spore similar to that described in the tetanus bacillus ;
this terminal spore giving to the bacillus the characteristic drum-stick shape.

(5) Bacillus or leptothrix epidermidis —Occurs in the fragments of
epidermis taken from between the toes; grows very sparsely on gelatine
and on agar-agar, where it forms only a superficial growth ; on potatoes, at
a temperature of 15 to 20°C., it occurs in the form of transparent fluid drops,
which gradually run together, become thicker, and form a characteristic
superficial skin ; also forms a similar skin on blood serum ; is a bacillus of
about 2.8 to 3 in length, and .3y in diameter ; forms spores from 1.2 to
1.5 in length, and .3 to .4# in breadth; this spore formation goes on best
at a temperature of 25°C.

c. Colonies form white nail-head projections on plates, and on the surface
of tube cultures.
a. Colonies microscopical with a granular border.
8B. Colonies with smooth borders, see p. 426,

a. Colonies microscopical with a granular border.

(1) Bacillus pneumonia (Friedlander).—Found in the lung and in the
rusty-coloured sputum of croupous pneumonia. Occurs in plates as small,
round, well-defined, darkish yellow or olive green granular colonies in the
deeper layers of the gelatine ; on the surface appears as white, thick, well-
defined projecting points; inthe gelatine needleculturesthe growth has the cha-
racteristic nail (with rounded head) appearance, the superficial growth almost
appearing like a very white, split, porcelain bead, that has been dropped
on the surface (after a time there is usually slight coloration of the gelatine,
and small bubbles of gas are formed if the gelatine is not too solid) ; grows
best at a temperature of from 16° to 43° C.; at the higher temperature (on
potatoes) it forms a moist, yellow mass, in which little bubbles of gas may
be seen ; grows very rapidly, and is not strictly zrobic. Under the micro-
scope seen as short, thick bacilli, with rounded ends, or oval cocci, which
are frequently arranged in pairs. When found in the lung tissue or sputum
it is usually surrounded by a delicate capsule, which gives it a very
characteristic appearance ; but this capsule is not, as a rule, found in
cultures. The organism is non-motile ; sometimes gives rise to pneumonia
in mice, guinea-pigs, and dogs, but does not affect rabbits. No spores
have been demonstrated.

(2) Bacillus crassus sputigenus,—Found in the sputum and in the ‘‘ fur”
scraped from the tongue. Forms on gelatine plates greyish-white viscid
drops, which project above the surface of the gelatine; colonies seen with
a lens are greyish brown, coarsely granular, and have a somewhat irregular
margin ; in needle cultures have the same characteristic nail appearance as
No. 1, and on potatoes also grow like No. 1; short, thick rods, with
slightly rounded ends, sometimes described as being like bent sausages ;
said to form spores at a temperature of about 35°C. Kills mice in about 48
hours, and in larger doses may kill rabbits and dogs in a very short time.

426 APPENDIX.

(3) Bacillus pseudopneumonicus.—Very like the true bacillus of pneu-
monia ; but it has been found in pus taken from abscesses. The colonies
seen through the microscope are dark grey in colour, and finely granular ;
puncture cultures in gelatine have the characteristic nail appearance ;
grows rapidly, and differs from the two previous forms in that it causes
rather dark coloration of the gelatine, and gives rise to a slight putrefactive
odour ; grows well on potatoes as a white, viscid layer, but no gas is formed
even at a temperature of 37°C. Requires air for its growth; micro-
scopically it is very like the pneumonia bacillus, 1.164 in length, and .op
in diameter. It is only slightly, if at all, pathogenic.

8. Colonies’ with smooth borders,

(1) Bacillus oxytocus perniciosus.—Obtained from milk that had been
allowed to stand for a considerable time. Occurs on plate cultures as
small colonies, with smooth borders and circular outlines ; under the micro-
scope appears to have a light brown colour ; growing on the surface may
attain a size of 1.5 mm. ; colonies are greyish white, and are usually round ;
needle cultures have, at first, the characteristic nail appearance but,
after a time, the growth along the needle track is comparatively small,
whilst the surface growth becomes very extensive; gives a peculiar acid
reaction to milk but no odour is developed ; under the microscope it
is seen asa short rod with rounded ends, somewhat thicker and shorter
than the lactic acid bacillus. In large doses is fatal to rabbits.

(2) Bacterium lactis aérogenes.—Found in the small intestine of mam-
mals and sometimes even in milk. Colonies have smooth borders ;
do not spread out much, but are usually of considerable thickness, like
little white porcelain points. In needle cultures it grows luxuriantly in
the nail form; along the line of the needle, small rounded points occur at
regular intervals, so that the growth looks almost like a string of beads ;
forms white layers on potate, in which bubbles of gas are frequently
developed, sometimes this layer has a peculiar creamy appearance ; grows
rapidly at about 37° C. ; causes diarrhoea and collapse in rabbits and guinea-
pigs, but does not affect mice; short thick rods with rounded ends I to 2p
long and .5 to .84 broad ; usually occurs in pairs, or may be arranged in
irregular masses ; the organism is non-motile.

(3) Weisser bacillus.—Obtained from water. Grows on gelatine plates
as round smooth white pin-head-like colonies; in gelatine tubes it grows

slowly, forms a whitish mass along the track of the needle and a white

head on the surface ; on potatoes it forms a yellowish-white growth ; grows
slowly; the organism is a short motile bacillus with truncated ends,
often joined to form chains.

d. Colonies are branched, not circumscribed.

(1) Bacterium Zopfiit.—First found in the intestine of fowls. It grows
on plates almost like a mucor ; in needle cultures appears as a thick, pale,
yellow string, from which white branches radiate into the surrounding
gelatine; is strongly wrobic, and grows very rapidly, especially at a
temperature of about 20° C.; spores are formed which are extremely
resistant to heat; the organism is from 2 to 5m in length and from .7 to Ip

in breadth; is motile; occurs in long threads, which in gelatine show - - - -

numerous bends or spirals. ; :
(2) Bacillus of Mouse Septicemia,—Originally found in garden earth and

.APPENDIX. 427

in putrifying fluids. Grows on plates, in the deeper Jayer of: thé gelatine
only, as exceedingly slowly growing delicate white clouds ; along the track
of a needle culture delicate, branching, almost cloud-liké growths are seen,
which are always more marked in the deeper part of the tube than in the
upper part. (N.B.—If the gelatineis exceedingly alkaline there may be slight
liquefaction of the medium.) On agar-agar, pale yellow sharply-defined
colonies are formed. The organism is non-motile, is exceedingly small,
being only about 1p in length and from .1 to. 2u in thickness ; two of them
are frequently adherent to one another; they contain spores. Mice
inoculated die in from 40 to 60 hours when bacilli are found in the
blood, especially in the capillaries of the kidneys and spleen.

(3) Bactllus of swine erysipelas (Schwein Rothlauf).—Has been obtained
from the spleen and blood of pigs that have died from this disease. It is
extremely like the previous organism except in the following points :—The
cloudiness in the needle culture is not quite so diffuse, and the bacilli are
slightly longer and thicker ; causes death of mice in from 2 to 3, pigeons
in from 3 to 4, and rabbits in 6 days; is also fatal to pigs.

B. Colonies colourless, nutrient substratum near growth stained.
a. Substratum stained greenish.
3 » blue or greyish brown.
Y- *% » Violet, see p. 428.
a The substratum is stained greenish.

(1) Bacillus fluorescens putidus.—Obtained from putrefying fluids.
In the deeper layers of gelatine plates it forms small dark colonies.
At the surface it appears as round wafers with irregular outlines; the
surrounding gelatine has a peculiar greenish fluorescent appearance ; in
needle cultures there is distinct cloudiness along the needle track and green
coloration of the gelatine, which is always more marked when oxygen has
access to the growth; strong herring brine odour; grows rapidly on
potatoes, forming a thin brown or greyish layer ; isa short, thin, very motile
bacillus with rounded ends. -
- (2) Bacillus erythrosporus. — Obtained from putrefying albuminous
fluids, drinking water, &c. Occurs on plates as whitish colonies, which
gradually spread over the surface ; around them in the gelatine a peculiar
fluorescence appears; the centre of the colony is usually opaque and
brownish, the outer zones are light yellowish green, not so opaque, and
there is a slight radiate marking ; along the needle track and at the surface
is a well-marked growth ; the surrounding gelatine is green by transmitted,
and yellow by reflected light; on potatoes forms reddish or nut-brown
localized patches ; grows moderately quickly, especially at the ordinary
temperature of the room; occurs as‘slender bacilli with slightly rounded
ends, single or in threads ; in these threads are from two to eight dirty red
spores, which are very distinctly seen, sometimes have almost the appearance
of a string of beads.

B. The substratum it stained blue or greyish brown.

(1) Bacillus cyanogenus (or Blue milk bacillus).—On gelatine plates
forms rounded, dirty white, finely granular colonies with smooth outlines ;
the surrounding gelatine takes on a light green or greenish brown colour ;
in, a needle culture in gelatine it has the ‘‘ nail” appearance, with
a milk white head, the surrounding gelatine becoming greenish blue or

428 APPENDIX.

even dark brown or black ; on agar-agar is seen as asimilar growth, which,
however, is somewhat grey in colour, and the green is never so well made
out as in the early gelatine cultures; on potatoes it forms a yellowish
layer near the point of inoculation, the surrounding potato being stained
greenish blue; on blood serum it gives rise to no coloration ; is an zrobic
and exceedingly motile bacillus from 1 to 4 in length and .3 to .5pin
breadth with slightly blunted or, if spores are being formed, club-shaped
ends, though very frequently spores appear to be formed in the middle of
the organism also ; in alkaline milk gives rise toa slate colour, but if grown
in the presence of lactic acid to an intense blue ; this colour is most freely
developed at from 15° to 18° C., at 37° C. no colour is formed at all.

y- The substratum is stained violet.

(1) Bacillus janthinus (Violet bacillus).—Differs from the bacillus
_violaceus which liquefies gelatine. (Grows very slowly, and later causes
liquefaction of the gelatine.) It was first found in water; when grown
on gelatine, milk white points appear, which later become violet, espe-
cially at the margins ; the surrounding gelatine also becomes deep violet,
but the organisms only develop the colour where there is a free supply of
oxygen ; in this case, as in the last, the colour is probably formed through
the breaking down of the proteid; under the microscope the organism is
found to consist of motile rods, some longer some shorter, but these
gradually break up into shorter lengths. ;

c. Colonies are cream coloured.

(1) Bacillus of septic pneumonia.—Poels has described an organism
as occurring in septic pneumonia of calves. It is a short rod-shaped
organism with a constriction in the middle, which gives it the appearance
of a diplococcus ; grows on‘gelatine as a rough layer ; forms small rounded
colonies around the point of inoculation, which gradually run together ;
along the track of the needle in puncture cultures small rounded whitish or
cream coloured colonies are formed ; on agar-agar it forms a shining
smooth layer exceedingly thin and sharply defined in from 10 to 15
hours ; on sterilized blood serum forms a creamy layer similar to that
already described, and on the surface of sterilized potatoes spreads over
a large area; kills rabbits, guinea-pigs, mice, calves, pigs; sheep and
dogs not affected, Poels thinks that it is closely allied to the mouse
septicemia group of bacilli.

p. Colonies of a yellow colour.

(1) Bacilius luteus.—Forms in the superficial layer of gelatine plate
cultivations small yellowish points 2 to 3mm. in diameter, often of a light
brown colour with a whitish translucent margin ; deeper down they are
much smaller, are usually the shape of a lentil, and can only be made
out with the aid of a microscope, when they appear to be irregular in out-
line and of a brown colour ; a yellowish growth is formed along a surface
needle track; it is a short non-motile bacillus of medium thickness. ;

(2) Bacillus fuscus.—Obtained from a putrefying infusion of maize, and
also found as an accidental impurity in certain cultures. Quickly forms
rounded brownish colonies; deeper it forms dark brown nodules sur-
rounded by a highly refractile border; at the surface of puncture cultures
it usually forms a wrinkled brownish red deposit around the point of
entrance of the needle. The organism is a motile rod,

APPENDIX. 429

(3) Bacillus Fitsianus.—Obtained from the dust of hay, and supposed to
be really a variety of the Bacillus subtilis. The colonies are brownish
yellow in colour with a dark opaque centre and sharp outline, those lying on
the surface of the gelatine are like brownish yellow gelatinous drops ;
organisms are from I to 24 and upwards in length and about rp in thick-
ness, the longer rods are frequently bent at the ends ; there is distinct spore
formation ; sets up zethylic alcoholic fermentation, especially when glycerine
is present. ’

II. The nutrient jelly is liquefied.

A. Colonies are white; nutrient substratum remains uncoloured.
B. Colonies or nutrient substratum coloured, see p. 433-

A. Colonies are white; nutrient substratum remains uncoloured.
a. Colonies branched or with processes.
6. Colonies circumscribed without branches, see p. 431.

a. Colonies branched or with processes,

a. Colonies are non-motile.
8B. Colonies motile and swarming, p. 430:
a. Colonies are non-motile.

