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LIBRARY

OF THE

UNIVERSITY OF CALIFORNIA.

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No.y/t/yb ... Class No. UBRAKY

BACTEEIOLOGY

ELEMENTS

OF

BACTERIOLOGY

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WITH ESPECIAL REFERENCE TO PRACTICAL METHODS

BY DR S. L. SCHENK
\\

PROFESSOR EXTRAORDINARY IN THE UNIVERSITY OP VIENNA

TRANSLATED FROM THE GERMAN

(BY THE AUTHOR'S PERMISSION)
WITH AN APPENDIX

BY W. B. DAWSON, B.A., M.D. UNIV. DUBL.

LATE UNIVERSITY TRAVELLING PRIZEMAN IN MEDICINE

WITH 100 ILLUSTRATIONS, PARTLYGOLOURED

LONDON
LONGMANS, GEEEN, AND CO.

AND NEW YORK : 15 EAST 16th STREET
1893

All rights reserved

UBRAftf
G

Dept.

TBANSLATOE'S PREFACE

THE present is practically a new edition in English of
Professor Schenk's Grundriss der Bakteriologie, published
in Germany some months ago, the scope and intention of
which the Author has explained so fully in his preface that
it is unnecessary to touch upon them here. Numerous
additions, many of which have been furnished by Professor
Schenk, have been made both in the body of the text and
in notes. Those by the Translator are distinguished,
where of any importance, by square brackets when in
the text, as on p. 92, and in the case of foot-notes by
square brackets with the letters * — TR.' following, as on
p. 21.

The Author originally intended to write an appendix, in
order to bring the English edition up to date, but this was
found unnecessary, owing to the short time since the ap-
pearance of the work in Germany, and the few additions
required for this purpose have been incorporated in the text.
It was, however, decided at the last moment, in view of the
attention lately attracted to those subjects, to furnish a
brief account of M. Haffkine's anti-cholera vaccination,
together with the results of recent research on the

vi BACTERIOLOGY

parasitism of protozoa, and on the action of light on
bacteria, and this has been done in an appendix, for
which the Translator alone is responsible. He has en-
deavoured to comply with the Author's intention by
describing practical details with considerable fulness, but
has thought it best to depart from the rule of not giving
references, the observations on these subjects being so
recent that certainty has not yet been attained. These
remarks apply also to the note at the end of Chapter X.
on Sabouraud's recent researches. A few additional
methods and formulae have also been given, the body of the
work has been divided into chapters, and the index greatly
extended.

The Translator has to express his thanks for assistance
on individual points from various sources, but especially
to Professor Schenk for his unvarying courtesy and readi-
ness in affording additional information ; and to acknow-
ledge occasional obligations to many of the leading English
and German text-books.

W. R. DAWSON.

DUBLIN : June 1893.

AUTHOR'S PREFACE

THE present work is intended to furnish the student and
the practitioner with a guide to the science of Bacteriology ;
consequently the methods of investigation have been dealt
with as thoroughly as possible, special attention being paid
to the elementary technique. The Author has thought it
best to describe the micro-organisms according to the loca-
lities in which they are met with, a plan which rendered
it possible to go thoroughly into the respective methods of
research in their proper places. Particular attention has
been given to the pathogenic micro-organisms, and so
much the more regard had to be paid to the chemical
relations of the life of bacteria, and to their biology gene-
rally, that recent events give us reason to hope for an ex-
tension of our therapeutic powers from this direction.

Engravings of the most important bacteria have been
provided, showing their form and the appearances pre-
sented by their growth. These are intended to serve the
reader as models of the typical forms, to which he may be
able to adhere in his own investigations.
""" The Author has endeavoured in the text to consider
the views of all the different schools, and to this end

viii BACTERIOLOGY

he has consulted other manuals and bacteriological publi-
cations. Conformably to the scope of a handbook like the
present, however, all references to the literature have been

omitted.

i

###**#

It is his hope that this manual may contribute to
preserve and promote the interest felt by practitioners
and students in the science of Bacteriology.

S. L. SCHENK.

1 The words omitted have reference to the German edition.

CONTENTS

CHAPTER I

GENERAL MORPHOLOGY AND BIOLOGY OF
MICRO-ORGANISMS

INTRODUCTORY Varieties of Micro-organisms — Bacteria — Motility of

Bacteria— Capsules of Bacteria — Multiplication of Bacteria — Pro-
ducts of Metabolism in Bacteria — Influence of Bacteria on the

Tissues Toxines, Toxalbumins, and Ptomaines— Moulds — Yeasts

— Al gag— Protozoa — Examination of Micro-organisms .

CHAPTER II

PRELIMINARY PROCESSES — APPARATUS AND REAGENTS

STERILISATION— Sterilisation by Heat— By Steam— Fractional Sterilisa-
tion Sterilisation by Steam under Pressure — Chemical Disinfect-
ants. APPARATUS AND REAGENTS — Microscope — Steam Steriliser
— Incubator — Thermo-regulator — Schenk's Thermo -regulator —
Meyer's Thermo-regulator — Gartner's— Altmann's— Petroleum In-
cubator— Miscellaneous Apparatus — Hot-water Filter — Apparatus
for Plate Cultivations— Moist Chambers— Plates— Petri's Capsules

Soyka's Plates Wire Crates — Reagents— Stains— Other Utensils

— Centrifugal Machines ...... •

CHAPTER III

NUTRIENT MATERIALS AND METHODS OF CULTIVATION

NUTRIENT MEDIA — Liquid Nutrient Media — Preparation of Meat
Bouillon — Preparation of Meat-extract Bouillon — Solutions of
White of Egg — Solid Nutrient Media — Preparation of Peptone
Bouillon Gelatine — Of Meat-extract Peptone Gelatine — Additions
to Nutrient Gelatine — Preparation of Urine Gelatine — Prepara-
tion of Nutrient Agar— Of Peptone Bouillon Agar — Modifications of

BACTERIOLOGY

PACK

Gelatine and Agar, &c.— Blood Serum — Modifications of Serum —
Eggs of Birds — Plovers' Egg Albumen — Hens' Eggs — Potatoes—
Eice, Bread, and Wafers. MODES OF CULTIVATION — Slide Cultures

Koch's Plate Process— Roll Cultures — Modifications of the Plate

Process — Plate Cultures on Serum and Plovers' Egg Albumen —
Cultivation of Anaerobic Micro-organisms— Permanent Cultures . 35

CHAPTER IV

EXAMINATION OF MICRO-ORGANISMS UNDER THE MICRO-
SCOPE AND BY EXPERIMENTS ON LIVING ANIMALS

EXAMINATION in the Fresh State— In the Hanging Drop — Staining of
Micro-organisms — Simple Staining of Cover-glass Preparations —
Preparation of Stain-Solutions—Staining of Flagella— Of Spores—
DECOLORISING AGENTS — Koch and Ehiiich Method of Staining —
Ziehl and Neelsen's Method — Ehrlich's — Giinther's — Weichsel-

baum's — Fraenkel's Gabbet's — Method of Pfuhl and Petri —

Method of Pittion — Arens's Chloroform Method — Gram's De-
colorising Method— Giinther's Modification of Gram's Process—
Weigert's Modification of Gram's Process— Impression Prepara-
tions— Examination of Micro-organisms in Sections of Tissue —
Examination by the Freezing Method — Hardening— Imbedding —
Imbedding in Gum Arabic — In Glycerine Jelly — In Celloidine — In
Parafime. ON STAINING OF SECTIONS — Unna's Drying-on Process-
Combination of Staining Methods — Kiihne's Methyl Blue Method
— Koch's Method — Loffler's — Chenzynsky's — Gram's — Kiihne's
Modification of Gram's — Kiihne's Dry Method — Weigert's Iodine
Method — Unna's Borax Methyl Blue Method — Unna's Methods of
Demonstrating the Organisms of the Skin — Noniewicz's Method.
EXPERIMENTS ON LIVING ANIMALS — Transmission of Micro-organisms
to Animals — Infection by the Air-passages — Infection by the Diges-
tive Canal — Subcutaneous Infection — Intravenous Infection — In-
fection into the Anterior Chamber of the Eye . . . . . 05

CHAPTER V

THE BACTERIOLOGICAL ANALYSIS OF AIR

MICRO-ORGANISMS IN THE AIR Simple Methods of Examining Air--

Pouchet's Method — Miquel's — Emmerich's — WTelz's — Hesse's —
Method of Strauss and WTurz— Petri's Method— Tyndall's Method

— Penicillium glaucum Brown Mould — Yeast — Micrococcus ra-

diatus — Micrococcus versicolor —Micrococcus cinabareus — Micro-
coccus flavus tardigradus — Micrococcus candicans — Micrococcus
viticulosus — Micrococcus Ureae — Micrococcus roseus — Diplococcus
citreus conglomerates — Micrococcus flavus liquefaciens and

CONTENTS XI

PAGE

Micrococcus desidens — Sarcina alba — Sarcina Candida — Sarcina
aurantiaca — Sarcina rosea — Sarcina lutea — Staphylococci —
Staphylococcus pyogenes aureus ; albus ; citreus — Streptococci — -
Bacillus subtilis— Bacillus prodigiosus — Potato Bacillus— Bacillus
mesentericus f uscus ; ruber ; vulgatus — - Bacillus liodermos —
Bacillus melochloros — Bacillus multipediculosus — Bacillus nea-
politanus — Atmospheric Spirilla 96