(1) Bacillus anthracis, see p. 272.

(2) Bacillus ramosus liquefactens.—Roundish colonies on plates with
radiating processes, the rounded disc looking as if it were surrounded by
a zone of hairs ; superficial colonies are oval or pear-shaped ; there is slight
liquefaction around the growth and a deep circular funnel is formed, which
is surrounded by concentric rings, which gradually increase in size ; run-
ning off from the funnel at right angles are a number of branches longer
near the surface and becoming shorter as the deeper layers are reached ;
the organism is a medium size slightly motile bacillus with blunted ends.

(3) Bacillus subtilds (Hay bactllus).—Obtained from hay infusion that has
been boiled. Grows on plate cultures as white rounded colonies with radiating
processes ; liquefies gelatine on plates rapidly ; along the track of needle
causes liquefaction which commences at the surface ; first occurs as small
whitish colonies, which under a low power have a yellowish brown colour
with the hair-like margin, outside this is a narrow clear zone, beyond which
again is a greyish layer composed of radiate lines; on potatoes and on agar
it forms a whitish moist creamy layer, which afterwards becomes somewhat
granular and dry; is dryer and more wrinkled looking on agar than on
potatoes. Liquefies blood serum ; grows rapidly about 30° C. ; is strongly
zrobic; is a motile organism about 6 in length and about 2p in breadth
with slightly rounded ends, it divides and multiplies exceedingly rapidly ;
large well. defined spores are “formed (when the supply of nutrition is
gradually cut off), about 1.2 in length and .6u in breadth.

(4) Bacillus pneumonicus agilés (or Bacillus of vagus pneumonia of rabbits).
—Grows on gelatine plates as round dark granular colonies with slightly
roughened surface and margins ; after from 20 to 24 hours there are marked
movements in the middle of the colonies, and liquefaction takes place rapidly;
in needle cultures in gelatine tubes rapid liquefaction of the medium occurs,
and a shallow funnel-like space, in which the gelatine is liquefied, is
formed ; growth on potatoes, spreads very rapidly over the whole surface as
a * chamois” red layer; on blood serum grows much more slowly and only

430 APPENDIX.

causes slight liquefaction ; the organism occurs as an elliptical coccus or
are tea or as ashort thick bacillus ; is fatal to rabbits when pure cultures
are used,

(5) Bactllus mesentericus fuscis.—On gelatine plates form whitish colonies
with sharp outlines, later these become yellowish brown and take on a
granular surface, rays running out from the periphery ; growth liquefies the
gelatine, especially near the surface, after a whitish opacity has grown along
the track of the needle ; the liquefied gelatine is at first turbid or has whitish.
flakes floating in it ; on potatoes a smooth yellowish growth appears on
the first day and spreads rapidly, this soon becomes dry and wrinkled ;
small and short motile bacilli usually occur in twos and fours and contain
small refracting spores, which as a rule are somewhat irregularly arranged ;
colonies pear-shaped with thick processes at the pointed end, in puncture
cultivations like “ sparks ” of fluid.

(6) Bacillus alvet.—(Cheshire and Watson-Cheyne) found in the disease
known as foul brood of bees. Grows on plates as small oval or pear-shaped
colonies, which under the low power appear to have thick processes at the
pointed end, presenting the appearances above described ; small lateral
projections make their appearance along the track of the needle in stroke
cultivations, these gradually curve and form well-marked circles, from these,
new circles or pear-shaped masses project, and so on ; the gelatine becomes
fluid immediately around these, forming canals, following the course of the
masses of bacilli; ultimately the gelatine becomes liquefied around the whole
colony; on potato the organism grows slowly in the form of a yellowish
deposit, best at the body temperature but even then somewhat slowly ;
liquefies gelatine very rapidly. The bacillus in the honey combs of foul
brood hives is about 3.6u in length and .8» in breadth; the cultivated
organism varies between 2.5 and 5. in length; the ends of the bacilli
are rounded or pointed; they are sometimes motile and form large spores
2.1p in length and 1.7p in breadth. : Ba

(7) Bacillus mycoides (Earth Bacillus).—Obtained from the surface
of the earth of cultivated fields or gardens. Colonies without distinct
centres in the form of a mass or mycelium-like network of threads ;
in gelatine plates we have a whitish turbidity, in which fine threads,’
irregularly branched and interwoven, may be seen; grows exceedingly
rapidly, and resembles the mycelium of a fungus, so much so that it is’
often mistaken for one; when they come to the surface the threads become
much thicker ; near the surface in needle cultures cause liquefaction of the
gelatine, but this is preceded by a growth of little spikes, which pass from
the track of the needle at right angles into the surrounding gelatine. On
potatoes rough granular parchment-like growths gradually spread over the
surface ; organisms are about the size of the anthrax bacillus, which they
resemble very greatly, and sometimes occur in threads; they are motile,
and contain highly refractile spores, which are usually situated in the middle
of the bacillus ; non-pathogenic.

B. Colonies motile and swarming, giving rise to rapid liquefaction.

(1) Proteus vulgaris—Found in putrefying organic matter, in ulcers, in
meconium-feces, in water, &c. Grows on gelatine plates very rapidly as
whitish-grey turbid masses which are distinguishable at the end of about eight.
hours. From the central colony little projections-pass outwards, the shape!
of these constantly varying as the bacilli are re-arranged ; the whole surface:

APPENDIX, 431

gradually becomes covered with these motile colonies, and then liquefaction
takes place rapidly ; this is completed at the end of forty-eight hours.
There is a foul odour and a marked alkaline reaction; in the track of the
needle in pure cultures colonies may be seen which have a peculiar radiate
formation, the liquefaction always extending wherever these colonies appear.
Liquefaction takes place more slowly when oxygen is cut off ; short ciliated
rods and threads 1°25 to 3.75 in length, and about ‘6m in thickness. ‘The
threads are usually twisted and convoluted ; grows at about 20° to 24° cc.,
and causes very rapid liquefaction of the gelatine ; no spore formation ; in-
volution forms are found~spherical bodies, about 1.6m in diameter; is
pathogenic. . ; :

(2) Proteus mirabilis.—Something like the preceding organism, but
liquefies the gelatine much more slowly. (No liquefaction takes place
when oxygen is cut off.) The threads are much longer, and the colonies
have a finely granular brownish appearance under the microscope, espe-
cially towards the centre ; the organism is about the same thickness as the
above, but may be somewhat shorter ; distinguished especially by the fact
that spherical or pear-shaped involution forms are more frequently met with,
these being from 3.75 to 7“ in diameter ; zoogloea forms are also very
numerous.

(3) Proteus Zenkeri.—In plate cultures forms thick whitish-grey layers,
but gives rise to no zoogloea forms ; in gelatine tubes a thick layer is formed
at the point of inoculation, which by regular steps becomes thinner towards
the periphery ; from the margin threads shoot out; at the end of twenty-
four hours there are large moving islands similar to those already examined,
but liquefaction only takes place immediately at the surface, the deposit
gradually becoming thicker and more opaque ; the long thread forms are
seldom met with; the bacilli are 1.654 in length and .4p in breadth, or
they may be more rounded, or even a little longer; they are motile.
(Although classed with the liquefying organisms, this liquefaction is some-
times so slight that it can scarcely be made out.) There is little or no
odour given off from gelatine or blood serum cultures, but there is a strong
smell given off when the organism is cultivated in meat infusions.

&. Colonies circumscribed without branches.

a. Bacilli 2.5 in breadth.
B. Bacilli at most 1p in breadth.

a. Bacilli 2.54 in breadth.

(1) Bacillus megaterium,— First found on boiled cabbage-leaves.
Occurs on plates as small round liquefying colonies ; grows on gelatine
very rapidly, liquefying in a funnel shape from the surface downwards ;
develops as a whitish layer on agar-agar, the surrounding material becoming
somewhat darker ; grows rapidly on potatoes at 20° C., as yellowish-white
cheesy points near the seat of inoculation ; proliferates by transverse division
and by end spores ; distinctly an zerobic organism ; occurs as slightly bent
motile rods, 1oz in length and 2.5 in thickness; the ends are somewhat
rounded ; sometimes form chains of from two to ten bacilli. The cell
contents are frequently granular.

GB. Bacilli at most 1p in breadth.

i, Development of clostridium forms before spore formation; see p. 432.
ii. No clostridium forms, see p. 432.

432 APPENDIX.

fii. Giving rise to the formation of bubbles of gas,
iv. Giving rise to a strong putrefactive odour, see p. 433-

i. Development of Clostridium forms before spore formation.

(1) Bactllus butyricus) (Hueppe).—One of the forms of bacilli giving rise to
butyric fermentation ; found in milk and in fleshy roots, such as turnips, &c.,
grows on plates in the deeper layers of the gelatine as delicate yellow masses ;
which later assume a brown granular appearance ; these rapidly liquefy the
gelatine and run together ; on agar-agar they grow as viscid superficial
yellow layers ; in gelatine tube puncture cultures rapidly cause liquefaction
along the track of the needle, the fluid becoming cloudy; the superficial
layer is greyish-white or yellow, forming a delicate felted mass; grows
very rapidly, especially at a temperature of from 35° to 40° C.; digests the
casein of milk, interferes with the lactic acid fermentation, and gives a
bitter taste ; large, thick, very motile rods, with rounded ends of from 3
to 10 in length, and 1m in breadth; frequently forms chains; gives rise
to well-developed spores.

(2) Zhe Clostridium butyricum or Bacillus amylobacter of Prazmowski
is morphologically exceedingly like the above organism, but has the charac-
teristic of giving off on solid nutrient media a large quantity of gas which
has the butyric acid smell ; it is also markedly anzrobic, transforms starch,
sugar, dextrine, and lactates into butyric acid, setting free CO, and
hydrogen; the threads may be unjointed ; gelatine is liquefied, a regular
felted scum forming on the surface; it grows at a temperature of from
35° to 40°C. Although one of the organisms first described, this bacillus
has not yet been fully investigated.

ii. No Clostridium forms.

(1) Bacillus mesentericus vulgatus.—(Potato bacillus.) Forms rounded
or oval colonies, with sharp margins; on plate cultivations, first some-
what transparent, afterwards slightly yellow; in needle cultures causes
liquefaction along the track of the needle from above downwards, this is
always more marked near the surface; a scum forms on the surface,
and the growths along the track of the needle sink to the bottom of
the funnel ; the fluid is usually turbid ; on potatoes grows extremely rapidly
in the form of a wrinkled moist layer; later becomes somewhat dried, and
rather like a crumpled felt; the organism, which is strongly zrobic, is
slightly motile, and occurs as small thick rods with rounded ends, arranged
in pairs, or sometimes in fours.

2. Bacillus Arophilus is very like the above as regards its growth in
gelatine, but the colonies are oval and have sharp margins ; on potatoes it
grows as a smooth yellow layer, which later becomes crumpled at the
margins; slender spore-bearing rods and threads, about 1.4 in diameter.

3. Bacillus liodermos (Described by Fliigge).—-Colonies form small irregular
heaps in gelatine, and on potatoes a smooth slimy layer; short bacilli with
rounded ends, which are actively motile; in other respects the growth is
very like No. x.

iii. Giving rise to the formation of bubbles of gas.

(1) Gasbildender (or gas-forming) bacillus.—An organism found in water.
It liquefies gelatine very rapidly, and on plates forms moderately large len-
ticulate spaces, in which greyish points may be seen; these sometimes.
contain gas; on the surface the gelatine becomes liquefied, the greyish
mass being seen in the centre; in gelatine tube cultures there is a tube-

APPENDIX. 433

shaped liquefaction area along the line of the needle, and in. the politine
that still remains solid, clear bubbles of gas may be seen, The organism
that produces these changes is a very minute, exceedingly motile rod.

iv. Giving rise to a strong putrefactive odour.

(1) Liguefying bacillus of Water (Verfitissigender bacillus of theGermans).
—Very rapidly forms round colonies with smooth walls; in the centre of
the liquefying: area is seen a white viscid mass; after a time an offensive
putrefactive odour is given off; in a gelatine tube puncture cultivation
there soon appears along the track of the needle a white granular mass,
which is followed by a funnel-shaped area of liquefaction; short, somewhat
thick rods with rounded ends.

B. Colonies or nutrient substratum coloured.
a. Colouring matter, red. .
6, Colouring matter green, see p. 434.
¢. Colouring matter violet, see p. 434.

a, Colouring matter red.

(1) Bacillus prodigiosus (Micrococcus prodigiosus, Monas prodigiosus).
—Grows very rapidly on plates at from 20° to 22° C., in the deep layers as
grey points, superficial growths small grey rounded colonies, about 1 mm.
in diameter ; these sink into the gelatine, which is rapidly liquefied, but
remains quite clear; under the low power deep colonies are seen to be
rounded or oval, and to have sharp outlines, but those at the surface are
granular and have an irregular outline; when liquefaction has set in a
beautiful red colour makes its appearance, but this is best seen on agar
cultivations, where there is free access of oxygen to the growing organism ;
on potatoes a beautiful moist blood-red layer is formed, which is perfectly
characteristic ; the bacilli themselves are colourless; the pigment is in-
soluble in water, soluble in alcohol ; occurs as egg-shaped non-motile cells,
about Ip in diameter, especially when the liquefaction is rapid ; sometimes,
when the growth is slower, the organism is distinctly rod-shaped, or it may
occur in the form of short threads.

(2) Bacillus indicus ruber.—Found in the contents of the stomach of a
monkey. Was the organism with which Koch made some of his experi-
ments in connection with the destruction of micro-organisms in the alimen-
tary canal. The colonies on plates are first of a yellow colour, and have
a wavy outline ; the superficial growths bring about the liquefaction of the
gelatine, this first appearing as a little zone round the colony, and giving
rise to a liquefied funnel-shaped area ; in gelatine needle cultures a waxy
or brick-red colour forms at the surface, but along the track of the needle
the growth is somewhat grey or white; on potatoes forms a localized brick-
red or waxy layer; grows best at a temperature of about 35° C., as a very
thin and short bacillus with rounded ends ; injected in large quantities into
rabbits it causes death from diarrhoea in from three to twenty hours.

(3) Red bacil/es:—Found in water. On plates forms finely granular
colonies with smooth surfaces; red coloured points accumulate in the centre
of small liquefied areas in gelatine tubes, as the gelatine is gradually
liquefied, and a brownish-red coloured mass sinks to the bottom; grows on
agar-agar, potatoes, and blood serum with the same characteristic brownish-
red colour; is an exceedingly motile bacillus of medium size, and with
somewhat truncated ends, sometimes united to form long threads.

29

434 APPENDIX.

6, Colouring matter green.

(1) Bacillus pyocyaneus (Bacterium aruginosum, Bacillus of blue or green
pus).—On plates forms microscopical colonies, which send out radiating
threads and give rise to funnel-shaped liquefaction of the gelatine about the
second or third day; the margin is clear and granular ; in tubes the gelatine
commences to liquefy at the surface in twenty-four hours ; there is a funnel-
shaped liquefying area limited to the neighbourhood of the needle-track, the
surrounding gelatine remaining solid, but assuming a beautiful fluorescent
colour ; on potatoes this organism grows as dry colonies with a dirty rusty
colour, the surrounding potato being stained slightly green ; if a drop of
ammonia is added to this, a green, if a drop of acid, a red colour is obtained ;
the organism grows extremely rapidly, and is strongly zrobic. It is a
very minute, short, thin rod, which is sometimes mistaken for a micro
coccus ; does not form any spores. -

(2) Bacillus fluorescens liguefaciens (Griingelber bacillus of the Germans).
—Is found in putrefying substrata, water, &c.; colonies seen under lens are
at first circular, later have irregular outlines; the centre is dark brown,
finely granular ; outside this is a transparent liquefying zone ; the whole
gelatine gradually becomes green ; in puncture cultures in gelatine a white
line is seen along the track of the needle, but near the surface there is a
little funnel-shaped depression, which gradually increases in size, a little
air-bubble frequently being formed near the surface; the gelatine around
the liquid has a greenish-yellow fluorescent appearance, an appearance that .
is not so marked in the liquefied gelatine itself; on potatoes a yellowish-
brown layer is formed, around which there is slight discoloration of the
potato; the organism consists of a short active bacillus, arranged in pairs
and usually constricted in the middle.

(3) Bacterium graveolens.—Found in the fragments of epidermis taken
from between the toes ; obtained by Bordoni Uffreduzzi. Grows at the
ordinary temperature of the room on gelatine plates in the form of irregular
whitish-grey specks, which rapidly liquefy the surrounding gelatine ; these
give off the peculiar smell of the feet, and give rise to a greenish-yellow
coloration ; on potatoes grow very rapidly, and form a greyish mass with
an exceedingly offensive odour ; these organisms are about .8p in length,
and nearly as broad as long.

; c. Colouring matter violet.

Bacillus violaceus.—Is found in water; on plates it grows as small
round colonies, which liquefy the gelatine very rapidly ; these are first
white, but they very rapidly assume a beautiful violet colour, the mass sink-
ing to the bottom of the liquefied gelatine; in tube puncture cultures the
gelatine is liquefied very rapidly, usually in a funnel shape, at the bottom
of which a beautiful violet granular mass collects, the liquefied gelatine
remaining clear; the same beautiful colour is formed on agar-agar, potatoes,
and blood serum; grows somewhat slowly and best at the ordinary tem-
peratire of the room; is a motile rod about four times as long as broad,
with rounded ends, and often contains spores; it also grows out into
onger threads.

III. Organisms do not grow on nutrient jelly, and only on
other media at higher temperatures and in the presence of
air.

APPENDIX. 435

1) Bacillus tuberculosis grows on blood serum at 37° C., see p. 206.

2) Bacillus septicus spuligenus (Diplococcus Pneumonia).—¥orms a
transparent layer on the surface of blood serum ; difficult to obtain a growth
of this organism in gelatine plates, as it requires about 20 per cent. of
gelatine to keep it solid at 24° C.; it then grows very slowly as small,
rounded, sharply defined, slightly granular, whitish colonies; is an oval or
coccus-like bacillus, resembling the pneumonia bacillus ; the short rods are
frequently joined together in chains of five or six links : are usually taken
from the sputa of cases of lung disease, from the rusty-coloured sputum of
pneumonic patients, from severe cases of empyzemia, and from the fluid from
cases of cerebro-spinal meningitis ; they have a kind of capsule, similar to
that met with round the pneumonia bacillus; this capsule never makes its
appearance in cultivations; mice, guinea-pigs, and rabbits die in 24 to 48
hours after inoculation with this organism, when the blood is found to
contain a large number of the encapsuled bacilli.

The other organisms belonging to this group may be cultivated at a
somewhat lower temperature, viz.:

3) Bacillus mallet (or Glanders bacillus), see p. 264.

4) Bacillus diphtheria (or Klebs-Lifiter bacillus), see p. 299.

'(5) Bacillus saprogenes.—Under this name Rosenbach describes three
separate bacilli:

No. 1.—Obtained from offensive secretions and from the white casts
taken from the recesses of the mucous membrane of the wall of the pharynx ;
grows slowly on the surface of agar-agar as a dirty grey opaque line along
the track of the needle ; it is, however, slightly transparent when held up
to the light; forms a growth of considerable thickness, and an opaque,
tenacious, viscid consistence ; later this surface assumes an almost shell-
like look ; causes putrefaction of albuminous substances, and gives rise to
a very offensive odour; for this, however, it apparently requires the presence
of oxygen; is a somewhat large bacillus, in which end spores may fre-
quently be seen; apparently non-pathogenic.

No. 2.—Obtained from the foul-smelling sweat of the feet; grows much
more rapidly than No. 1 as a delicate superficial transparent growth, which
gradually becomes whitish-grey and of a tough, gelatinous consistence ; is
also an zrobic organism, but it can give rise to its peculiar foul sweaty odour
even when oxygen is excluded ; is a bacillus thinner and shorter than No. 1;
when injected into serous cavities of rabbits it sets up suppurative inflam-
mation.

No. 3.—Obtained from cases of suppuration of bone in a patient suffer-
ing from septic poisoning ; grows moderately rapidly, the growth at the
temperature of the room taking about eight days to form an ash-grey,
almost fluid, semi-opaque layer, with wavy outlines; isa short, thick bacillus,
with rounded ends. qalihsugt it is erobic, it gives rise to putrefactive
chahges much more rapidly when growing anerobically.) On injection
into a joint it induces a peculiar yellowish-green infiltration, with surround-
ing inflammation, giving off at the same time a very offensive putrefactive
smell.

(6) Bacillus necrophorus.—Found by Léffler when he inoculated small
particles of flat condylomata into the anterior chamber of the eye of a
rabbit. It does not grow on gelatine or agar-agar, and only very slowly
on blood serum, but in neutralized rabbit broth it forms a white filuffy
mass around the particles of the substance that has been implanted in the

436 APPENDIX.

broth, from these smaller masses break off and float in the fluid ; the organism
occurs as long threads consisting of bacilli, these bacilli of various lengths
are usually of the same thickness, and are frequently very much bent, or
curved, or even intertwined ; causes death of rabbits in about eight days,
giving rise to a peculiar necrotic process at the seat of inoculation ; kills
white mice in about six days. a

(7) Bacillus of puedo septicemia of mice.—Described by Bienstock. An
organism found in faeces. Grows on agar-agar very slowly, forming a
scarcely visible layer on each side of the inoculation stroke; is a non-motile
organism very like the bacillus of mouse septicaemia, but is somewhat
thicker, being sometimes half as broad as long; is occasionally mis-
taken for a micrococcus ; found mostly in the cedematous fluid and not in
the blood of animals that are inoculated ; kills both rabbits and mice.

(8) Bactllus of conjunctivitis.—Was been described by Weeks as obtained
from the conjunctival sac in cases of conjunctivitis ; will not grow on
gelatine, but in .5 per cent. of agar-agar it -grows slowly on plates, form-
ing in about forty-eight hours small pearly growths at the point of inocula-
tion ; about the fifth or seventh day the growth is complete, and at the end
of a month the organisms appear to have lost their power of growth; it
grows exceedingly well in fluid flesh broths, but not in solid media; grows
best at from 34° to 37° C.; a bacillus from 1p to 24 in length and .25u in
thickness ; like the tubercle bacillus, often forms long threads. As
another organism was present with this when inoculations into the human
subject were made it is not yet quite proved that this organism is the actual
cause of conjunctivitis.

(9) Xerose bacilius.—From the xerotic masses of the conjunctiva of a
child suffering from keratomalacia, conjunctivitis and hypersecretion of the
Meibomian glands. This organism does not grow on gelatine or potatoes,
but grows at the temperature of the body on blood serum, and afterwards
(though not primarily) on agar; on blood serum it forms fine dull grey
streaks along each side of a surface needle track ; is sometimes floated off
by the water of condensation ; is a bacillus about the length of the mouse
septiczemia bacillus; usually collected into little groups; a peculiar fatty
substance forms a kind of capsule which makes it appear to vary in thick-
ness according to the method of staining used.

(10) Bacillus der Akne contagiosa des Pferdes (Bacillus of horse-pox).—
Grows best on blood serum at 37° C. ; on the surface along the stroke track
of the needle grows as white points, which later become yellowish-grey,
some of this growth is washed away by the condensation water, but sinks
to the bottom leaving the water quite clear ; also grows 7 gelatine as small
white round points, which gradually increase in size, and om gelatine as
minute strongly refractile points; small rods 2u in diameter, usually straight
or slightly curved ; when injected subcutaneously the bacillus gives rise to
the so-called acne in the horse, calf, sheep, and dogs ; causes erysipelatous
swelling and pus formation in the rabbit ; when injected subcutaneously is
fatal to guinea-pigs and mice.

IV. Organisms will only grow under conditions of anzro-
biosis.

(1) Bacillus of malignant ede (Bac. odematis maligni, vikrion
eptigue).—Grows deep down in gelatine as small “ bubbles” with fluid

APPENDIX. 437

contents ; in agar-agar as smoke-like opacities not sharply defined from the
surrounding agar ; in needle cultures there is a similar turbidity along the
track of the needle ; grows best in this latter substance when one or two per
cent. of grape sugar is added, and at the temperature of the body ; liquefies
gelatine, and is an exceedingly motile organism from 3 to 3.5 in length,
and 1.tmin breadth ; usually a couple of rods are linked together, or they
may form threads 14 to 4ou in length; have rounded ends, are compara-
tively stiff, may be broken or looped and twisted around each other ; spores
are formed either at the end or in the middle, giving rise to drum-stick
shape or spindle shaped forms, these spores are most readily formed at the
temperature of the room; they differ from the anthrax bacillus in having
the rounded ends, being somewhat smaller, and in being strongly anzerobic.
2 doede Pseudotdem) bacillus (Liborius).—Found in the cede-
matous fluid of the tissues of a mouse that had been inoculated with garden
earth. On plates forms small globes with fluid contents, at the lower part
of which there are usually white deposits ; above this is fluid and then a little
bubble of gas; in agar-agar containing sugar little oval or lentil-shaped
bubbles with irregular outlines are formed ; in puncture cultures in agar-agar
there occurs a cloudiness along the needle track and gas is formed, this fre-
quently bringing about the cleaving of the medium; the organism grows
slowly, and does not liquefy the gelatine; under the microscope it is a bacillus
somewhat thicker than the cedema hacillus; one or two spores may be
formed in each bacillus; when injected into the veins or into the sub-
cutaneous tissue of mice and rabbits it causes death in a very short time.

(3) Bactllus of symptomatic anthrax (Charbon symptomatique, Rausch-
brand bacillus).—Is found in the serous fluids, bile, and muscular tumours
in cases of ‘‘ quarter evil.” Can only be cultivated anzerobically ; it has been
cultivated in fowl broth to which small quantities of glycerine and sulphate
of iron have been added, the air being driven from the upper part of the
vessel in which the culture is made by means of CO, or hydrogen; grows
best at the temperature of the body; is a motile organism from 3 to 5p in
length, and from .5 to .6u in breadth; the organism very frequently con-
tains spores at the ends, and is usually motile.