CHAPTER VI

THE BACTERIOLOGICAL, ANALYSIS OP WATER

MICRO-ORGANISMS OF WATER — Filtration and Filters —Variations in
Water Depending on Source— Examination of Water — Pfnhl's
Method — Kirchner's Method — Other Methods — Micrococcus aqua-
tilis — Micrococcus agilis —Micrococcus f uscus — Micrococcus luteus
— Micrococcus aurantiacus — Micrococcus fervidosus — Micrococcus
carneus — Micrococcus concentricus — Diplococcus luteus — Bacillus
tluorescens liquefaciens and Bacillus nivalis (Glacier Bacillus)
— Bacillus fluorescens non-liquefaciens — Bacillus Erythrosporus —
Bacillus arborescens— Bacillus violaceus — Bacillus gasoformans —
Bacillus phosphorescens (indigenus ; indicus) — Bacillus ramosus
— Bacillus aurantiacus — Bacillus aureus — Bacillus bruneus — Ba-
cillus aquatilis — Bacillus aquatilis sulcatus — Bacillus aquatilis
radiatus — Bacterium Ztirnianum — Bacillus membranaceus ame-
thystinus — Bacillus indigoferus — Bacillus ianthinus — Bacillus
ochraceus — Bacillus gracilis — Bacillus sulphydrogenus — Bacillus
of Asiatic Cholera and Allied Micro-organisms— Vibrio proteus —
Vibrio Metschnikoffi — Bacillus of Typhoid Fever — Bacterium Coli
commune —Spirilla in Water — Other Micro-organisms of Water . 122

CHAPTER VII

BACTERIOLOGICAL ANALYSIS OF EARTH AND OF PUTRE-
FYING SUBSTANCES

MICRO-ORGANISMS IN THE SOIL — Method of Examination — Bacillus
mycoides (Earth Bacillus) Bacterium mycoides roseum — Bacillus
radiatus — Bacillus spinosus — Bacillus liquefaciens magnus —
Bacillus scissus — Clostridium foetidum — Bacillus oedematis ma-
ligni — Bacillus of Tetanus— Streptococcus septicus — Bacillus An-
thracis — Plasmodium Malaria? — Other Micro-organisms of the
Soil. ANALYSIS OF PUTREFYING SUBSTANCES — Differences in Putre-
factive Processes — Bacillus f uscus limbatus — Proteus — Proteus
vulgaris — Zenkeri — Mirabilis — Hominis — Capsulatus — Bacillus
saprogenes— Spirillum concentricum— Spirillum rubrum . . 155

xii BACTERIOLOGY

CHAPTER VIII

MICRO-ORGANISMS IN ARTICLES OF DIET

PAGE

Methods of Examining Different Foods. EXAMINATION OF MILK —
Methods— Bacillus lacticus (Bacillus acidi lactici) — Micrococcus
acidi lactici — • Clostridium butyricum (Bacillus amylobacter) —
Micrococcus acidi lactici liquefaciens — Oidium Lactis — Bacillus
butyricus — Bacillus Butyri viscosus ; fluorescens — Spirillum tyro-
genum — Bacillus Lactis viscosus— Bacillus Lactis pituitoSi— Bacil-
lus actinobacter — Bacillus foetidus Lactis — Bacillus cyanogenus —
Bacterium Lactis erythrogenes — Sarcina rosea — Micrococcus of Bo-
vine Mastitis — Other Pathogenic Bacteria in Milk — Saccharomyces
ruber — Bacillus caucasicus (Dispora caucasica, or Kephir Bacillus).
EXAMINATION OF OTHER ARTICLES OF DIET — Bacillus megaterium —
Bacillus Aceti — Bacillus indigogenus— Pediococcus Cerevisiae —
Sarcina Cerevisiae — Micrococcus viscosus — Bacillus viscosus Cere-
visiae— Bacillus viscosus Sacchari— Moulds on Articles of Food —
Aspergillus niger; albus; glaucus ; flavescens ; fumigatus — Mucor
Mucedo ; rhizopodiformis ; corymbifer ; ramosus .... 178

CHAPTER IX

BACTERIOLOGIAL EXAMINATION OF PUS

Properties and Composition of Pus — Actinomyces — Bacillus pyocy-
aneus a and )8— Staphylococcus cereus albus ; flavus ; aureus —
Streptococcus pyogenes — Micrococcus of Gonorrhoea — Bacillus of
Syphilis — Bacillus Tuberculosis — Bacillus of Glanders — Other
Microbes of Pus 196

CHAPTER X

BACTERIOLOGICAL EXAMINATION OF THE ORGANS AND
CAVITIES OF THE BODY AND THEIR CONTENTS

MICRO-ORGANISMS OF THE LIVING BODY — I. THE SKIN — Micro-organisms
of the Skin— Methods of Examination — Diplococcus subflavus —
Micrococcus lacteus faviformis — Diplococcus albicans amplus —
Diplococcus albicans tardus — Diplococcus citreus liquefaciens —
Diplococcus flavus liquefaciens tardus — Micrococcus haematodes
— Micrococcus of Trachoma — Diplococcus of Acute Pemphigus —
Vaginal Bacillus — Bacillus of Symptomatic Anthrax — Lepra Bacil-
lus—Bacillus sycosiferus foetidus — Ascobacillus citreus— Bacillus
Xerosis — Trichophyton tonsurans — Fungus of Favus (Achorion
Schoenleinii) — Microsporon furfur. NOTE : Trichophyton micro-
sporon-Macrosporon 222

CONTENTS xiii

CHAPTER XI

THE ORGANS AND CAVITIES OF THE BODY AND THEIR
CONTENTS — (C0nt.) II. THE DIGESTIVE TRACT

PAGE

THE CAVITY OP THE MOUTH — Micro-organisms of the Mouth and their
Examination — Leptothrix — Bacillus buccalis maximus — lodococcus
— Micrococcus salivarius septicus and Bacillus salivarius septicus —
Bacillus ulna — Bacillus Gingivae — Bacillus Diphtherias — Spirillum
Miller— Spirochtete Dentium (Denticola) — Vibrio rugula— Fungus
of Thrush — Other Bacteria of the Mouth. THE TYMPANUM. THE
STOMACH — Micro-organisms of the Stomach — Sarcina Ventriculi —
Micrococcus tetragenus mobilis Ventriculi — Bacterium Lactis
aerogenes — Bacillus indicus — THE INTESTINE —Intestinal Micro-
organisms— Micrococcus aerogenes — Bacillus putrificus Coli —
Bacillus coprogenes foatidus — Bacterium Zopfi — Bacterium aero-
genes, Helicobacterium aerogenes, and Bacillus aerogenes — Bacillus
Dysenteriae — Bacillus of Fowl Cholera —Other Intestinal Bacteria . 237

CHAPTER XII

THE ORGANS AND CAVITIES OF THE BODY AND THEIR
CONTENTS — (cont.) III. THE F.ECES AND URINE

THE FAECES— Composition and Modes of Examining — Bacillus subti-
liformis— Bacillus albuminis — Bacillus cavicida — Micrococcus
tetragenus concentricus. THE URINE — Micro-organisms of the
Urine — Yeasts and Moulds in Urine — Urobacteria— Staphylococcus
Ureaa candidus — Liquefaciens— Micrococcus Ureas liquefaciens —
Bacilius Ureas Urobacillus Freudenreichii — Madoxii — Micrococ-
cus ochroleucus — Streptococcus giganteus Urethra — Bacterium
sulphureum Bacillus septicus Vesiose —Urobacillus liquefaciens . 254

CHAPTER XIII

THE ORGANS AND CAVITIES OF THE BODY AND THEIR
CONTENTS — (cont}. IV. BACTERIOLOGICAL EXAMI-
NATION OF THE RESPIRATORY TRACT, AND (v.) OF
THE BLOOD

THE NASAL SECKETION — Micro-organisms in the Nasal Secretion —
Micrococcus cumulatus tennis— Micrococcus tetragenus subnavus —
Micrococcus Nasalis— Diplococcus Coryzge— Staphylococcus cereus

aureus Bacillus fcetidus ozasnae— Bacillus striatus albus et flavus

— Bacillus of Rhinoscleroma— Bacillus capsulatus mucosus — Vibrio
Nasalis — Other Nasal Bacteria. THE KESPIRATORY PASSAGES —
Micro-organisms of the Respiratory Passages — Sarcina Pulmonum

BACTERIOLOGY

PAGE

Sarcina aurea — Diplococcus Pneumonia — Pneumobacillus

Friedlaenderi — Micrococcus tetragenus — Bacillus aureus — Tubercle
Bacillus and Actinomyces — Bacillus Tussis con vulsivae— Bacillus
pneumosepticus. V. BACTERIOLOGICAL EXAMINATION OF THE BLOOD —
Micro-organisms in the Blood — Methods of Examination — Influ-
enza Bacillus — Bacillus Endocarditis capsulatus — Bacillus of
Swine Erysipelas — Bacillus murisepticus— Spirocheete Obermeieri
— Protozoa in the Blood . . . . . . . , 264

APPENDIX (by the Translator)

A. VACCINATION AGAINST ASIATIC CHOLERA— Principle of Anti-cholera
Vaccination — Preparation of Vaccine — Results of Vaccination.
B. PARASITIC PROTOZOA — Pathogenesis in Protozoa — Coccidium
oviforme — Amceba Dysenteriae — Protozoa in Carcinoma— Protozoa
in other New Growths, &c. C. THE ACTION OF LIGHT ON MICROI-
ORGANISMS — Action of White Light — Action of Coloured Light —
Mode of Action of Light — Applications in Nature. D. AD-
DITIONAL METHODS AND FORMULAE — Fixing Methods — The Gum
Freezing Method — Staining Formulas 283

INDEX . 303

OF THB

UNIVERSITY

BACTEBIOLOGY

Errata

Page 11, line 5 from bottom, for CRENOTHI.E read CRENOTHRIX

, 236, line 4, the words NOTE BY TRANSLATOR refer to what follows, not to what
precedes.

„ 283 line 4, from bottom (note 1), for Klemperei read Klemperer.