(4) Bacillus butyricus (Liborius).—Is an anzrobic organism very like the
bacillus butyricus of Prazmowski, even as regards the method of formation
of the spores ; when grown in gelatine from which the whole of the oxygen
has been driven off, it appears as whitish, not very sharply defined, colonies,
which on about the third day are surrounded by a narrow zone of liquefac-
tion, this gradually increases in size, and the whitish mass sinks to the
bottom of the globe so that we have a clear globe with a small precipitate
at the bottom, bubbles of gas gradually passing into the upper layers of
gelatine, driving out the oxygen and allowing the organism to grow in this
position, which it never does in the first instance; this gas has a disagreeable
odour.

(5) ? Clostridium butyricum or bacillus butyricus of Prazmowski, see p. 432.

(6) Bacillus of tetanus, see p. 287.

V. Organisms described in the tissues, but has not yet
been artificially cultivated outside the body, z.e., they do
not grow under ordinary conditions.

(1) Bacillus leprae, see p. 247.

438 APPENDIX

(2) Zhe bacillus of syphilis.—Has been demonstrated in tissues by Lust-
garten, who stained his sections of syphilitic new growths in Weigert’s aniline
gentian violet solution (see p. 412), decolorized-by means of a solution of per-
manganate of potash, and then washing with sulphurous acid, this is repeated
until the sections are colourless, when the bacilli stand out prominently.
The bacilli of leprosy and tubercle are stained by the same method, but
they may be distinguished from the syphilis bacillus by the fact that the
latter loses its stain on the sections being washed with a mineral acid. The
organisms are somewhat S-shaped, are about 4.5m in length, and have
frequently a slight swelling at the ends; they are somewhat wavy or
slightly indented, and along the line of the bacillus may be seen two or
four clear spaces, probably spores. It has more recently been found, how-
ever, that other bacilli take on a similar stain.

(3) Bactllus of rhinoscleroma.— An organism found in the tissues of patients
suffering from a disease very rarely met with in this country. In sections
of the thickened skin or mucous membrane stained in methyl violet for
forty-eight hours and then decolorized for forty-eight hours in absolute
alcohol a bacillus may be demonstrated ; short rods, 1.5 to 3 in length, and
5 to.8y in breadth, with rounded ends, each containing stained granules,
and surrounded by an oval capsule which stains much more delicately thar
the organism itself.

(4) Bacillus septicus, see p. 344.

(5) Bacillus diphtheria vitulorum.—Described by Loffler as occurring in
the diphtheria of calves. Long bacilli 2.5 to 3.6 in length, and .5 to .6u
in breadth, frequently united to form long threads; found in the deeper
tissues under the diphtheritic deposits in the mucous membrane.

The Organism is a Spirillum.

The more important of the spirella may be distinguished by their appear-
ance as they occur in nutrient gelatine at a temperature of from 20° to 24° C.

I. The gelatine is liquefied.

II. The gelatine is not liquefied, see p. 439.

Ill. The organisms have not yet been cultivated on
artificial media, see p. 440.

I. The gelatine is liquefied.

(1) Koch's cholera bacillus or spirillum cholere Asiaticie.—Plate cul-
tures, colonies light yellow, have irregular outlines liquefying the gelatine
slightly and sink to the bottom, leaving a clear surrounding space. Grows
on potatoes as a brownish film at 30° C. or higher, see p. 153.

(2) Finkler and Prior bacillus.—On gelatine plates grows rapidly and
appears as small white points; under a lens these are yellow or yellowish-
brown in colour, and have a sharp well defined circular outline; the surface
is not so refractile nor so granular as in the case of the true cholera colonies,
Liquefaction takes place at an early date and goes on very rapidly. The
liquefied fluid becomes turbid, whilst in the cholera bacillus it will be noted
that the upper part remains perfectly clear. In gelatine tube cultivations
thisis still moremarked. Liquefaction occurs in the form of a funnel-shaped
tube, the fluid gelatine being exceedingly turbid. On nutrient agar
yellowish white films are formed, Blood serum is rapidly peptonized and

APPENDIX. 439

liquefied. This organism grows on potatoes at the ordinary temperature of
the room as a yellowish white layer. Organisms slightly larger than
Koch’s bacillus, frequently somewhat pointed at the ends. The spirals are
as a rule not so long and not so perfect as the cholera bacillus, involution
forms being more frequently met with. The odour is very disagreeable. The
organism is more resistant than the cholera bacillus; it is not nearly so
fatal to guinea-pigs, though a certain number usually succumb to its action.
It was supposed to be obtained from cases of cholera nostras, but it is
probable that it is merely one of those spirilla which are met with in the
alimentary canal under ordinary conditions, similar to those described as
organisms of the mouth.

(3) Deneke’s cheese bacillus (Spirillum tyrogenum).—Plate colonies
under microscope similar to those of the Finkler and Prior bacillus,
but brownish in colour. It grows very rapidly as plate cultivations,
and gives rise to liquefaction of the gelatine, not so rapidly as the Finkler
and Prior bacillus, but more rapidly than the cholera bacillus. It
also forms a yellowish-white layer on agar-agar and blood serum. It
was first described as giving rise to no growth on potato at any tempera-
ture, but is now found to grow as a yellow Jayer on this medium.
This bacillus is somewhat smaller than the cholera bacillus. Forms long
spiral threads, in which the spirals are close and very perfect; like the
previous bacillus, it is exceedingly motile. When introduced into the
duodenum of the guinea-pig, according to Koch’s method (p. 164), kills
about twenty per cent. of the animals, but the organism under ordinary
conditions is probably non-pathogenic.

(4) The Vibrio Metschnikoff was first observed by Gamaleia in the con-
tents of the intestine of a fowl. Its growth on gelatine plates resembles
that of the Finkler-Prior bacillus; although it does not liquefy gelatine
quite so rapidly, it gives rise to the same peculiar cloudiness. It
sometimes resembles the Finkler-Prior, at others the Koch comma bacillus,
and sometimes the cheese bacillus cultures. It grows in gelatine, on
agar, and on potatoes, as does the cholera bacillus, but in bouillon it
causes turbidity of the fluid at an early date, and a thin film soon appears on
the surface. It gives the cholera red reaction on the addition of sulphuric
or hydrochloric acid, just as does the cholera bacillus. It is an organism
very like the cholera bacillus in many respects, and is certainly closely
related to it. It occurs as a somewhat curved bacterium, shorter and
thicker, but rather more bent, than Koch’s comma bacillus. In fluid
media it forms regular spirals. It is provided with long delicate wavy
flagella, and is motile. Metschnikoff has described it as being capable of
passing through a whole series of changes in form, and as being one of the
best examples of polymorphism. Apparently it does not form spores. It
is pathogenic it: the case of hens, guinea-pigs, and pigeons, but does not
affect mice. : 7

II. The gelatine is not liquefied.

(1) Emmerich’s bacillus is a special form of bacillus which was de-
scribed in the cholera epidemic of Naples (1884). It occurred along with
the true cholera organism, and was obtained in the alimentary canal and
was said by Emmerich to occur in the blood of patients who had died
from cholera.

On plate cultivations colonies grow deep down as small white points.

440 APPENDIX

These come to the surface, spread out as thin yellowish opalescent growths,
which do not liquefy the gelatine. Under the microscope small charac-
teristic deep growths, with a dark brown colour and having somewhat the
concentric appearance of the Spirillum concentricum, are seen. The super-
ficial growths are usually paler in colour, and are slightly yellow in the
centre. The margins are toothed, and the whole surface has somewhat the
appearance of a network.

In tube cultures it grows almost like the typhoid bacillus. Yellowish-
white points make their appearance along the line of the needle, whilst on
the surface there is a greyish-white glistening layer with scalloped margins,
The surrounding gelatine becomes somewhat cloudy, but there is no lique-
faction. On agar-agar it grows as a white moist layer, which has no special
characteristics. On potatoes it forms a yellowish-brown viscid layer which
has a somewhat characteristic appearance.

It occurs as short rods with rounded ends, which usually remain single,
but are sometimes united into long threads. It forms spores, and can grow
anzrobically.

It is said that it is also found in the feeces of healthy individuals, in the
air, and in putrefying fluids, and that it is not necessarily found in cases of
cholera. It is certainly not found in all cases of cholera, and it is now
thought that it may be nothing more than the ordinary feces bacterium.

(2) The Spirillum rubrum was first obtained by Von Esmarch from the
body ofa mouse. It grows extremely slowly, and cannot be made out in plate
cultivations for about five days. Under the microscope the points, which
are exceedingly small, have a yellowish-red appearance, are finely granular,
and have sharp margins. In the presence of oxygen no colouring matter is
formed, but in the deeper ‘part of the needle track in a puncture culture
there is a beautiful wine colour developed, the surface growth having a
moist appearance and well-defined margins. It is'a somewhat thick,
transparent, homogeneous, screw-like spirillum with three or four to
forty spirals ; is extremely motile, and is provided with flagella. Its vege-
tative multiplication is by transverse division. It probably forms spores,
as it is resistant to the action of drying, although it cannot withstand
a temperature of morethan 50°C. It multiplies at a temperature of between
16° and 40°C., but most actively at the temperature of the body. In solid
cultures the rods are short, but in fluid media the spirals are well developed.
It is non-pathogenic.

(3) Sperillum concentricum occurs in putrefactive blood ; it was first
described by Kitasato, and probably belongs to the same group as the
larger spiral bacteria that are frequently found in that substance. It grows
rapidly at the temperature of the room on plate cultivations, forming
greyish-white, round, smooth, well-defined colonies, each of which appears
to grow with concentric marking, and looks almost like a cockade; hence
the name. In gelatine it gives rise to no liquefaction, and grows better on
the surface than deeper down along the needle track. It does not grow on
potatoes, and is apparently non-pathogenic. : :

Like the Spirillum rubrum, it forms short spirals on nutrient media, but
long spiral threads on fluid media. It is motile, and is provided with
flagella.

III. The organisms have not yet been cultivated on artifi-
cial media.

: APPENDIX. 441

(1) The Spirillum Obermeteri is an exceedingly delicate, flexile
spiral of from ten to twenty turns. It is found in the blood of patients
suffering from relapsing fevers. It is from 16 to 4ou in length, and is
usually less than half the diameter of the cholera bacillus. According to
Koch and Vandyke Carter these organisms can only grow in the blood
inside the body, although they may be preserved alive for a considerable
length of time in blood serum or in normal salt solution. When the blood
of patients suffering from relapsing fever (during the febrile stage) is inocu-
lated into the long-tailed macacus monkey an attack of fever is set up,
during which the spirillum can be demonstrated in the blood. It may also
be demonstrated in the blood vessels of organs removed from animals killed
during this febrile attack. There are no relapses in the monkey as in man,
but one attack of the disease does not protect against a second.

(2) Spirochete plicatilis is an organism of extraordinary length—110 to
225u. It is extremely motile, and occurs in stagnant pools in which there is
decaying organic matter. The threads are arranged in long wavy lines, the
long waves appearing to be cut into shorter waves; these, of course, are
merely the spiral turns.

(3) Sperilum tenue is an exceedingly short spiral of from 1.5 to 5 turns
of a screw. The length of the organism is from 4 to 15u. It usually
occurs in vegetable decoctions, in which it darts about with very great
rapidity

(4) Spirdllum serpens consists of thin threads, which interlace to form a
kind of network. The organisms are from 11 to 28 in length, and
usually have from three to four regular wave-like turns. It is often found
in stagnant pools and where there is decaying vegetable matter. It is
about 1p in thickness. Where the felting is very marked it may some-
times appear to be united in chains. It moves about rapidly.

(5) Sé2rdl/em undula.—Sometimes thicker than the above, but not so
long, though the average length is greater. Length 8 to 124, and breadth
1.1 to 1.4”. The spirals are well marked, but they seldom consist of more
than about three turns. There are usually distinct flagella at the ends
(fig. on p. 29). Like the other forms, it occurs in putrefying fluids.

(6) Spiril/em volutans is much longer and thicker than any of the pre-
ceding forms. It is 1.5 to 24 in thickness and 20 to 30 in length. The
ends are somewhat thinned and rounded. The protoplasm contains a
number of dark granules; there is a distinct flagellum at each end. This
spirillum may be motile, but very frequently it is perfectly motionless. It
has been found in marsh water, and also in a decoction of dead fresh-water
snails. :

(7) Spzrillum rugula consists of short cells or single wavy bacilli 6 to 8
in length and .5 to 2.5 in thickness. They have usually a single bend or
a short flat turn ; may hang together in chains or form a felted mass. They
are extremely motile, and rotate round their own axis. They are provided
with flagella, and form spores at their ends very much as does the tetanus
bacillus, having then very much the appearance of 2 comma. Found in
marsh water and very frequently in the alimentary canal ; probably anzrobic
in character, and, according to Prazmowski, sets up decomposition of cellu-
lose.

442 APPENDIX.

LITERATURE.

To Zopf, Fligge, &c., the reader is referred for full de-
scriptions of Crenothrix, Beggiatoa three forms (B. alba, B.
roseo-persiciana, and B. mirabilis), Cladothrix, Dichotoma,
Streptothrix Foersteri, and various forms of Monas, Spiro-
monas, and Rhabdomas.

In addition to the works on general bacteriology already
quoted—Brefeld, Cornil and Babes, Crookshank, Fligge,
C. Fraenkel, Koch, Pasteur, Salomonsen, Bordoni - Uffre-'
duzzi, and Woodhead and Hare—the following may be re-
ferred to:

EIsENBERG.—Bacteriologische Diagnostik. -Hamburg and
Leipzig, 1888.

GtnTHER.—Einfiihrung in das Studium der Bakteriologie.
Leipzig, 1890.

Hueppe.—Die Formen der Bakterien Wiesbaden, 1886, and
loc. cit.

Kte1n.—Micro-Organisms and Disease, 4th Edition. London,
1889. ;

Ktune.—Prakt. Anleitung zum mikroskopischen Nachweis
der Bacterien im tierischen Gewebe. Leipzig, 1888.

Mutter.—Micro-Organisms of the Human Mouth. Phila-
delphia, 1890. ;

Zorr.—Die Spaltpilze ; Die Pilze, 1890.

INDEX TO SUBJECTS.

—++_

Abiogenesis, 55, 59
Acetic acid ferment, 10
3 », fermentation, 132, 138
Acidification, 138, 139
Acclimatization of bacteria, 35, 327
ais tissues, 327, 377,
378

yeasts, 121
Actinomycosis, 253

4 cultivation, 258

59 different forms, 256

Px in animals, 253

39 in Denmark, 259

es inoculation forms,
258

a mode of infection,
259

‘3 nature of, 260

- sheath of club, 257

summary, 257
Acute articular rheumatism, 81
Erobic organisms, 19, 137
Agar-agar peptone meat jelly, 403
Agar and gelatine medium, 392
Agar serum, 405
Air bacteria, 381, 383 .
»» examination, 384, 386
yy organisms carried by, 381
»» Solid particles in, 382
», of wards, tubercle bacilli in, 381
Albuminoids in fermentation, 66,
116
chapter on, 356
Albumoses, 363, 364, 375, 376
% poisonous, 358, 363-5
oa protective, 366, 376,

377
Alcoholic fermentation, 94, 142
inkephir, 13

” ”»

Algze, 42
Alkalies in cholera, 164

‘ Alkaloids in urine, 357

» relations of ptomaines to
vegetable, 358

» ue 357» 359: 364,
366

Amido compounds, decomposition
of, 142
Ammoniacal fermentation, 132, 135
Aniline dyes, 25, 411 (see Staining)
Antagonism of bacillary products,
3771 379
drugs, 379
Anthrax ’bacillus, asporogenous, 275
structure, 273
condition of deve-
lopment, 275
cultures, 272, 277,
278, 283
development, 275
examination, 273,
278
exciting cause of
anthrax, 271
inoculation, 279

” ”
” ”

a? ”

5 o spore formation,
275, 276, 283

», inman, 283

»» inoculation experiments,

374 :
2» susceptibility of animals to,

281, 282
»» Vaccine, protective, 372,
_ 373: 375
Aneerobic cultures, 408
33 fermentation, 121, 122,
137, 172
35 organisms, 411

444

Anzrobic organisms, cultivation of,
4073 409

i vibrio, 67
Antiseptic surgery, 8,72
Artificial cultivation media, 67
Arthrospores, 34-37, 168
Arthrostreptococcus, 45
Ascobacteria, 43
Ascococcus, 40, 43-6
Asporogenous anthrax, 275

Bacillus acidi lactici, 423
» erophilus, 432
1, _ alvei, 340, 430
»» amylobacter, 15, 37) 39, 84,
89, 126, 135, 145s 432) 437
» anthracis, 75, 76, 148, 270,
271, 274, 276,
372, 373, 375s
_ 429
limiting mem-
brane of, 27
modification of,

284, 373) 375
os By positions, 279,
280

1, of blue or green pus, 434

», of Brieger, 423

», buccalis maximus, 339

»» butyricus, 15, 37, 39 84,
89, 126, 135; 136, 145,
432, 437

3» cavicida, 423

»» of cheese (Deneke), 439

» Cholera, 31, 150, 152-4,
158, 159, 161, 163, 4i3,

43
5» cholerz gallinarum, 422
» of conjunctivitis, 436
», coprogenes foetidus, 424
3. crassus sputigenus, 425
3, crystallosus, 414
‘4, cyanogenus, 427
», of diphtheria, 297, 299, 300,

435
» diphtherize columbarum,

423
of diphtheria of rabbits, 424
», diphtherize vitulorum, 438
3» Emmerich, 439
», epidermidis, 425
3, erythrosporus, 427

INDEX TO SUBJECTS.

Bacillus Finkler and Prior, 438

» Fitzanius, 429

», fluorescens liquefaciens, 434
‘putidus, 348,

350, 427

> fuscus, 428

5» gas producing, 432

” glanders, 263-5, 414, 435

1, horsepox, 436

3; iodococcus vaginatus, 339

». indicus ruber, 433

5 janthinus, 428

1, leprosy, 245, 412, 437

» leptothrix innominata, 339

3, liodermos, 12, 432

», luteus, 428

,»» malignant oedema, 436

» — Mnallei, 263-5, 414, 435

x» megaterium, 431

+s _Mesentericus fuscus, 430

vulgatus, 12,

__ 432

ss mouse septiczemia, 426

4, mycoides, 430

yy necrophorus, 435

4, oxytocus perniciosus, 426

»» photo-bacterium ‘balticum,

353,

Fischeri, 353
Fluggeri, 352
indicum, 354
luminosum, 354

_ phosphorescens,

: - 351-4
», panificans, I1
yy | pneumonize, 425
y» pneumonicus agilis, 429
»» prodigiosus, 9, 12, 347, 433
, proteus mirabilis, 431
vulgaris, 414, 430
3 . zenkeri, 431
» pseudoedema, 437
sy» pseudopneumonicus, 426
ij » _ septicus of mice, 436
4, putrefactive, 425
» pyogenes foetidus, 424
., ramosus liquefaciens, 429
” red, 433
5, rhinoscleroma, 438
+» Saprogenes, 435
», septic pneumonia, 428
» septicus agrigenus, 423

” ”

” ”

” ”

INDEX TO SUBJECTS.

Bacillus septicus sputigenus, 435
8

” 43
» Spirillum sputigenum, 339
” subtilis, 37, 38, 429
», Swine erysipelas, 427
s, symptomatic anthrax, 437
1, syphilis, 438
4 tetanus, 286, 287, 437
5» tuberculosis, 219, 381, 412,

435
» typhoid fever, 21, 194, 203,
414, 423
» ulna, 340
1 «rez, 424
»» Violaceus, 434
1», water, liquefying,
3» Weisser, 426
3, xanthogenus, 61
xerose, 436
Bacteriology, history of growth, 1
Bacteria, action on albuminoids, 130
of light on, 199, 201

433

33 ”
Bacteriacez, 44
Bacteria in air, 382-4

»» biological characters and

classification, 5

», in cavities of the nose, 345

»» examination of soil for, 393

si a ace of species,

» in fpcapbiatica: 84
»» modification of function, 6,
129, 130
mouth, 337, 340, 343, 344
Bacterial pathology, 83
Bacteria pigments, 348, 350
»» poisons antagonistic action,
379 F
33 ” summative action,
379
pure cultures, 2-5, 76,
155, 157, 196, 250, 300,

391
saprophytic organisms, 148
y+ as scavengers, 15
+i vegetable organisms, 60, 62
»» in water, 178, 387, 388,
392, 393, 433
Bacterium eruginosum, 434
On coli commune, 154, 423
3 graveolens, 434,
55 lactis eerogenes, 426

445

Bacterium merismopedioides, 31, 40,

ae termo, 62, 340
54 zopfii, 35, 426
Baden, tuberculosis in, 221
Baking, Il
Beef broth media, 399
Beer, muddiness of, 108, 109
Beggiatoa, 40, 44-6, 349-51
os alba, 441
rf mirablis, 442
5 roseopersicina, 348, 442
Benzol, 359
Betaine, 359
Biogenesis, 55, 56
Biological characters and classifica-
tion of bacteria, 5
Biuret reaction, 365
Bladder, bacteria in inflamed, 81
Bleeding bread, 9, 11, 22, 61, 347
Blood, bacilli in, 76, 270, 271, 274
Blood poisoning, 81
Blood serum, sterilized, 404
Blue milk, 22, 84, 348
Blue pus, immunity, 378
Black torula, 418
Bloody sweat, 347
Bouillon media, 399
Bread, bacteria formed inunsound, 11
Bread crumb media, 400
Brewing, 10, 97, 99, 120
Brieger’s bacillus, 423
Brownian movement, 30, 61
Butyric acid formula, 136
»» fermentation, 135
Bye products of fermentation, 94

Cadavarine, 185, 359, 360, 362
Cancer, monad of, 64
Cape meal orange ferment, 22
Capillary tubes, 406
Carbol-methylene blue, 411
Carbuncles, 282
Caries of the teeth, 147
Cattle inspection for tubercle, 225
Cellulose in bacteria, 26
Chain bacteria, 31
Chain cocci, 31
Charbon symptomatique bacillus,437
Cheese, 9, 15, 22,59

»» bacillus, 439
Cholera, 65, 83, 150, 369

446

Cholera alkalies in, 164
» inanimals, 162
s» bacilli, conditions of
growth, 175,
176, 178, 180,
186

flagella on, 28
pathogenic para-
site, 147
in sewage, &c.,
161
temperature rela-
tions, 174, 175
+ and Chinese vegetables,
182
»» causation of, 152, 153, 183
» comma bacillus, 31, 150,
152-4, 158, 159, 161,
103s 438
», cultures, 3, 154, 155, 157-
60, 168 54, 155, 15
1» epidemics, 150, 156
9 ” period of, 174
», due to ferment, 64
», hygiene of, 190
»» infection, 161, 171, 172,
188
ss inoculation against, 185
39 sa ee ap 147, 162,
173, 183-5
+, morphia in, 164
», pathology of, 166, 167
»> poison, 164
»,» predisposing causes, 181,
189
prophylaxis, 188, 191
» regions, 159, 177, 179, 181
»— Spirilla, 168 ©
»» spread of, 151, 189
toxalbumens, 363
Choline, 185, 361, 362
Chromogenic bacteria, 39, 43, 347
Cilia, 2
Cladothrichez, 44.
Clahothrix, 27, 31, 40, 42-7, 442
Classifications of bacteria, 38-47,
53», 54. 60
Classification of yeasts, 102, 106-11
Clathrocystis, 40, 46
Clostridium, 39, 44, 45, 89, 432,
437
Coccus, 44, 47

INDEX TO SUBJECTS.

Ccelosphzerium, 40

pelos particles in bacteria, 27,
34

Conjunctivitis, bacillus of, 436

Contagium vivum, 53

Copenhagen Milk Supply Associa-
tion, 225

Coverglass preparations, 206, 299,
4ll

Cowpox, inoculation for smallpox,

309

Crassuiscula, 54

Creatine, 185

Crenothrix, 27, 40, 42-5, 441

Cultivation of anzerobic organisms,
407

Cultivations fractional, 83

Cultivation media, 4, 67, 301, 391,
399; 400-5

Cultures, 2, 21, 35, 76, 155, 1575
159, 196, 214, 250, 278, 300,
301, 391, 394, 410, 411

Decomposition, 142
” ofammonianitrogen |
compounds, 142
Deneke’s cheese bacillus, 439
Dentine tubules, bacteria in, 341
Desmo-bacteria, 38
Development of bacillus subtilis,

Diastase, action on starch, 115
»» ferment in the mouth, 340
Digestion by bacteria, 15
Diphtheria, 81, 83, 297
” antiseptics, 309
sa bacillus, 297-302
cultures,
301, 307
in man and
animals, 303,
304, 306
modification of
virulence,
ZIT, 312
‘in mouth, 310
products, 363
of rabbits, 424
staining of, 299
Diptheretic paralysis, 302, 30% 306
Ph poison, 303, 305-8, 363
Diphtheria, predisposing cause, 309

” ” 300,

” yw

a ”
” ”
cal ”

INDEX TO SUBJECTS.