„ 298, note 1, for quoted without reference, &c., read Pfluger's Archiv, xxx,
p. 95.

„ 302, lines 5-3 from bottom should read as follows : minutes in Loffler's or
Kiihne's methyl blue, washed, dipped for an instant in 10 per cent, tannin
solution, slightly decolorised in feebly acid water, washed in water,

iiie oungate or strict parasites
being those which grow exclusively in the living body and
perish apart from it, whereas the facultative parasites have
the power of adapting themselves to altered conditions of life,
and of flourishing externally as well as internally. With fur-
ther reference to their relation to the human or animal frame
we speak also of ectogenous micro-organisms, which occur
outside the body, of endogenous, which exist in its interior,
and of ambigenous, which are capable of life either within
or without. Again, the majority are unable to live without
oxygen, and these are termed aeroles ; but a large number

B

xiv BACTERIOLOGY

PAGE

— Sarcina aurea — Diplococcus Pneumonias — Pneumobacillus

Friedlaenderi Micrococcus tetragenus — Bacillus aureus — Tubercle

Bacillus and Actinomyces — Bacillus Tussis convulsivae — Bacillus
pneumosepticus. V. BACTERIOLOGICAL EXAMINATION OF THE BLOOD —
Micro-organisms in the Blood — Methods of Examination— Influ-
enza Bacillus — Bacillus Endocarditis capsulatus — Bacillus of
Swine Erysipelas — Bacillus murisepticus— Spirocheete Obermeieri
—Protozoa in the Blood ...... ,264

APPENDIX (by the Translator)

A. VACCINATION AGAINST ASIATIC CHOLEKA — Principle of Anti-cholera
Vaccination — Preparation of Vaccine — Kesults of Vaccination.

BACTERIOLOGY

CHAPTER I

GENERAL MORPHOLOGY AND BIOLOGY OF MICRO-ORGANISMS

Introductory.— Varieties of micro-organisms. — Within and
without the human body there exist countless organisms of
microscopic minuteness, micro-organisms, which, alighting
upon the surface, may effect an entrance in various ways
into the interior ; they belong partly to the vegetable,
partly to the animal kingdom, and have the property of
developing on animal and vegetable bodies. According
as they are capable of growth upon dead substances or
living matter, we distinguish respectively saprophytes and
parasites ; the latter of which are subdivided into obligate
and facultative parasites — the obligate or strict parasites
being those which grow exclusively in the living body and
perish apart from it, whereas the facultative parasites have
the power of adapting themselves to altered conditions of life,
and of flourishing externally as well as internally. With fur-
ther reference to their relation to the human or animal frame
we speak also of ectogenous micro-organisms, which occur
outside the body, of endogenous, which exist in its interior,
and of ambigenous, which are capable of life either within
or without. Again, the majority are unable to live without
oxygen, and these are termed aerobes ; but a large number

B

2 BACTERIOLOGY

can thrive whether .it is absent or not and are called
facultative anaerobes ; while those micro-organisms whose
growth can make no progress in the presence of the gas
are denominated obligate anaerobes.

Micro-organisms belong to the following different
classes — viz. bacteria, moulds, yeasts, alga, and protozoa.

Bacteria, schizomycetes, or fission-fungi, are colourless
cells of a glassy transparency, possessing an enveloping
membrane with protoplasmic contents but no nucleus,
and having a length which amounts in general to a few
thousandths, and a breadth of some ten-thousandths, of a
millimetre. The interior of the bacterial cell has usually a
homogeneous appearance, but sometimes shows oil-like
granules. Bacteria are distinguished according to their
form as cocci, bacilli, and spirilla.

Cocci are globular in shape, and are found either singly
or united in groups. If they lie singly they are called mono-
cocci; if grouped in masses, stapliylococci ; if the elements
are joined in pairs and fours we distinguish respectively,
according to the number, diplococci and tetracocci ; if each
eight is so united as to resemble a bale of goods, they are
named sarcince ; and if they are strung together in chains,
streptococci.

Bacilli are minute straight rods, the smallest discovered
up to the present time being the influenza bacillus. Their
ends are sometimes sharply cut across, sometimes rounded
off, and the rodlets themselves are in some cases thin, in
others stout and thick, in others again swollen in the centre,
and so forth.

Spirilla are spirally curved rods, and are subdivided into
comma-bacilli or vibrios, spirilla in the more restricted
sense, and spiroch&kz. The vibrios usually form strings of
cells which strongly resemble spirilla ; the spirochaetse are
distinguished by their flexibility.

MOTILITY— CAPSULES

Motility of bacteria. — A large number of bacteria pos-
sess the power of movement, accomplished by means of
cilia or flagella, forming spiral processes, of which some-
times one is present, sometimes several, and which keep
up an incessant vibration.

This motility must not be confounded with oscillatory
movements (also, however, to be understood as originated by

• sKTSfc

»!</•'•

FIG. 1.— FORMS OF BACTERIA. Magnified about 700 times. (After Baumgarten.)

the bacteria themselves) , nor with such motion as occurs in
inert particles suspended in a fluid medium, and which is
known as the ' molecular movement of Brown ' (Brownian
movement} .

Capsules of bacteria. — Capsules are formed by swelling
of the membrane, and are best seen in those micro-
organisms which are found in groups — viz. the diplococci,
streptococci, tetracocci, and sarcinae ; but various bacilli
also, such as Pfeiffer's capsule-bacillus and the Bacillus

B 2

4 BACTERIOLOGY

capsulatus mueosus of Fasching, are furnished with cap-
sules. By coalescence of the cell-capsules conglomerations
of cells are formed which are called zooglcea, and may
spread over the surface of fluids in the form of a pellicle.
This pellicle sometimes serves to distinguish between
micro-organisms which strongly resemble each other — for
instance, cholera bacilli form a pellicle, whereas Finkler-
Prior bacilli do not.

Multiplication of bacteria takes place by fission, hence
their name of Schizomycetcs, or ' fission-fungi.' As soon as
the individual organism has attained its normal size, there
appears in the centre a clear line which forms the sign of
division, the two individuals so produced then breaking free
from one another and forming again independent or-
ganisms. If, however, the daughter- cells do not become
disjoined, groups are formed in which the cells remain
connected in strings, in clusters (staphylococci) , in chains
(streptococci), and so on, the spiral strings formed by
the vibriones being often wrongly described as spirilla.
If the division of cocci takes place in one direction of
space, diplococci are formed ; division in two directions
yields as a result tablet-cocci (merismopedia, tetracocci) ;
while division in three directions gives packet-cocci or
sarcinse1.

A second mode in which bacteria propagate is multi-
plication by the development of spores. These are dis-
tinguished by their very remarkable power of resisting the
influences of temperature and the action of chemicals, and
are therefore called also permanent forms. The spores in
most cases occupy a position in the centre of the bacterial
cell, but in a few varieties they are at the end. Sometimes
they cause a bulging of the centre of the cell, so that the
latter becomes spindle-shaped, a form which is known as
clostridium (see fig. 1). In the cases in which they

SPORES— PRODUCTS OF METABOLISM 5

occupy one end, as in the Tetanus bacillus, the cells show
some resemblance to a drum-stick.

Formation of spores in the interior of the mother-cell
is described as proper or endogenous, while arthrogenous is
the term applied to that which takes place when single
portions separate from the cell and develop gradually into
independent individuals (arthrospores).

Bacteria require for their growth a certain amount of
moisture, many of them speedily perishing if dried.

Products of metabolism in bacteria, — The biological
properties of bacteria are, next to their morphological
peculiarities, of special importance.

A large number of micro-organisms have the property
of generating colouring matter, though not chlorophyll.
The bacteria are themselves colourless and transparent,
and the pigment is merely formed as a product of their
metabolism, especially under the influence of light.

Many bacteria throw off odorous products, and some
anaerobic micro-organisms generate very foul putrefactive
gases (ammonia, sulphuretted hydrogen, scatol, &c.) The
bacillus of Asiatic cholera exhales a pleasantly aromatic
odour, and the Bacillus procligiosus a smell resembling that
of trimethylamine.

Micro-organisms have also the property of producing
changes in the medium on which they are cultivated by the
products of their metabolism, so that albuminoid sub-
stances are peptonised by some of them and gelatine
liquefied. Many have the faculty of resolving organic
bodies into their simplest elements ; but some possess
the power of forming nitrates by the conversion of am-
monia into nitrous and nitric acids. Certain microbes
break up the chemical combination of albuminoid bodies,
causing putrefaction, while others induce fermentation ;
and a small number, again, have the property of becoming

6 BACTERIOLOGY

luminous in the dark (phosphorescence), in consequence of
the molecular activity of their protoplasm.

Influence of bacteria on the tissues. — The influence which
bacteria exert on the tissue of the bodies of men and
animals depends both on the qualities of the bacteria and
on the nature of the tissue. The action of the pathogenic
bacteria is not alike in all animals, and those which are
insusceptible to certain bacteria are said to be immune in
their relation to those particular organisms. Animals,
however, which are commonly immune towards a patho-
genic micro-organism may, under altered conditions, lose
their immunity ; for example, the frog, although usually
immune to anthrax, becomes susceptible to it at a higher
temperature.

The virulence of pathogenic bacteria may be diminished
by various factors, amongst which are included sojourn in
the body of immune animals, increased atmospheric pres-
sure, the action of higher degrees of temperature, the
influence of sunlight, &c. This weakening is dependent
on an alteration in the products of metabolism.

Toxins, toxalbumins, and ptomains, — As products of
the metabolism of those bacteria which effect an entrance
into the body substances are formed, some of which exert
violent toxic action, and which are divided into toxins and
ptomains, or cadaveric alkaloids, and to these is ascribed
the action of the pathogenic bacteria in originating morbid
processes. The toxins are further subdivided into tox-
albumins and proteins.