Diphtheria, streptococcus exciting
cause of, 298, 305
” treatment, 308-310
Diplococci, 31
Diplococcus albicans amplus, 416

albicans _ tardissimus,
_ 415

+ citreus conglomeratus,
417

vs intracellularis meningi-
tidis, 421

35 lacteus faviformis, 416

se * der , Pferdepneumo-
nie,’ ” 416

3 of pneumonia, 26, 435

as subflavus, 420

of trachoma, 415

Disinfectants for tubercle bacilli,
221

Dispora caucasica, 12

Division of bacteria, 30-2

Drugs, antagonistic, 379

Drumstick bacillus of putrefaction,
16, 22, 287, 425

Drying effect on various bacilli, 168,
202, 219, 269, 302

Dyes, aniline, 25 (see Staining)

Earth bacillus, 284, 430
» examination for bacteria, 393
» asa filter, 1
’ Eberth-Gaffky bacillus, 203
Egg albumen, cultivation on, 401
Ehrlenmeyer flasks, 388
Emmerich’s bacillus, 154, 439
Endospore formation, 34, 37
Endostreptococcus, 45
Haymes, 13, 14, 127, 131, 142, 293,

305
Enzyme function, special, 129, 141
Evaporation of toxalbumens in vacuo,

305
Exanthemata, 369
Excretory theory of fermentation, 124
Exhaust pump, 409

Facultative zrobic organisms, 19
" parasites, 146
” pathogenic parasites, 148
” saprophytes, 146, 147
False branching, 31
-Farcy pipes, 263

447

Fat in myco-protein, 25
Fatty acids, 135

», acid fermentation, 136, 142
Fermentation, 64, ie 72, 92-4,

II
% albuminoids in, 66,

116

i alcuholic, 142

8 ammoniacal, 132

ia anzerobic, 121, 122

iy artificial fluids, 117

"i butyric, 8, 67, 94,
132, 135

3 bye products, 94

yi digestion, relation
to, 128

is in fruits, 119

a heat, action on, 91,
93

6 “high,” 98

$i by hydration, 132

<i lactic, 9, 10, 66, 132,

133,135, 142
" = formula, 136
# light, action on, 91
a ‘low, ” 98, 99
mannitic, 132
3 milk-sugar, 130
* nature of, 87, 93,
94, 116
6 by oxidation, 138
” processesillustrated,
8-91
‘i products (interme-
diary) of, 94, 142
43 tartaric, 67
9 theories of,
125,127
” urea, 64, 134
Be vitalist theory, 97
see yeast cells, condition
in, 141
yeast, 95
Filling’ of vessels with fluid media,
405
Film formation in fermentation,
104, 105
Filter, cotton wool, 58
», Chamberland, 409
2 earth as a, 19
» for gelatine, 402
» Kitasato’s, 410

124,

448

Filter, Miquel’s, 410
1» Pasteur-Chamberland, 393
3» placenta as, 77
Filtration of potable water, 393
Finkler-Prior bacillus, 340, 438, 439
Fish, putrefaction of, 356
Fisson fungi, 24, 63
Flagella, 28, 29, 413
Flax, maceration of, 15
Fluid culture media preparation, 399
Fogs, 382
Formula butyric fermentation, 136
Fowl tubercle, 217
Fractional cultivation, 33
French Cholera Commission, 152
Fraedlander, 26, 27, 425
Frog-spawn masses, 26
Function, modification of, 6, 129,
130
“ specilization of, 6, 16
Fungus protein, 25

Gadinine, 362

Gangrene of the lung, 8r

Gas forming bacillus, 432

Geissler’s water exhaust pump, 409

Gelatine filter, 402

Gelatinized meat peptone medium,
402

Gelatine method, 4, 402

Gelatinized milk serum, 402

Generatio eyuivoca, 60

German Cholera Commission, 152

Germ free substances, 58, 59
3, theory, early observations, 52

Germicides, 71, 186, 197, 221, 270,
276, 277, 309, 310

Gleeocapsa, 40

Gleothece, 40

Glanders bacillus, 263-5, 270, 414, |

435
46 55 staining, 264
i distribution, 269
5 and farcy in man, 268
- nature of the disease, 262

sa poison, 80, 263

+3 susceptibility of animals,
266, 267

3 temperature, effect on,
269

Glass needles, 407
Globulins, poisonous, 358

{INDEX TO SUBJECTS.

Glycerine agar, 404
” meat extract, 400
Glycocol, 359
Glycogen converted into sugar, 116
Grape sugar, 92
Gram’s staining method, 26, 195,
412
Ground water, effect on cholera
bacillus, 172
Gypsum blocks, 103

Hanging drop cultures, 158, 410,
- 4I

4II
Hay bacillus, 429
Heat action on fermentation, 91,

»» effect on spores and germs,
35-71 59, 201, 269
Hesse’s apparatus, 385, 386, 398
‘* High ” fermentation, 98°
1» «yeast, 98, 106
History of growth of bacteriology,
I, 49 e¢ seg.
Hog cholera, 375
Horsepox bacillus, 436
Hot air sterilizing apparatus, 397
Hydration of urea, 135, 137
Hydrocollidine, 361
Hydrolisis, 142
Hydrophobia, protection from, 321,

. 327 ;
‘5 immunity against,
. 373 ,
+ incubation period,
318
55 infection, 317-20
- inoculation at Pas-
teur Institute, 328
sis micro-organisms in,

315, 316, 328

a nature of, 317

en researches on, 314,
315

4 treatment of, 323-6

Pe virus, modification
of, 320, 321

Identification of species of bacteria,
414 et seg.

Immunity, 239, 266, 321, 323, 364,
368-71, 375, 376, 378

Incubation apparatus, 398

INDEX TO SUBJECTS.

Indiarubber sterilized, 409

Infusoria, 54

Ingrafting of smallpox, 369

Inoculating apparatus, 406

Inoculation, 217, 232

of various media, 212,
406, 407

Instruments, sterilization of, 135,
406

Intermediary products of fermenta-
tion, 142

Internal metabolism, 125, 139

Intestine bacillus, 424

»» its condition and relation

to cholera, 165

Intoxications, 198

Inversion a chemical process, 128

of saccharose by invertin,

* 115, 128

Invertin, 115, 127

Todine, 25

Iodococcus vaginatus, 339

Isolation apparatus, Hallier’s, 79

a

a”

Jequirity poison, 379

Kephir grains, 12
Kidney, bacteria in abscesses of,
81

Lactic acid ferment, 9, 94, 126, 132,
133, 135, 136, 142, 145
» fermentation, 9, 10, 66,
142, 340
acting on teeth, 9

”

” ”
Lenses, 49
Lepra cells, 248
Leprosy bacillus, 246, 247, 250

¥ in tissues, 248-50
alleged causes of, 252
communicability of, 250
distribution, 244, 245
and tuberculosis, differences
between, 248
Leptothrix, 40, 42-7
buccalis, 147, 342
epidermidis, 425

7 innominata, 339
Leucomaines, 362
Leuconostoc, 34, 35

”
”
”
”
”

”

”

449
Leuconostoc mesenteroides, 27 _
Light, action on bacteria, 91, 199,
200
fermentation, 91,

” ”

201

» bacillus, 36, 351 e¢ seg
Limiting membrane, 26-8
Lineola, 54, 60
Living contagium element, 53
Lockjaw, 22
Logwood, 25
‘* Low ” fermentation, 98

9» yeast, 98, 107, 108
Lungs, mouth bacteria in, 344
Lupus, tubercle bacilli in, 208

Magenta micrococcus, 9, 12, 347; 348
Malaria, 78
Malignant pustule, 76, 77
» cedema, 77, 295
Malting, 97
Mammitis, tubercular, 227
Mannitic fermentation, 132, 134
Meat, tuberculous, 228
Media, 4, - 399, 400-2, 404,
4
»» inoculation of, 212, 406, 407
Merismopedioides, B., 31, 40, 44
Meristze, 43, 4
Metabolism, internal, 125, 139
Methylene blue, 411
Methylguanidin, 185
Micrococci, 29, 38, 40, 42-6, 81-3
9 found in pyzmia, 82
Micrococcus zerogenes, 419
candicans, 345, 416
ofcattle pneumonia,415
cinnabareus, 418
coronatus, 420
flavus desidens, 419
»» liquefaciens, 420
»» tardigradus, 417
gonorrhoea, 421
mastitis, 415
osteomyelitis, 9
prodigiosus, 9, 12, 347,
350, 433
pyogenes tenuis, 421
radiatus, 420
roseus, 418
of sputum septicemia,

344

(?

a

30

450

Micrococcus tetragonus, 348, 416
3 of trachoma, 415
o ures, 134, 416
” » liquefaciens, 418
” vesicolor, 417
Milk, bacilli in, 224, 228, 413
1», Cholera bacilli in, 159
», ducts, tubercule bacilliin, 228
», Sterilized, 59, 400, 402
x» supply, 225
», typhoid bacilliin, 197
3, yellow organism in, 84
Mineralization, 17, 68
Mixed infection, 199
Millon’s reagent, 365
Modification of function, 6, 9
35 »» Virulence, 311, 320,
321
Moist chamber, 410
Monas, 54, 442
4, prodigiosus, 61, 433
Monospora, Metschnikovi, 106, 111,
112
Mother of vinegar, 63
Mouse septicemia, 412, 426
Mouth bacteria, 63, 147, 337 ef s¢g.,
343, 345
Movements of bacteria, 28
Muddiness of beer, 108
be ea of bacteria, 30, 32,

3
Muscarine, 360-2
Muscle sugar, 134

Mycoderma aceti, 10, 69, 126,
138-40
” pastorianum, 10

Mycophycez, 62
Myconostoc, 40, 43, 46
Mycoproteim, 25
Mydaleine, 360, 362
Mytiloloxine, 361-2

Necrotizing substance in tubercle
bacilli, 234
Neurine, 360-2
Nicotine, 250, 359
Nitrification, 17 a:
Nitrogen clo ane, decomposition
of, 142

», trichloride, go

1 teriodide, 90
Nitro-glycerine, 91

INDEX TO SUBJECTS.

Nonpathogenic bacteria, 146
Nose cavities, bacteria in, 345
Nosema bombycis, 63, 70
Nostocacese, 42

Nuclei in yeast-cells, 113

Obligate parasites, 146

Occurrence of capsule in B. anthra-
cis, 27

(Edematis maligni, bacillus, 436

Ophidiomonades, 41

Organic nitrogen not necessary for
yeasts, 118, 119

Organized ferments, 72, 142

Orleans process of vinegar-making,
139

Oscillaria, 42

Osteo-sarcoma, 253

Oxidation by bacteria, 119

sy in soil, 18
Oxygen necessary for film formation,

104
‘eae a
Panhistephyton, 70

Papain, 379

Paralysis, diphteritic, 302, 303, 306
Parasites, facultative, 146
Parasites, obligate, 146

Particulate virus, 80

Parvoline, 361

Pasteuria, 45

Pasteur Institute, description of, 329
Patteson, Bishop, death from tetanus,

2m on
Pathogenic bacteria in mouth, 343,

344
Pathology of cholera, 166
Pathogenic parasite, 147
Pathologia animata, 49
Pediococci in beer, 10
Pentamethyline diamine, 359
Peptonization, 14, 15
Petruschky’s flask, 389
mobi method of collecting water,
309
Phasoeylen theory of Metschnikoff,
112
Pharmidiothrix, 44
Phosphorescence, 346
Phosphorescent micro - organisms,

351

INDEX TO SUBJECTS.

Photo-bacterium Balticum, 353
Fischeri, 353
Fluggeri, 352
Indicum, 354
Luminasum, 354
‘9 >». Phosporescens, 351
Phthisis, 83
Phycochrome, 40
Pigment forming bacteria, 145
Pink yeast, 111 .
»» torula, 418
Pipettes, graduation of, 389, 406
Placenta as filter, 77
Plate cultures, glass dishes for, 389,
390, 39%
Pleomorphism, 43, 47
Pneumonia diplococcus, 26, 435
Poisoned weapons, 294, 295
Poisonous bye products, 82
Poisonous substances in putrefying
fish, 356

Poison, production of, by tetanus

bacilli, 293
Polybacteria, 43
Polymorphism, 78-80
Potato bacillus, 12, 340, 432
»» culture media, 4o1
Preparation of sterilized potato
chambers, 401
Products of bacteria, 95, 136, 362,
363, 368, 409 .
Prognosis in diphtheria, 300
Protective albumoses, 238, 366
Proteus, 54
3» mirabilis, 431
»» Vulgaris, 414, 430
», Zenkeri, 431
Protoplasm of bacteria, 24
Pseudo-cedema bacillus, 437
3: Septicsemia of mice bacillus,

436

»» typhoid bacillus, 202, 423
Ptomaines, 357-9, 361, 362
Puerperal fever, 81, 83
Punctula, 43
Pure cultures, 2-5, 76, 155, 157,

196, 250, 300, 391°

3, lactic fermentation, 133
Pus, organisms in, 63, 84
Putrefaction and disease-germs, 43,

52, 53, 5
Putrefactive bacillus, 425

451

Putrefactive bacteria, 145
” decomposition, 142
is processes, 68-70
Putresceine, 185, 360, 362
Pyzmia, 77, 82, 83
Pyelo-nephritis, 83
Pyocyanin products, 238
Pyridine, 359
Rauschbrand bacillus, 437
Ray fungus, 27, 254, 255
Red bacillus, 433
Respiration in yeasts, 124
Resting spore, 32
Rhabdomonas, 442
Rhinoscleroma, bacillus of, 438
Rice medium, 401
3, water stools, 166
Rinderpest, 81, 83
Rivolaria, 40
Ropiness in white wines, 132
Rose Hefe, 111
Rugula, 54, 60
Rye-bread ferment, 11