Toxalbumins are albuminoid bodies formed during the
growth of the bacteria upon culture-media, especially in
bouillon. Their activity depends upon certain definite
degrees of temperature, and they decompose at the boiling-
point. Our knowledge of them is due to the researches of
Eoux, Yersin, Brieger, and C. Fraenkel.

TOXINS— TOXALBUMINS— PTOMAINS 7

Proteins are albuminoid bodies which are contained in
the actual substance of the bacteria, and whose chief point
of difference from toxalbumins consists in their not being
decomposed by boiling, even if kept up for hours. They
were discovered by Nencki, and further studied by Buchner.
Proteins can be abundantly obtained by boiling pure
cultures of bacteria in their bouillon or mixed with water.
Koch's tuberculin belongs to this group of substances.

Toxins, when injected in small quantity into animals,
have the property of affording immunity from infection
with the corresponding bacterium.

According to Brieger, the cultures or juice from the
tissue should be filtered by means of earthenware cells, so
as to free the liquid from living germs. The albuminoid
substances are then precipitated with ten times the quantity
of absolute alcohol, redissolved in dilute alcohol, and pre-
cipitated a second time with an alcoholic solution of cor-
rosive sublimate. The mercury is next removed by means
of sulphuretted hydrogen, the residue dissolved in water,
and again treated with sulphuretted hydrogen ; this process
having been several times repeated, the toxins are finally
precipitated from the aqueous solution by absolute alcohol.

Scholl produced a toxopeptone from cultures of cholera-
bacilli in hens' eggs, without filtration, by the following
process : — The albumen liquefied by the bacteria was poured
into ten times its quantity of absolute alcohol, and the
precipitate washed with alcohol, digested with water, and
filtered. The aqueous solution was then repeatedly added
to ether and alcohol slightly acidulated with acetic acid,
decanted each time from the residue, and the latter re-
dissolved in water rendered alkaline, after which a final
addition to pure ether, which was then evaporated off,
resulted in the obtaining of the poisonous substance.

To procure ptomains from the actual bacterial cells

8 BACTERIOLOGY

after the method of Buchner potato cultures are prepared,
and the mass of bacteria is then scraped from them, and
rubbed up in a mortar with a little water, mixed with
fifty times its volume of a half per cent, solution of caustic
potash, and then digested in a water-bath until the greatest
possible degree of fluidity is reached, after which it is
filtered through several small filters. Dilute acetic or
hydrochloric acid is next added to the filtrate until, while
avoiding any excess of acidity, the reaction becomes dis-
tinctly acid. The protein precipitated in this way is
collected on a filter, washed, and dissolved in water which
is feebly alkaline.

Koemer procured his extracts of the Bacillus pyocyaneus
and pneumobacillus in the following manner : — The masses of
bacteria are carefully scraped from well-developed cultures
on potato and rubbed to a fine emulsion with ten times
their bulk of distilled water. The emulsion, having been
sterilised by boiling for several hours, is left for about four
weeks in the incubator, during which time it must fre-
quently be boiled for an hour or two, so that in the course
of the process it is boiled for from thirty to forty hours in
all. When the four weeks have expired the emulsion is
filtered through a tubular filter made of kaolin (Chamber-
land's candle), or through one of infusorial earth, and the
resulting filtrate is a clear brownish or yellowish fluid con-
taining albuminoid substances.

Koch obtained his tuberculin by extraction from pure
cultures of tubercle bacilli, which he grew upon a feebly
alkaline infusion of veal containing an addition of one per
cent, peptone and four to five per cent, glycerine. The
culture-vessels are inoculated by floating a fairly large
piece of the seed-culture on the surface of the fluid, and are
then kept at a temperature of 38° C. In from three to four
weeks the surface is covered with a tolerably thick mem-

KOCH'S TUBERCULIN— MOULDS

brane, dry above, and often thrown into folds, which in
two or three weeks more becomes moistened by the fluid,
and finally breaks up into ragged pieces and sinks to the

- Columella

Acicular crystals of
calcium oxalate
Sporangium

Hyphe

Mycelium

FIG. 2. — MUCOR MUCEDO. (After Baumgarten.)

bottom. The cultures (which thus require from six to
eight weeks for their growth), when fully ripe, are evapo-
rated to a tenth of their bulk in a water-bath, and filtered
through earthenware or infusorial earth.

Sterigmata with

spores
Fructification

X

Hyplie

Mycelium

FIG. 3.— ASPKRGILLUS G-LAUCUS.

Moulds. — These are for the most part saprophytes,
though pathogenic varieties are also to be found among
them. They form spores which, like those of bacteria, are
marked by the strong resistance they offer to external
influences, and which develop under favourable circum-
stances into complete individuals. They sometimes con-
tain shining drops like fat-globules.

10 BACTERIOLOGY

A tubular bud pushes out from the enveloping mem-
brane of the spore, lengthens by growing at the end, and
quickly forms a very freely-branching network of fibres,

Mycelium

FIG. 4. — PEXICILIUM GLAUCUM. Magnified 4(50 times. (After Baumgarten.)

spoken of as mycelium, and possessing special seed-bearing
organs called hyphce or thallus, from which the moulds
derive the name of HyphomyceUe. According to the form
of the seed-organ they are divided into muconneae, aspcr-
gillinece, penicilliacece, and oidiacecc.

Couidia
Hyphe

FIG. 5, — OIDIUM LACTIS. (After Baumgarten.)

In the mucorinece, or headed moulds, the ends of the
hyphae swell into knobs (columella), around which a seed-
capsule, or sporangium, forms. In this the spores develop
in such a way as to burst the enveloping epicarp membrane
when fully ripe (fig. 2).

The aspergillima (knob-moulds) have the knobbed ends

MOULDS

11

of the hyphae covered with a variable number of spore-
carriers, or sterigmata, from the extremities of which the
spores divide off in rows (fig. 3) .

The hyphae of the pcnicilliacece (pencil-moulds) are
branched, which is not the case with the mucor and asper-

G-roup of buds -
(gemmation)

Mother- cell ^ ^

- Vacuole

FIG. 6. — YEAST CELLS (Saccharomyces Cerevisice). Magnified 900 times.

gillus varieties, and on the terminal twigs of the tuft so
formed (the basidia) are seen the sterigmata, from which
the spores, or conidia, are separated off in the form of
chains (fig. 4).

Arthrosporus

- Single segments

— Common sheath sur-
rounding the sepa-
rate spores

Fia. 7.— CBEXOTHI^E KUIIMAXA. Magnified 600 times. (After Zopf.)

The oidiacea are distinguished by the fact that the
hyphse form no special spore-bearing organs, but become
articulated at their extremities, and so divide off the spores
in the form of segments.

12 BACTERIOLOGY

Yeasts. — These possess neither spore-bearing organs
nor spores, but multiply by gemmation, which consists in
the budding out of daughter- cells in different places from
the gradually enlarging mother-cell, these in their turn be-
coming mother-cells, thus forming groups of buds. The
individual yeast-cells are round or elliptical, and often
display in their interior colourless lacunae, which are not
spores, but may perhaps consist of minute drops of fat,
and are called vacuoles. The yeasts play an important
part in nature in causing fermentation. Several species
of them form pigments.

Algae, — Of these the cladothrix, crenothrix, and leg-
giatoa varieties belong to the micro-organisms. They are
jointed filaments, which multiply not by fission but by
germination at their extremities (fig. 7).

Protozoa, — Of the protozoa those important as regards
bacteriological investigation are the sporozoa, which include
the gregarince, psorospemnii, and coccidia. They are
unicellular organisms which can only live in a moist or
liquid medium, and in the absence of water, nutrient
material, or oxygen, are transformed into roundish durable
cysts. They possess a sort of larval condition, consisting
of irregular and roundish little masses of protoplasm,
which move by means of processes projecting oat like
limbs (pseudopodia), or by flagella, and often, losing their
mobility, take up a permanent residence in other cells.
The contents of the cyst separate by division or gemmation
into particles called sporocysts or pseudonavicella, the
contents of which, again, break up into a number of sickle-
shaped germs. Pfeiffer considers the plasmodia of malaria
to be also cysts of this nature containing crowds of spores.

Examination of micro-organisms, — Microscopic examina-
tion alone is not sufficient to establish fully the properties
of micro-organisms in their morphological and biological

EXAMINATION OF MICRO-ORGANISMS

13

relations. The attempt must be made to obtain pure
cultivations, which are then, on the one hand, to be sub-

Segmeutatioi
into rods

Spirilla-like

coils

Branch resembling
spirocha;ta

FIG. 8.— FOKMS OP VEGETATION OF CLADOTHRIX DICHOTOMA. (After Zopf.)

mitted to microscopic examination, and on the other
transferred to substances liable to fermentation and putre-

14 BACTERIOLOGY

faction, and used for experiments on animals. In order to
procure pure cultures, however, the instruments and
utensils employed must be freed from the micro-organisms
adhering to them, or sterilised. It seems, therefore,
advisable first to discuss the methods of sterilisation, and
then the preparation of culture-media, passing on after-
wards to microscopic examination, and, finally, the methods
of transmission to living animals.

15

CHAPTEK II

PRELIMINARY PROCESSES APPARATUS AND REAGENTS

Sterilisation is the process by which both instruments
and culture-media are freed from living germs, and is
carried out in different ways.

Sterilisation "by heat, — Articles capable of withstanding
a very high temperature are best sterilised by being held
in the flame of a Bunsen burner or spirit-lamp until a
red or white heat is reached, a method which is especially
applicable to small platinum wires or plates.