Saccharomyces, 96, 106

35 apiculatus, 111

¥y cerevisex, 102, 103,
105-7

- conglomeratus, III

0 ellipsoideus, 102,
105-8

5 exiguus, II0

53 Ludwigii, 109, 110

25 Marxianus, 110

53 membraneeaciens,
110 3

“a mycoderma, 104

Pastorianus, 102,

104-6, 108, 109, 118
Saccharose, 134
i with invertin, 115, 128
Saponification, 142
Saprine, 360, 362
Saprophytes, facultative, 145-7
Sarcina, 10, 40; 43-6, 63, 34, 147
» alba, 419
>) aurantiaca, 417
” lutea, 417
»» — ventriculi, 32, 146
Scarlatina, 81, 368
Scirrhous cord, 253, 254, 259

452

Schizomycetes, 24, 63
Schizophytes, 39
Self-digesting yeasts, 126
Senescent yeast, 123
Separation of organisms from pro-
ducts, 128, 409
Sepsines, 81, 184, 356, 358
Septiceemia, 77, 81, 83
Septic tooth disease, 346
Serum medium, 405
Sewage, bacteria in, 21, 383, 387
Silk-worm disease, 63, 64, 69
Size of bacteria, 24
Smallpox, 83, 369
Snake poison, 376
Soil, bacteria in, 393
Solid culture media, 78, 400, 407
Soluble products, anthrax, 375
Pe ‘i immunity by, 375,
376
53 95 of yeasts cells, 127
Soyka’s ground rice medium, 401
Specialization of function, 6, 16,
14!
Specific fermentation, 126
-y, Characters of yeasts, 101
3, Germs of disease, 52
»» immunity, 368, 371
Spheero-bacteria, 38
Spirillum, 40, 43-7, 54, 60, 63
” cholerze Asiaticze, 438
4 concentricum, 413, 440
7 Obermeieri, 441
5 rubrum, 413, 440
. rugula, 441
x serpens, 441
7 sputigenum, 339, 340
3 tenue, 441
” tyrogenum, 439
9 undula, 441
33 volutans, 441
3 spirochete, 40-6, 60
Spirocheete denticula, 340
3 plicatalis, 62, 441
Spiromonas, 41, 442
Spirulina, 40, 41, 45, 47, 62
Spontaneous generation, 54-7, 60
Spores, effects of heat on, II, 35-7
Spores, 32-71 471 54, 96, 102-4,
109, 148, 167, 207, 275, 287,290,
292, 302
Spring water, bacteria in, 20, 387

INDEX TO SUBJECTS.

Sputum, phthisical, 220
Sputum septicaemia, micrococcus of,

344

Staining methods, 25-6, 36, 154,
166, 195, 206, 246-8, 264, 299,
315, 411, 412

Staphylococcus albus, 416

“a cereus flavus, 417

pyogenes albus, 340,
418

or pyogenes aureus,
340, 419

< pyogenes aureus,
toxalbumens from,
363 u

” pyogenes citreus,

420

Starch, action of diastase on, 115
Starved yeasts, 117

Steam sterilizer, 398

Sterilization of clothing, 221

3 discontinuous, 400
” of glass, 389, 390, 391,
397, 398 -
33 of indiarubber, 409
a5 of instruments, 135, 401,
406

Sterilized vessels, 399
Sterilizer, steam, 398

” hot air, 397
Stock flasks, 405
Streptobacteria, 31, 40
Streptococcus, 31, 40, 44.

55 articulorum, 415

“ coli gracilis, 420

” diphtherize, 305

3 erysipelatosus, 414

- Forsteri, 442

5 pyogenes, 414

3 es malignus,
414

” septicus, 415
Streptothrix, 40, 46
Structure of bacteria, 25 et seg.

> gy: Yeast-cells, 113

33.» flagella, 28
Succinie acid, 94 .
Sugar fungi, 96

xy Of milk, 134

Summativeaction ofbacteria poisons,

379
Surface water, 19

INDEX TO SUBJECTS.

Synechococcus, 39
Syphilis, bacillus of, 438
Swine erysipelas, bacillus of, 427

Tartaric acid ferment, 67
Teeth, decay of, 63, 342, 346
Tetanus, 22, 286, 2904
ss bacillus, 287, 289, 290,
293, 295, 437
growth in wounds,
291
cultivation, 288,
290
distribution of
spores, 292
- experiments with earth,
289, 292
a” poison, 291, 293, 294, 357,
358, 362, 363, 365, 366
‘s pus, experiments with, 289
<5 spore formation, 287, 290
Tetraspora zoogloea, 62
Thallophytes, 39
Thebaine, 358
Torula, 113, 418
Toxines, 184°
Toxic saliva, 343
Trachoma, micrococcus, 415
‘« Trigger” theory of fermentation,
125
Trimethylamine, 350, 359
Tube plate inoculations, 391-4
Tubercle, an infectious disease, 221
re inspection of cattle, 225
3 statistics of, 221, 222
sy bacilli, 219, 381, 412, 435
in animals, 215,
217, 229, 233
cause of tubercu-

” 2”

” ”

” »

” ”

losis, 209
” » cultivation of,
210-14, 216, 217

examination of
sputa for, 218
of fowls quite dis-
tinct, 217
in lupus, &c., 208
» modification of
virulence, 217,
219, 229, 230
spores in, 20, 21
staining of, 412

453

Tubercle bacilli, temperature rela-
tions, 215
in tissues, 208,
209, 230, 231
9 in water 211
Tuberculin, 235-8, 242
Tuberculosis of the breast, 224
@ disinfectants, 221
i and leprosy, difference
between, 249

” ”

5 in milk, 224, 413
iy predisposing cause,
223
a treatment, 222, 231
233
of the bladder, 225-7

Tuberculous meat, 228
Turbidity of beer, 109
Typhoid bacillus, 21, 194, 195, 203,
414
in animals, 198
effect of carbolic
acid on, 197
flagella on, 28
cultivation in ge-
latine, 196
in milk, 21, 197
products, 197,
198, 363
(pseudo) 202
staining of, 195
position in tissues.
195
” in water, 21, 197
7 cultures, 196, 199
” fever, 65, 81, 83, 369
Typho-toxine, 357, 362
Tyrothrix bacillus, 15, 59

” ”

Udder, tubercle of, 225

Unorganized ferments, 96, 142

Unsound bread, 11

Urea, hydration of, 135, 137
1, fermentation of, 134

Vaccine lymph, 80

Vacuoles in yeast-cells, 113

Vegetable ferments, 67

Vegetative multiplication, 30, 31

Vibrio, 40, 43-6, 54, 60, 62, 63, 67,
68, 75, 152

454

Vibrio ee! 185, 413, 439 -

Vibrion Septi re 436
Vinegar, 63, 68, 139

Viscous fermentation, 132

Vitalist theory of fermentation, 97
Volvox, 54

Water bacteria, 62, 161, 178, 197,
211, 381, 387, 388, 392
‘Water, examination of, 387-90, 392
» exhaust pump, 409
+ filtration of, 393
x» supply, I9, 20
"Weisser bacillus, 426
Wens, 253
Wild yeast, 107, 108
“Wine diseases, 69
»» fermentation, 108, 120
3» Hower of, 68
Wool-sorters’ disease, 273, 280
Wooden tongue, 253, 254

Yeasts, 96, 108, 117, 145

INDEX TO SUBJECTS.

Yeasts, acclimatization of, 125
» cells, 95, 96, IOI, 113, 122
128, 141.
5 », cause of ermentation

63, 64, 92, 94
», Classification (Hansen) 102

I
»» fungus in kephir, 13
», growth of, 96, 105, 119
122, 127
» high,” 98, 106
a “low,” 98, 107, 108
» pink (Rosa Hefe) 111
1 pure, 100
oy wild, 108
wine, 108
Yellow colour organism in milk, 84

Ziehl-Neelsen, carbol-fuchsin method
of staining, 412

Zoogloea mass, 12, 26, 43-5, 141

Zymogenes, 43

INDEX TO AUTHORS.

—\_+—

ABBOT, 346

Acland, 261

Aitken, 382, 394
Andry, Nicolas, 51, 73
Arloing, 295

Arning, 250, 252

BABES, 34, 36, 48, 154, 160, 168,
191, 249-51, 252, 259, 305, 313,
316, 326, 335, 367, 442

Bang, 8, 224, 242, 261

Bassi, 64, 73

Bastian, 59, 73

Baumgarten, 47, 206, 242

Bechamp, 71, 125, 143

Behring, 275, 313, 365

Belfanti, 295

Bendall, 268, 270

Bergmann, 356, 367

Berthelot, 115

Beyerinck, 351, 353-4, 355

Bienstock, 16, 22

Biondi, 346

Birch-Hirschfeld, 83-4, 85

Blunt, 199

Bochfontaine, 161, 164, 191

Boehm, 152, 191

Bollinger, 242, 253, 261, 284

Bolton, 387, 394

Bonnet, 55, 78

Bonome, 295

Bossano, 292, 295

Bostrom, 261

Bouchard, 263, 270, 357, 367

Boutroux, 143

Brauell, 77, 85, 271, 284

Brefeld, 4, 80, 85, 143, 442

Brieger, 184, 191, 291, 295, 313,
357-9) 365, 367

Brittain, 152, 191

Brown, Graham, 152

Brunton Lauder, 129-131

Burrows, 356

Buchner, 36, 47, 191, 275, 284, 316-
7s 324, 326, 370, 879, 409, 411

Bujwid, 191, 325

Bunge, 92-3, 143

Busck, 225.

CAGNIARD-LATOUR, 64, 66, 70, 72,
73, 95, 148

Cantani, 191, 406

Capitan, 263

Carle, 296

Carnelley, 383, 394

Cassedebat, 202, 203

Castor, 250-1

Cattani, Mdlle., 287

Cazeneuve, 58, 73

Chamberland, 315, 333, 385, 3755
380, 399, 409

Chantemesse, 143, 197-8, 293, 296,
313, 334

Charrin, 263, 334, 380

Chauveau, 7, 8, 80, 85, 224, she,
270, 284, 370, 373, 375, 380

Cheshire, 430

Chevreuil, 58

Cheyne, Watson, 8, 58, 73, 152, 191,
207, 242, 411, 430 -

Chiene, 58, 13

Clarke, 296

Coats, 194

Cohn, 3,.8, 22, 32, 38-40, 47, 62,
73, 80, 85

Cohnheim, 242

Cornet, 219, 221, 243

Cornil, 34, 48, 154, 168, 211, 249,
316, 326, 335, 442

Creighton, 380

456

Crookshank, 261, 380, 442
Cunningham, 152, 191

DALLINGER, 28

Davaine, 4, 8, 60, 73, 75-7, 80, 83,
85, 119, 143, 192, 271-2, 284

David, 846

de Bary, 12, 13, 14, 16, 31, 34; 47—

J 2
Delafond, 76, 85
Delbruck, 9
Delepine, 261
Deneke, 192
Desmaziéres, 95, 143
Dolan, 335
Donne, 63
Dowdeswell, 335
Downes, 199
Dubrunfant; 143
Duclaux, 15, 18, 23, 59, 68, 73, 201,
3301; 334
Duguid, 375
Dujardin, 40, 62, 48, 73, 75, 85
Dunnenberger, 11, 23
Dusch, 74

EBERTH, 194
Ehrenberg, 48, 60-2, 73
Ehriich, 36

Eisenberg, 442
Emmerich, 192

Engel, 11, 103, 110, 143
Englemann, 201, 355
Escherich, 34
Esmarch, 391, 394, 449
Etard, 361

Ewart, 58, 73

FABER, 296

Finkler, 192

Fischer, 36, 351, 355

Fitz, 15,23 #

Filigee, 43, 46, 47, 48, 148, 154, 176,
188, 192, 195, 208, 243, 278, 282,
296, 432, 442

Fol, 315, 335

Formad, 298, 305, 313

Forster, 351, 355

Fraenkel, Carl, 388, 442

Fraenkel, 195, 198, 305, 311, 343:
346, 363-5, 394, 407

INDEX TO AUTHORS.

Frangois, 132
Frankland, 382, 394
Friedlander, 26, 27, 344, 425

GaFFKY, 156, 192, 195-6, 243

Galippe, 342, 346

Galtier, 219, 326, 335

Gamaleia, 185-6, 192, 313, 333-4,
335

Gautier, 319, 326, 361-2, 367

Gerlach, 224, 248

Gibbes, 152

Gibier, 315, 317, 335

Globig, 35

Goodenough, 294

Goodsir, 32, 48, 84, 85, 146

Grancher, 313, 324, 334

Grawitz, 370

Greenfield, 8, 280, 284, 347, 355,
372, 380

Griffiths, 367, 383

Giinther, 442

HALDANE, 383

Hall, Marshall, 296

Halliburton, 367

Hallier, 40, 78-81, 153, 192

Hankin, 238, 305, 363-4, 367, 375-6

Hansen, A., 245 :

Hansen, Chr., 8, 23, 100, 102-111,
113-4, 133, 143, 184, 252

Hare, 383, 442

Hart, 394

Hartsocker, 55, 73

Harvey, 59

Hauser, 53

Hayem, 152

Heiberg, 83, 85

Henle, 65, 73

Hesse, Frau, 403

Heuter, 83, 85

Hime, 275, 335

Hirsch, 252

Hoffmann, 58, 71, 73, 80, 85, 311

Holm, ro4, 143

Hoppe-Seyler, 115, 143

Hubermaas, 224, 243

Hiippe, 9, 15, 28, 34, 36, 45, 47
73, 126, 129, 131, 148, 168-9,171
173,179, 192, 284, 340, 346, 376-
7, 380, 401, 405, 422-3, 432, 442

ISRAEL, J., 259, 261, 270

INDEX TO AUTHORS.

Jenner, 369, 380

Joblot, 55

Johne, 243, 261
Jorgensen, 9, 11, 23, 143

Katz, 355

Kern, 12, 14

Kerner, 356

Kieser, 143

Kircher, A., 49, 51

Kirk, 346

Kitasato, 3, 287, 290, 296, 410,

440

Klarindero, 250, 252

Klebs, 3, 4,8, 23, 58, 74, 81-3, 85,
194, 298, 305, 313, 369

Klein, 8, 152, 161, 164, 194, 192,
199, 215, 277, 284, 304, 313, 339,
343, 346, 375, 380, 442

Klob, 153, 192

Koch, 4, 7, 8, 23, 65, 76, 78, 85,
150-2, 155, 161-4, 172, 176-8,
180, 183, 185, 192, 206, 210-1,
214, 217, 221, 229, 232-3, 239,
241, 243, 271, 275, 285, 296, 340,
380, 384, 394, 398, 402, 404, 407,
438-9, 442,

Kolisko, 313

Krause, 9

Krebbe, 270

Kiihne, 195, 411-2, 443

Kunz, 351

Kussmaul, 296

Kiitzing, 95

LANCISI, 52

Lange, Christian, 49

Lankaster, 348, 365

Laurent, II

Ledantec, 294, 296

Leeuwenhoek, 49, 51, 54, 63-4, 74,
147, 337, 346

Leplat, 76, 84, 85

Lehmann, 275, 351, 355

Leloir, 252

Lemaire, 71

Leube, 58, 134, 143

Levy, Alex., 14

Lewis, 152, 192, 339

Liborius, 407, 437

Liebig, 93, 96-7, 118, 143

Lindner, 10

457

JACQUEMART, 361, 367

Jaillard, 76, 84, 85

Janowski, 201

Linnaeus, 52-3

Lister, 4, 7, 8, 23, 58, 72, 74, 83,
143, 406

Livon, 58, 73

Loffler, 28-9, 48, 52, 62, 73, 79,
85, 195, 263, 266, 270, 298, 300—-
2, 304-5, 311, 313, 362, 367-8,
405, 435, 438

Liiders, Johanna, 79, 85

MACLEOD, 151-2, 165, 167, 177,
179-81, 192

Macnamara, 161, 167, 192

M’Fadyean, A., 130-1

M’Fadyean, J., 224, 226, 228, 243,
253, 255, 256, 260-1, 285

Maddox, 384

Maffucci, 240, 241, 243

Manassein, 80

Marchand, 58

Martin, Sidney, 364-5, 367

Mayer, 139, 143

Mitscherlich, 68

Metschnikoff, 111-2, 230, 248, 331,
370, 380, 439

Migula, W., 393, 394

es 192, 339-4C, 342, 346, 349,

Miller, 442

Miquel, 383-4, 386, 394, 410

Montague, Lady Mary Wortley,
6

309
Miiller, O. F., 53-4, 60, 62, 74

NAEGELI, 4, 23, 39, 63, 70, 73

Needham, 55, 74

Neisser, 36, 48, 250, 252

Nencki, 25, 357, 367

Nicati, 164, 180, 192

Nicolaier, 286-7, 295, 296, 394

Nocard, 152, 228, 248, 296, 326,
335, 404. :

Northrup, 305

OERTEL, 313
Orth, 83, 85

PACINI, 152, 192
Panum, 8, 81, 85, 225, 356, 367

458

Pasteur, 2, 4, 7-9, 22, 23, 58-9, 66-
70, 74, 76, 78-9, 83, 8B, 93, 97,
99, 113, 115, 118-9, 122-3, 132,
134, 139 143,
271-2, 285, 314-5, 317, 319, 321,
323-4, 326-8, 335, 343, 357) 367,
369, 372-5, 380, 393, 406, 442

Pawlowsky, 215, 243

Perdrix, 375

Perroncito, 261

Perty, 62

Petri, 383, 389, 391

Pettenkoffer, 150, 171, 175, 183, 192

Pfeiffer, 156, 192, 198

Pfuhl, 394

Plenciz, 52

Poels, 428

Pollender, 75, 85, 271, 285

Ponfick, 261

Pouchet, 152, 184, 192, 357, 362

Poulsen, 104

Prazmowski, 275, 285, 432, 437

Prudden, 303, 305

RAKE, BEVAN, 251, 252

Ransome, 219

Raskina, Madame, 403

Rayer, 75, 271

Raynaud, 152

Reaumur,.55 |

Recklinghausen, 81-2, 86, 194

Reess, 96, 102, 106, 107, III, 144

Richards, 162

Rietsch, 164, 180, 296

Rindfleisch, 58, 74, 80, 85

Roberts, 3, 23, 58, 73

Roger, 285

Rose, 296

Roux, 152, 154, 216-7, 243, 275,
296, 299, 300, 302, 306~7, 310-13,
315, 326, 330-1, 335, 375, 404,

fe)

4
Roy, 152, 192
Ruffer, 313, 380

SALISBURY, 78, 80, 85

Salomonsen, 4, 8, 23, 352, 880, 397,
399, 402, 409, 411, 442

Salomonsen, Madam, 352

Sanchez, Toledo, 296

Sanderson, Burdon, 8, 58, 74, 80,
85, 162, 375

147, 184, 238,

INDEX TO AUTHORS,

Saxtorph, 296

Schmelk, 394

Schmiedeberg, 356, 360

Schottelius, 9, 154, 192, 424

Schroeder, 58-9, 74

Schulze, 56-7, 74

Schiitz, 263-6, 285

Schutzenberger, 125, 127, 135-6,
138-9, 144

Schwann, 57-8, 64, 66, 74, 79, 35,
95, 144

Selmi, 357, 367

Sewall, 376

Sherrington, 152

Simmonds, 198

Simpson, 159, 296

Smith, Angus, 395

Sonnenschein, 81, 86, 357, 367

Spallanzani, 54-6, 74

Stanley, 295

Sternberg, 343, 346

Straus, 152, 154, 193, 334, 395

Swaine, 152, 193

TALAMON, 313

Thiersch, 162

Thuillier, 152

Tieghem, Van, 15, 23, 41-2, 47, 48,
102, 144 :

Tilanus, 351

Tizzoni, 287, 296

Tollhausen, 351

Toussaint, 243, 285, 372

Traube, 81 :

Trecul, 84, 86, 144

Treves, 261

Tulasne, 78, 86

Turpin, 97, 144

Tyndall, 58, 74, 199, 383, 400

UNNA, 252, 412

VAILLARD, 293, 296
Van der Broek, 58, 74
Varro, 52

Vaughan, 361
Verhoogen, 296
Verneuil, 296

Vesta, 326

Vignal, 340, 342, 346
Villemin, 217, 248
Villiers, 184, 193

INDEX. TO

Vincent, 293, 296

Virchow, 152, 169, 241, 252
Von Dusch, 58

Vulpian, 270, 324.

WADDELL, 313
Wagner, 64, 74
Waldeyer, 82

Walley, 227, 243
Wassilieff, 220
Weichselbaum, 270
Weigert, 82, 86, 243, 315
Wasserzug, 193

Widal, 197-8, 293, 334
Wiedermann, 296
Wiesner, 102-3, 243
Williams, 219, 243
Willis, 97, 144
Winogradsky, 355

AUTHORS. 459

Winter, 44, 47

Wolff, 259, 261

Wolffhiigel, 197

Wood, Cartwright, 9, 129-31, 141,
143-4, 169, 171, 173, 185, 193,
284, 318, 376-7, 380 .

Woodhead, 193, 224, 226, 238, 243,
285, 327, 377, 380, 395, 442

Wooldridge, 375, 380

Wyssokowiez, 300

YERSIN, 299, 300, 302, 306-7, 310-
12,313”

ZAGARI, 326
Zarniko, 313

Zopf, 31; 34, 43, 44, 47-8, 111, 442
Ziilzer, 81, 86

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LIBRARY OF
HUMOUR.

EDITED BY W. H. DIRCKS.

Each Volume will contain from 50 to 70 [llustrations and
Srom 350 to 500 pages.

In each of these volumes the object will be to
give an anthology of the humorous literature
of the particular nation dealt with. France,
Germany, Italy, Russia, Spain, and Holland

will each have their respective volumes; England,

Ireland, and Scotland will each be represented,

as will also America and Japan. ‘From China
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with the amusing Fabliaux of the thirteenth century; in Germany
from Hans Sachs; characteristic sketches, stories, and extracts
from contemporary European and other writers whose genius is
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of the development of a.special and important phase of national
spirit and chgracter,—namely, the humorous. Proverbs and
maxims, folk-wit, and folk-tales notable for their pith-and humour,
will have their ‘place ; the eccentricities of modern newspaper
humour will not be overlooked. Each volume will be well and
copiously illustrated ; in many cases artists of the nationalities of
the literatures represented will illustrate the volumes. To each
volume will be prefixed an Introduction critically disengaging and
marking the qualities and phases of the national humour dealt
with; and to each will be appended Notes, biographical and
explanatory.

LIBRARY OF HUMOUR

Cloth Elegant, Large 12mo, Price $1.25 per vol.

VOLUMES ALREADY ISSUED.

THE HUMOUR OF FRANCE. Translated, with an

Introduction and Notes, by Elizabeth Lee. With numerous
Iustrations by Paul Frénzeny. ; ie

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papers fresh from the kiosk, we have a tremendous range of
selections.” —Birmingham Daily Gazette.

‘French wit is excellently represented. We have here examples
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La Rochefoucauld, Regnard, Voltaire, Beaumarchais, Chamfort,
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. . .» The book sparkles from beginning to end.”— G/obe (London).

THE HUMOUR OF GERMANY. | Translated, with
an Introduction and Notes, by Hans Miiller-Casenov. With
numerous Illustrations by C. E. Brock,

An excellently representative volume.— Daily Telegraph (London).

‘* Whether it is Saxon kinship or the fine qualities of the collec-

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Its riotous absurdities well overbalance its examples of the oppres-

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— Atheneum (London).

** Apart from selections of writers of classical reputation, the book
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THE HUMOUR OF AMERICA. Selected, with a

copious Biographical Index of American Humorists, by James Barr.

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THE HUMOUR OF HOLLAND. Translated, with

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THE HUMOUR OF IRELAND. Selected by D. J.
O’Donoghue. With numerous Illustrations by Oliver Paque.

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Trish Humour as it is, or has been, written, from the twelfth century

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THE HUMOUR OF RUSSIA. Translated, with Notes,

by E. L. Boole, and an Introduction by Stepniak. With 50
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THE “HUMOUR OF JAPAN. Translated, with an
Introduction, by .A. M. With Illustrations by George Bigot
(from Drawings made in Japan).

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XXV. THE EVOLUTION OF MODERN CAPITALISM.
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XXVIT. AN INTRODUCTION TO COMPARATIVE
; PSYCHOLOGY. By Professor C. LLoyp Morcan. With
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Ibsen’s Famous Prose Dramas.
Edited by William Archer.

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Vo.. I “A DOLL’S HOUSE,” “THE LEAGUE OF
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Vou. II. “GHOSTS,” “AN ENEMY OF THE PEOPLE,”
and “THE WILD DUCK.” With an Introductory Note.

VoL. III. “LADY INGER OF OSTRAT,” “THE VIKINGS
AT HELGELAND,” “THE PRETENDERS.” With an
Introductory Note and Portrait of Ibsen.

Von IV. “EMPEROR AND GALILEAN.” With an
Introductory Note by WILLIAM ARCHER.

Vout. V. “ROSMERSHOLM,” “THE LADY FROM THE
SEA.” “HEDDA GABLER.” _ Translated by WILLIAM
ARCHER. With an Introductory Note.

Vow. VI. “PEER GYNT: A DRAMATIC POEM.”
Authorised Translation by WILLIAM and CHARLES ARCHER.

The sequence of the plays ¢# each volume is chronological ; the complete
set of volumes comprising the dramas thus presents them in chronological
order.

‘* The art of prose translation does not perhaps enjoy a very high literary
status in England, but we have no hesitation in numbering the present
version of Ibsen, so far as it has gone (Vols. I. and II.), among the very
best achievements, in that kind, of our generation. ’—Academy,

*©We have seldom, if ever, met with a translation so absolutely
idiomatic.”— Glasgow Herald.

New York: CHARLES SCRIBNER’s SONS.