Bodies which have no great power of resistance, and
instruments which could not be exposed to so intense a
heat without impairing their efficiency and sharpness, are
subjected for a longer time to a temperature of 150° C., by
which both the micro-organisms and also their spores are
destroyed. For this purpose a box made of sheet iron,
with double walls, is best employed, which has thus a
layer of air between the walls (hot-air steriliser). It must
be put together with rivets, not with solder. By means of
a single powerful burner or a number of small ones placed
beneath, the temperature of the interior is rapidly brought
to 160°- 170°, after which half an hour suffices for sterilisa-
tion. In the top of the box are openings for two ther-
mometers, one of which extends into the interior, while
the other registers the temperature of the space between
the inner and outer walls ; and there is also a valve for

16

BACTERIOLOGY

the purpose of regulating the temperature. This arrange-
ment is suited for the sterilisation of instruments, apparatus,
glassware, &c. (fig. 9).

To prevent flasks and test-glasses from becoming re-
infecfced after sterilisation they must be closed with a plug
of cotton-wool before being placed in the steriliser, as such
a plug, while allowing the air to enter the vessels, keeps

back the organisms floating in it.
The plug can be further covered
with a cap of indiarubber.

Instead of plugging with
cotton, Staff- surgeon Schill re-
commends the use of double
test-glasses, consisting of two
test-tubes made of stout glass
and with smooth even edges, one
of which is pushed over the
other as a cover. The for-
mer should be only so much
wider as to leave a space the
thickness of a sheet of paper
between the two, and should
be but half as long as the lower.

Sterilisation by steam. — Articles which cannot be ex-
posed to so high a temperature are sterilised by the
vapour of boiling water at 100° C. For this purpose a
cylindrical vessel of sheet copper is used, measuring nearly
a metre in height and about 20 cm. in diameter, covered
with felt or asbestos to prevent loss of heat, and capable of
being closed with a lid similarly protected. The latter is
provided with an opening for the introduction of a ther-
mometer, and does not fit quite air-tight (fig. 10). The
bottom of the vessel is double, the inner bottom consisting
of a grating fixed about 30 cm. above the outer, and the

FIG. 9. — HOT-AIR STERILISER.

STERILISATION BY STEAM 17

space between the two is about half filled with water, the
height of which is observed by a gauge-tube at the side
(Koch's Steam Steriliser).

The articles to be sterilised are placed in a tin vessel
provided with a lid, and the bottom of which is also grated,
and are left in the steriliser for from half an hour to an
hour (from the moment when an abundance of steam is
given off), which suffices for complete sterilisation.

Water-gauge

King of flames — -~«r-=

FIG. 10.— KOCH'S STEAM STERILISER.

For laboratories which are not supplied with gas,
Bude?iberg's steam generator offers great advantages. In
it the disinfecting cylinder communicates through a tube
4 cm. in diameter with a flat evaporating vessel, completely
closed all round, in which water is heated to generate the
steam. The disinfecting chamber is covered with a bell-
shaped cylinder furnished with a thermometer, and serving
the purpose of condensing the steam. The water so formed
drips back into a dish, filled with distilled water before

18 BACTERIOLOGY

heat is applied, which rests upon the upper wall of the
evaporating vessel, and is connected with it by some small
apertures.

Many substances, such as nutrient materials, are, on
account of their albuminoid constituents, unable to stand
the action of a temperature of 100° C. for any length of time
without undergoing changes; gelatine, for example, loses
the power of solidifying, which alone renders it applicable
to bacteriological purposes. Hence it is advisable to expose
all culture-media to a current of steam for not more than
a quarter of an hour daily on three successive days. The
heating on the first day kills all the micro-organisms
present and most of the spores ; but some of the latter
still remain and develop by next day into micro-organisms,
which perish on heating for the second time. Any that
remain are destroyed on the third day.

Fractional sterilisation. — Certain substances, however,
particularly the serum of blood, undergo so many changes
even during a short sterilisation in the steam-current, as to
be no longer suitable for use as culture-media, and in such
cases recourse must be had to the process of discontinuous
or fractional sterilisation, introduced by Tyndall. This con-
sists of heating to a temperature of 54° to 56° C., for three
or four hours daily during one week, in a chest with double
walls between which there is a layer of water, the tem-
perature being kept at a constant height by means of a
thermo-regulator ; or, according to Heim's method, the
test-glasses can be placed in the warm water of a bath, the
temperature of which is kept continually at the height
mentioned above.

Sterilisation by steam under pressure. — High-pressure
steam, applied by means of autoclaves, acts with greater
rapidity than ordinary steam at 100° C.

Chemical disinfectants. — Besides high temperatures,

CHEMICAL DISINFECTANTS 19

various chemical substances are employed for the purpose
of sterilisation. Those possessing the greatest germicide
power of all are carbolic acid in strong solutions, corrosive
sublimate in 1 in 1,000 solutions, and quicklime; but next
to these chlorine, iodine, and bromine waters, 1 per cent,
solutions of osmic acid, 1 per cent, solutions of potassium
permanganate, oil of turpentine, iron percliloride , &c., have
a more or less energetic disinfectant action. Of the above
disinfectants, corrosive sublimate in 1 in 1,000 solution is
at present most in use.

Heider states that the efficacy of a large number of
disinfectants is very markedly increased by moderately
raising the temperature.

Chloroform is recommended by Kirchner as an excellent
disinfectant, having the advantage of great activity com-
bined with a low boiling-point, so that it can be driven off
with certainty from other fluids by heating after sterilisa-
tion is complete. It is particularly suitable for sterilising
blood serum, which cannot be exposed to a high tempera-
ture, and which, therefore, as Globig has shown, it is im-
possible to free by the method of discontinuous sterilisation
from the germs of such micro-organisms as do not grow
below 50° C., and are capable of withstanding a tempera-
ture of 70° C. To sterilise by this method the fluids under
treatment are shaken up with excess of chloroform, and
allowed to stand for some days, after which they are freed
from the chloroform before use by heating for an hour at
62° C., the boiling-point of chloroform being 61-2°.

In bacteriological work it is often necessary to com-
bine several modes of sterilisation in order to secure com-
plete destruction of germs, but the method chosen varies
continually according to the bodies to be so treated. For
the sterilisation of instruments, boiling for five minutes
in water is sufficient, according to Davidsohn ; and plates,

c 2

20 BACTERIOLOGY

at all events, may be sterilised over a gas or spirit flame
after being cleansed with alcohol and corrosive sublimate,
after which they are laid, with the heated side uppermost,
on a sheet of clean paper and merely protected with a glass
cover which has been likewise cleaned by means of alcohol
and corrosive sublimate, or even with a cleaned soup-plate.
Cleanliness of all objects coming in contact with the
bacteria is of particular importance ; and therefore it fol-
lows that in the practical application of bacteriology to
surgery there is need of the utmost care in the cleansing
of hands, instruments, and dressings, in order to render
an aseptic procedure possible. For this purpose a thorough
brushing of the hands (which have first been carefully
cleansed with soap), followed by rinsing with alcohol and
ether and washing in a -nj-th Per cent, solution of corrosive

sublimate, is absolutely necessary.

•

APPARATUS AND EE AGENTS

A microscope provided with Abbe's illuminating appa-
ratus, ordinary objectives of various powers, and an oil-
immersion lens.

The steam steriliser described above, with the corre-
sponding gas-burner and a thermometer.

Incubator. — The warm chamber or incubator consists of
a quadrangular chest of stout sheet metal with double
walls, the space between which is filled with water and has
two apertures, one for a thermometer dipping into the
water, while into the other a tliermo-r emulator is inserted.
The chest is closed above by a suitable lid, and the whole
apparatus is covered with felt, with the exception only of
the lower surface, to which heat is applied. The interior
space may be subdivided by partitions.1 A glass gauge,

1 [The incubator chiefly used in this country differs slightly from that
described in the text, as it opens at the side instead of above, and is closed

INCUBATOR

21

fixed to the outside, indicates the height of the water.
The heating of the apparatus is carried on by small gas-
flames (micro-burners), protected from draughts by cylinders
of mica. The incubator serves the purpose of keeping-
cultures of micro-organisms at a fixed uniform temperature
in cases where they will not grow at higher or lower degrees
of heat (fig. 11).

In order to secure an even temperature a thermo-
regulator is employed, which (with the very slightest varia-

Felt coating

Thermometer

- Water-gauge

FIG. 11.— INCUBATOR.

tions) maintains the thermometer at a constant temperature.1
It has the function of increasing the flame when the tempera-
ture falls, and diminishing it when the temperature rises by
regulating the supply of gas. For this purpose Bunsen took

by means of double doors, one or both of which is made of glass, in order
that the cultures, &c., in the interior may be observed without the loss of
heat which must necessarily follow the opening of the apparatus. The two
instruments are, however, identical in principle and in all other essential
details.] — TK.

1 [The terms ' room temperature,' * ordinary temperature,' &c., which
will be frequently met with in the following pages, denote a temperature of
about 20° C., while by ' incubation temperature ' is meant one of about the
heat of the human body, i.e. 37° C. (German, Zimmertemperatur and Brut-
temperatur respectively). The Incubator is usually kept at the latter tem-
perature.]— TK.

22 BACTERIOLOGY

advantage of the rise and fall of mercury, and, in case the
opening for the passage of the gas should become com-
pletely closed, he devised a safety aperture which allows
just so much gas to pass through as keeps the flame from
being extinguished. Various thermo-regulators have been
constructed on this principle.

Schenk's thermo-regulator, — A regulator in use for the
incubator in the author's Institute is constructed as fol-
lows (it can be procured from Siebert, 19 Alserstrasse,
Vienna) : A piece of glass tubing is sealed into a vessel of
glass shaped like a test-tube, in such a way that one end
of the former, which is widened out, adheres air-tight to
the sides of the latter vessel, while the other end reaches
nearly to the bottom. If now mercury be poured into this
apparatus a portion of it will sink through the narrow tube
to the bottom of the wider vessel, while the rest fills the small
tube and extends above it to such a height as to allow of the
test-tube being closed with a cork which is perforated with
one aperture. The air contained in the apparatus renders it
more sensitive by causing the mercury to rise and fall more
rapidly with the alterations in its volume produced by
variations of temperature. Into the cork is fitted a second
glass tube, which is expanded in its upper half, this ex-
panded part being closed by a cork perforated twice and
traversed by two glass tubes bent at right angles. One
of these, which extends down as far as the constriction
of the upper vessel, is ground away obliquely at the end,
and has a minute- safety aperture in one side ; the other is
simply a bent tube reaching to the lower surface of the
cork only.

In actual use the apparatus is interposed between the
supply-pipe of the gas and the burner by attaching to each
angular tube a piece of india-rubber pipe, on the one side
from the burner, on the other from the gas-tap. The test-

SCHENK'S THERMO-REGULATOR 23

tube-shaped vessel, supplied with a suitable amount of
mercury, is placed in the water surrounding the incubator.
On warming the water the column of mercury ascends so
long as the gas flows from one angular tube to the other ;
when, however, the temperature becomes so high that the
mercury reaches the longer angle tube which is obliquely
ground, and closes the end, then the limit of temperature
is fixed at which the incubator can be kept constantly.

Safety aperture

Mercury

Glass tube sealed in
Air-space

Mercury
FIG. 12. — SCHENK'S THERMO-REGULATOR.

Now, as soon as this opening is covered with mercury, the
passage of gas would necessarily cease and the flame of the
burner be extinguished, did not enough gas escape through
the lateral aperture to keep it burning. Accordingly,
the filling of the regulator with mercury must be done in
such a way that this limit is reached at the incubating
temperature. By pushing in or withdrawing the glass
vessel above the test-tube the temperature can be raised or
lowered a few degrees according to need.

If the temperature of the water in which that part of
the apparatus containing the mercury is placed rises, then

24 BACTERIOLOGY

the opening for the passage of the gas must become con-
tinually smaller, and this is followed by cooling of the
water and sinking of the mercury, so that the aperture
transmitting the gas again enlarges, and therewith the
flame increases and the temperature ascends, but cannot
pass beyond the limit for which the regulator is set. With
good management the variations are only very small.

Meyer's thermo-regulator. — Another thermo-regulator,
constructed by Victor Meyer, which is extensively used and
can be highly recommended on account of its sensitiveness,
consists also of a glass vessel like a test-tube, and which
can be closed with a rubber cork. It is furnished with a
small side-tube in the upper part, and is divided into
two sections by a capillary funnel of glass, the end of
which is just above the bottom. The lower division is
filled with mercury, the surface of which is only some three
cm. distant from the edge of the funnel, and the interspace
thus left is occupied by a mixture of alcohol and ether. In
the upper part is fitted a glass tube, cut off obliquely below,
and passing through the rubber cork; it ends a little
above the capillary funnel, and is pierced in one side above
the lower opening with a hole the size of a pin's head.

In order to graduate the regulator it is immersed in
a water-bath, the temperature of which is controlled by
an accurate thermometer ; even a slight increase of heat
volatilises the ether and drives the mercury up, so that it
comes to stand above the capillary funnel. If now the
water has reached the temperature fixed on, the obliquely-
cut glass tube is so far introduced into the mercury that
the lower opening is quite covered and only the safety
aperture at the side remains pervious. The regulator is
so connected with the flame under the incubator that this
receives only such a quantity of gas as can traverse the
regulator. When the water attains too high a temperature,

ALTMANN'S THERMOKEGULATOR 25

the ether vaporises and causes the mercury to rise, so as
more and more to close the obliquely-cut tube until only
the lateral hole remains open, and the discharge of gas is
reduced to the minimum amount. If the temperature falls,
the mixture of alcohol and ether again contracts, the mer-
cury sinks, the supply of gas increases, and the temperature
of the water rises once more.

Gartner's thermo-regulator. — Gartner has constructed a
thermo-regulator which, instead of a safety-aperture, pos-

Aperture of exib

X 1350T* J

Aperture of entrance

Regulating screw

Mercury vessel

FIG. 13.— ALTMANX'S THKU.MO-KEGULATOR.

sesses a by-road for the gas, consisting of a rubber gas-
tube, compressible by means of a screw, to prevent the
minimum flame, which must still burn even when the
regulator proper has completely shut off the gas-supply,
from proving too large if the pressure of gas increases.

Altmann's thermo-regulator. — The thermo-regulator of
Altmann is constructed on Gartner's principle, being pro-
vided with a horizontal tube which can be closed by a tap.

26

BACTERIOLOGY

On each side of this tap a lateral tube proceeds in an
oblique direction from the horizontal one, to unite at an
acute angle with a vertical tube, which contains a vessel
drawn out to a capillary termination, and filled with mer-
cury in such a way that the convex surface of the metal
begins at once to close the lumen of the angle formed by the
union of the oblique tubes. If the lower end of the regu-
lator is placed in too warm a medium the mercury expands
in the capillary, and so permits the gas to pass only
through the horizontal tube, where the supply can be still

Thermometers

Bearing of lever
Float

i !i i n

Wooden casing -| J§_ Water.Te8sel

Chimney

Rubber tube

Petroleum lamp

w

FIG. 14. — BAUJIEYER'S PETROLEUM INCUBATOR.

further reduced by means of the tap. The height of the
mercury in the perpendicular tube can be regulated by a
screw at the side.

Petroleum incubator, — If the laboratory is not provided
with a gas-supply, incubators heated by petroleum can
be employed, which are also provided with contrivances
arranged for regulating the temperature in the chamber.
Such an incubator is made of wood, and contains within it
a chest of sheet metal, to the bottom of which is fitted a
heating canal furnished with a chimney which runs per-
pendicularly up the outside wall (fig. 14). The flame of a
petroleum lamp plays into the heating canal and warms

HOT- WAI ER FILTER

27

the water circulating in six rubber pipes connected in a
water-tight manner with the metal chest. A vertical tube
is so attached to the outside that the surface of the water in
it ascends as the temperature rises, and it contains a float
furnished with a lever which presses upon a bar connected
with the lamp and made to regulate the flame of the petro-
leum. When the surface of the water sinks the bar is
lifted and the flame increased ;
when the water ascends the
flame is diminished (Bau-
meyer's apparatus).

Water-baths and sand-
baths, and such contrivances
for warming and boiling, with
suitable stands, gas-burners,
an ice-tank for refrigerating,
flasks, test-glasses, and funnels
and dishes of the most various
kinds are also included in the
equipment necessary for a
bacteriological laboratory.

Hot-water filter. — A kind
of funnel known as the hot-

Water filtering' funnel is fre-

quently in use when it is
necessary to filter in the hot state substances which become
solid at ordinary temperatures. Such a funnel is made of
copper, brass, or sheet-iron, with double walls, and fitted
with an appendage at the side which is warmed by means
of a flame ; but those hot-water funnels in which the
warming is managed by a ring burner surrounding the
lower part of the external surface are also very efficient.
By filling the space between the walls through an opening
above with water, which is then. heated, masses of gelatine

IS.— HOT- WATER FILTER (HEATED BY
A RING BURNER).

28 BACTERIOLOGY

and agar can be filtered while warm through a suitable
glass funnel inserted into the hot-water filter (fig. 15).

Apparatus for plate-cultivations, — For the purpose of
spreading nutrient gelatine upon plates a levelling apparatus
is in use which must be so arranged that the plate lies
•horizontally, in order to prevent the gelatine from easily
running off it when poured out. The apparatus consists of
a levelling- stand in the shape of a wooden triangle with
feet formed by levelling- screws, upon which rests a rather

Bell-glass

Thick plate of MiH llH^Hilk ^ --— ' Glass Plate

glass

Levelling screw
Levelling stand

FIG. 16.— LEVELLING APPARATUS FOR MAKING PLATE-CULTIVATIONS.

large glass dish filled with water and pieces of ice and
covered with a thick glass plate or a sheet of iron. The
latter having been brought into a horizontal position with
the help of a spirit-level, glass plates to receive the
gelatine can be laid upon it and protected with a bell-glass
(fig. 16).

Moist chambers are employed for the further carrying
out of the cultures } they are made of glass, and have a
diameter of about 24 cm. and a height of 6 to 7 cm. (see
p. 56).

Instead of the ordinary glass plates, which are the size
of a photographic quarter-plate, round glass dishes are
also used. Petri's capsules consist of flat double dishes of
glass, of which the lower has a diameter of 10 cm.

REAGENTS USED IN BACTERIOLOGICAL RESEARCH 29

Soyka's plates are similar to Petri's capsules, but differ
from them in having eight to ten depressions ground in the
lower plate, which resemble the ' wells ' in hollowed slides.

In addition to the above, all articles employed in
microscopic investigation are required. The slides used
for examining micro-organisms in the ' hanging drop ' have
a well ground in the centre (fig. 17) . This is covered with
a cover- glass the lower surface of which has been prepared
with the micro-organism.

Crates of galvanised wire are used for holding glass
utensils, especially test-tubes, while being sterilised (fig. 18).

Reagents, — It is scarcely possible to give a complete list

Well

FIG. 17. — HOLLOW SLIDE.

FIG. 18.— WIRE CIIATE.

of all the reagents used in bacteriological research, since
recourse must be had as much as possible, according to the
nature of the particular investigation, to the province of
the auxiliary sciences, including, of course, chemistry.
Speaking generally, the reagents used are acids, salts, disin-
fecting fluids, various oils, colouring matters, and other drugs.

Of acids, those most used are sulphuric, nitric, chromic,
acetic, and oxalic, and less frequently also dilute osmic acid.

Of alkalis and salts, solutions of caustic potash and
soda and lime water are employed, also the bicarbonates of
potassium and sodium, sodium chloride, potassium iodide,
iron perchloride, ammonium carbonate, and potash-ammonia
alum.

30 BACTERIOLOGY

Iodine is used both solid and in solution.

Chloroform is an important disinfectant, particularly for
sterilising blood-seruin.

Corrosive sublimate in solutions of 1 to 1,000 and
carbolic acid are necessary reagents for the laboratory
table.

The oils employed are aniline, cedar, and those of
bergamot and cloves, used partly for clearing the microscopic
preparations, partly as solvents.

For imbedding, a harder and a softer variety of parqffine,
and celloidine, are used.

Canada balsam, and less frequently glycerine, are em-
ployed in the preparation of permanent microscopic objects.
The latter finds, however, its most extended application in
preparing nutrient materials.

Gelatine, agar-agar, blood-serum, albumen from the eggs
of hens and of insessorial birds, potatoes, starch, paste,
milk, rice, and bread are all used for making nutrient sub-
stances. Their application will be gone into in detail in
treating of culture-media.

Stains, — The following is a list of the colouring matters
which are indispensable in making bacteriological pre-
parations : — Fuchsine, methyl blue, gentian violet, Bismarck
brown, methyl violet, malachite green, cosine, safranine, and
dahlia are aniline colours which are sufficient for nearly all
investigations. Besides these, however, carmine, picro-
carmine, picric acid, hcematoxyline, and Magdala red are re-
quired for preparations of tissues ; and for certain methods
of staining still other dyes are used, such as extract of
logwood,1 &c.

Distilled water, alcohol, ether, xylol, and oil of turpentine
complete the equipment of reagents.

Other utensils. — Platinum wires with loops sealed into

1 [Practically identical with Extractum Hamatoxyli, B. P.] — TE.

MISCELLANEOUS APPARATUS 31

glass rods — best done over a B arisen burner or with the
aid of a gas blow-pipe.

Of instruments, scalpels, scissors, forceps, different kinds
of needles, hooks, inoculation needles, and hypodermic
syringes, or better still Koch's syringe (fig. 19), are required ;
and, in the preparation of nutrient media and their use for
cultivation, flasks, test-tubes, dishes, plates, pipettes, blocks
or benches of glass, potato-knives, &c., are employed.

— Needle

Tap

Rubber ball

FIG. 19. — KOCH'S IXJKCTIXG SYRINGE.

With the instruments now specified, a laboratory is
in a position to begin bacteriological work, and it need
only be mentioned that the articles necessary for all
scientific work must also be at hand, as, for example,
working benches, cupboards for reagents, test-tube stands,
corks, meat-presses, scales, different kinds of glass and
metal vessels, bibulous paper, &c.

Centrifugal machine. — In order to examine fluids which
are poor in corpuscular elements, Stenbeck has introduced

32

BACTERIOLOGY

a centrifugal machine, or centrifuge, to be driven by hand.
This contrivance carries a metal frame or a disc with several
apertures in which are fixed metal cases for the reception
of small glass tubes. The fluid to be examined is poured

FIG. 20.— STEXBECK'S CENTRIFUGE (after Jakscb).

into the small tubes, which are provided at their lower end
with a little reservoir communicating with them through
a conical constriction, and in which the precipitate gathers
when thrown to the bottom. This centrifugal machine
has been several times modified by Von Jaksch (fig. 20).

CENTBIFUGAL MACHINES 33

Gartner's centrifuge consists of a case of sheet brass
with a movable cover. The bottom is shaped like the surface
of a very flat cone, and carries clamps for small test-tubes,
which are laid in with their mouths towards the centre,
and are so far filled with the fluid to be treated that
none flows out when the tubes are placed in the slanting
position. Six to eight samples can be centrifuged at the
same time. As soon as the glasses are in position, the
cover is lowered and fastened down by means of a bayonet-
catch. The centrifugal machine has for its axis a spindle

Cover

— - Hole for the end
of the catgut

FIG. 21.— GARTNER'S CENTRIFUGE.

revolving with very little friction in sockets in a cast-iron
frame, which serves to fasten the entire apparatus to a
table or a window-sill. A hole is perforated in the lower
part of the spindle, into which the end of a catgut string
is introduced, the string itself being wound round the shaft.
By pulling away the string the apparatus is put in rotation
after the manner of a child's top, the number of revolutions
at starting reaching over 3,000 in the minute, and the
motion keeps up with gradual slackening for ten to fifteen
minutes (fig. 21). For decanting the supernatant liquid

34 BACTERIOLOGY

Gartner uses a little contrivance consisting of a cork with
two glass tubes which is inserted into the mouth of the
test-tubes, and converts them into miniature wash-bottles.
The fluid is forced out by blowing and the sediment remains
behind on the bottom.

Less recently Csokor constructed a large centrifugal
machine marked by its fixed and perfectly even rate of
rotation. It is driven by water-power, and makes over
3,000 revolutions per minute.

35

CHAPTEK III

NUTRIENT MATERIALS AND METHODS OF CULTIVATION

Nutrient media.— In order to observe the growth of
micro-organisms, it is absolutely necessary to provide
a number of nutrient materials in which the individual
microbes may multiply into larger masses, so that their
peculiarities can be more thoroughly made out. Some of
these culture media are so prepared as to approximate more
or less closely to the natural soil of the micro-organisms, and
others in such a manner as to render them suitable for use
as general media on which the most widely differing varieties
may be cultivated. They are divided generally into liquid
and solid media.

Liquid nutrient media. — Fluid media fall rather into the
background in use compared with the solid, since the con-
ditions of growth and characteristic pecu-
liarities in the shape of the colonies come
out less strongly on them than on the
latter.

They are employed either in sterilised
test tubes closed with a plug of cotton-
wool, or in little flasks, of which those FIG. 22.

ERLENMEYEK'S FLASK.

of Erlenmeyer are particularly usetul (ng.
22). The media, especially bouillon or broth, after beinpj
distributed into such smaller vessels, must be carefully
heated in the current of steam for 15 minutes daily on
three to five successive days, in order to sterilise them. The

D 2

36 BACTERIOLOGY

inoculation and further cultivation of the micro-organisms
depends on the particular kind under observation.

Preparation of meat bouillon. — The following recipe gives
Loffler's method of preparing a liquid medium (broth or
bouillon) which has come into general use : — A half-kilogram
weight of meat freed from fat is chopped fine in a mincing
machine. (Such meat cannot, strictly speaking, be termed
free from fat, because only the masses of fat are cleared
away, whereas that which exists in the substance of all
meat is not removed.) A litre of ordinary water is poured
over the meat and the whole is allowed to stand in a cool
place for twenty-four hours. In this way the albuminoid
bodies and other substances soluble in water are dissolved
out from the meat, the result being an aqueous extract,
coloured in most cases with haemoglobin, and which is
separated from the residue by squeezing it through a cloth.
About a litre of fluid is thus obtained, which must now be
freed from albuminoid bodies by heating in a water-bath or
in the steam-steriliser. The heating must be kept up until
a sample, when filtered and boiled, no longer shows any
turbidity, which usually takes about half an hour. The
fluid is then filtered, and to the filtrate are added one per
cent, of dry colourless peptone and 0-5 per cent, common
salt, which is equivalent to 10 grams peptone and 5 grama
salt to the litre of water. After this the solution is boiled,
and, as it has a feebly acid reaction, is neutralised with a
saturated solution of sodium carbonate, until it causes
in litmus paper a alight blue coloration, which afterwards
passes into a faint red. It is not essential to add water to
make up what has been lost through evaporation, but it
will do no harm to do so if desired. The broth thus prepared
is boiled once more and filtered after boiling ; it should
not become turbid, either during sterilisation or on standing.
The filtrate must be clear, pale yellow, and of neutral

MEAT EXTRACT BOUILLON 37

or feebly alkaline reaction. Turbidities are either caused
by the reaction being strongly alkaline, and are in that case
removed when this is corrected, or are due to a finely floccu-
lent precipitate of albuminates, which are cleared away by
adding the white of a hen's egg and boiling for a quarter of
an hour [with subsequent filtration].

Preparation of meat extract bouillon, — A second mode of
making bouillon consists in the combination of meat-extract
and sugar to obtain a liquid culture medium. The fol-
lowing is Hueppe's process : — To a litre of water are
added ^ per cent (5 grams) of extract of meat and 3 per
cent. (30 grams) of dry peptone, or instead of extract of
meat and peptone, 2 to 3 per cent. (20-30 grams) of
peptone of meat. A further addition of five grams of grape
or raw sugar is then made, and the liquid boiled, carefully
neutralised with solution of sodium carbonate, and subse-
quently sterilised. Admixture of glycerine with the bouillon
is also advantageous, and tubercle bacilli grow excellently
on the medium thus obtained.

Solutions of white of egg. — Solutions of white of egg are
well suited to form fluid culture-media after they have
been completely freed from germs by the discontinuous
method of sterilisation. The white of plovers' eggs lends
itself well to this purpose, being clear and transparent, and
capable of dilution with water, and of being filtered ; it
admits also of the addition of dextrine, sugar, or other
ngredients according to need. The albumen when sterilised
affords for a considerable time a suitable medium for culti-
vations in test tubes or on glass plates placed in the
moist chamber.

Solid nutrient media. — Owing to the introduction of the
use of solid culture media in bacteriological research,
a series of micro-organisms have been more thoroughly
examined, and it has thus been possible to observe that

38 BACTERIOLOGY

certain characteristics in the growth and multiplication of
the elements in this way are more distinctly brought out and
that the individual forms are thereby specially distinguished.

Preparation of peptone bouillon gelatine. — The Koch-
Loffler peptone bouillon gelatine is that most in use, and is
prepared in the following manner : — 500 grms. of meat freed
from fat are minced up fine, mixed with a litre of water,
and allowed to stand for twenty-four hours, after which the
mass of meat is squeezed out, the filtrate boiled in a water-
bath for three quarters of an hour until all albuminoid
bodies are precipitated, and then filtered again. Another
method is to place the meat, over which a litre of water has
been poured, at once upon the fire, between the flame of
which and the vessel a plate of asbestos must be interposed.
The broth is made to boil for several hours and then let
cool, in order to separate the fat, after which it is filtered
and sufficient water poured in to replace that lost by
evaporation. 100 grms. gelatine, 10 grins, colourless pep-
tone, and 5 grms. common salt are next added to the
filtrate, and this mixture is allowed to stand for some time
and then heated in the water-bath until all the gelatine is
dissolved. In order that the gelatine may be colourless,
the solution must not become concentrated while being
heated in the water-bath, but from the very commencement
as much water must be added, from time to time, as it ap-
pears to have lost by evaporation. The reaction of gelatine
is always acid, and this is also the case with broth, so that
it must be neutralised with a concentrated solution of
sodium carbonate or with solution of caustic soda.

The entire mass is next filtered through a creased filter
paper in the hot-water funnel.1 The creased paper must
be moistened with warm water before filtering, as otherwise

1 [The paper is not folded in the manner usually adopted, but creased in
folds radiating from the centre, somewhat like a circular fan ; the piece

NUTRIENT GELATINE 39

the pores would be clogged by solidification of the gelatine.
To avoid using the hot-water funnel, Kirchner suggests
allowing the gelatine to cool slowly in the steam steriliser,
after turning out the flame ; it is then after a few hours
perfectly clear and can be filtered with facility. Instead
of the folded paper, a thin layer of cotton- or glass-wool may
be used for filtering.

If a sample of the filtrate is taken in a test-tube and
heated until it boils, it must remain clear and should also
not become cloudy while cooling. Any turbidity, if such
occurs, may possibly be due to the gelatine having been
rendered too strongly alkaline in neutralising ; as in
heating such a solution the carbonic acid is driven off,
and then compounds are thrown down which cause the
turbidity. It need hardly be said that care must be
taken in such cases to neutralise exactly in order to
get an efficient gelatine. That perhaps other faults, such
as inferior paper, dirty vessels, &c., may be to blame, is
also evident, and such must be avoided in the preparation
of nutrient media. Cloudiness is most easily dealt with by
adding the white of a hen's egg to the lukewarm gelatine
while it is still fluid, and shaking so as to divide it finely,
after which the solution is again boiled and filtered in the
hot-water filter. Indeed it is the rule to add the white of
an egg to nutrient gelatine immediately after neutralising,
so as to ensure the avoidance of all faults of turbidity.

The gelatine when ready should be clear and of an
amber-yellow colour, and should not become cloudy on
heating. Carefully cleaned test-tubes are filled with about
10 cubic centimetres each and plugged with cotton wool, or
Schill's double test-glasses may be used (see p. 16). The

should be about eighteen inches square, and folding is begun by doubling it
down the centre. The creased paper is finally gathered up, inserted into
the funnel, and the superflous part cut off.] — TB.

40 BACTERIOLOGY

test-tubes are sterilised before filling, by placing them one
over the other in a wire crate lying on its side, in. which
they are introduced into the hot-air steriliser and exposed
for an hour to a temperature of 100° C. [The cotton- wool
plugs should be inserted before sterilising.]

The gelatine is introduced into the test-tubes with the
aid of a pipette, care being taken that it does not soil the
edge of the tube, and least of all comes in contact with that
part of the inner surface which supports the cotton plug.
In this manipulation the plug must be seized on the dorsal
surface of the hand between two fingers, and extracted from
the tube with a twisting motion ; the pipette is then filled
and closed with the forefinger, which is only raised when
the gelatine is to be allowed to run out into the tube.
After this procedure has been repeated a few times, each
worker in his owh way acquires such a degree of expert-
ness, that the greatest possible celerity is attained in filling
the tubes with gelatine and quickly reclosing them.

Instead of the pipette, the use of which always demands
a certain amount of skill, small glass funnels may aptly be
employed, or a glass tube capable of being closed by a tap
may be attached to the funnel through which the gelatine
is filtered and introduced into the test-tube to be filled.

It is particularly to be observed that the gelatine
must not be kept continuously at a high temperature, lest
it should lose its power of solidifying when cold. It
must, therefore, be heated in the steam apparatus for
fifteen minutes on several — about three to five — days in
succession, in order that the culture-medium preserved in
the test-tubes may be completely sterile, and capable of
being stored for future use in all bacteriological experi-
ments.

Preparation of meat extract peptone gelatine. — Hueppe's
meat-extract peptone gelatine is a 10 per cent, solution of

MEAT EXTRACT PEPTONE GELATINE 41

gelatine, to which 5 grm. extract of meat, 5 grm. grape-
sugar, and 30 grm. peptone have been added. As soon
as the gelatine is dissolved in water the other ingre-
dients are mixed in and the whole boiled, after which the
solution is filtered off by means of the hot-water funnel
and neutralised. Should clouding by any chance occur,
recourse must be had to those measures described in speak-
ing of the preparation of peptone bouillon gelatine. When
this has been sterilised — which must be done with par-
ticular care, as the extract of meat contains many germs —
a culture-medium is obtained which can in most cases be
used exactly like the preceding. The finished gelatine is
stained a brownish colour, owing to pigments derived from
the meat extract.

Both these modes of preparation yield gelatines which
are most extensively used in all bacteriological researches.
Attempts, are now made to alter the culture-media by
adding various substances to the gelatine, such as grape-
sugar (up to 2 per cent.) and dextrine or glycerine (4 to 6
per cent.). By means of these different admixtures, the
nutritive value of the gelatine for certain micro-organisms
is said to be increased.

In summer the gelatine has a tendency to liquefy, and
its strength must accordingly be increased from 10 to 15
per cent. ; while for cultivating anaerobes a 7J per cent,
gelatine is required.

Additions to nutrient gelatine. — A modification deserving
of special notice is litmus gelatine, which is prepared by
mixing a tolerably concentrated solution of blue litmus
with the gelatine, thus obtaining the substance to which
this name is given. Its importance lies in the fact that the
acids or alkalies formed by micro-organisms in their growth
can thus be qualitatively demonstrated.

It is advisable to add the most widely different sub-

42 BACTERIOLOGY

stances to the gelatine, so as to meet individual require-
ments in cultivating micro-organisms, and accordingly the
greatest multiplicity of admixtures have been recommended
by approved investigators. For example, Koch uses mixtures
of gelatine with blood-serum, aqueous humour, infusion of
hay and of ivheat, decoction of horses' dung, and decoction of
plums.

Miquel uses, instead of meat bouillon, a solution of 40
parts peptone, 10 parts common salt, and 1 part carbonate
of potash in 1,000 parts of water, to which the further addi-
tion of 4 parts of gelatine can be made.

Holtz has devised a potato-gelatine, for use in growing
typhoid bacilli. The potatoes are grated and squeezed
through a straining-cloth, the liquid which flows away is
allowed to stand for twenty-four hours, and is then boiled
with 10 per cent, of gelatine.

Preparation of urine gelatine. — A very cheap and easily-
prepared gelatine is the urine gelatine recommended by
Heller. Urine is caught in sterilised vessels, and its
specific gravity having been brought to 1010 by dilution
with sterilised water, it is rendered feebly alkaline with soda
solution and filtered. After 1 per cent, of peptone, ^ per
cent, of common salt, and 5 to 10 per cent, of gelatine have
been added, it is next boiled and filtered, and the fluid so
obtained is poured into test-glasses and sterilised, a single
sterilisation being said to be sufficient. This process can
be modified by filtering the urine through animal charcoal
before diluting it with water, in order to remove part of the
urinary colouring matter.

Preparation of nutrient agar. — Agar-agar is a vegetable
jelly procured from different algse growing in the East
Indies and Japan, and was introduced into bacteriology by
Hesse because of its distinctive property of remaining in
the solid state at 40° C., and only melting completely at

NUTRIENT AGAR-AGAR 43

90° C. Hence this jelly is well adapted for use as a culture-
medium for those micro-organisms which must be grown
at the higher temperatures in the incubating chamber.
Agar-agar appears commercially in the form of transparent
strips, or four-cornered pieces, or as a white powder, and
swells up in water.

Preparation of peptone bouillon agar. — To make nutrient
agar (peptone broth agar), 500 grm. of meat free from fat
are taken, minced up, and mixed with a litre of water, and
after standing for twenty-four hours in a cool place the
liquid is filtered through a cloth and squeezed out from the
mass of meat. The albuminoid bodies are precipitated by
boiling the meat infusion, and are removed from the liquid
by filtration, the result being ordinary nutrient bouillon.
This is rendered feebly alkaline with sodium bicarbonate,
and mixed with 10 grm. peptone, 5 grm. common salt, and
20 grm. agar-agar cut up small. The agar swells up in the
broth, and is then boiled over a sand-bath, in the steam
steriliser, or even over the naked flame, until only small
flakes and slight turbidities are observed, the fluid lost by
evaporation being then made up by the addition of water.
It is seldom necessary to neutralise the fluid a second time
with sodium bicarbonate, as the agar has of itself a
neutral reaction, so that it only remains to filter the solu-
tion, though this is in some cases difficult to do suc-
cessfully. The filtration is carried on through a double
layer of filter-paper, by means of the hot-water funnel, or
in the steam steriliser, and is a very slow process. After
addition of white of egg it sometimes happens that when
the agar mass has been boiled the small particles are
gathered into lumps by the albumen, and the filtration is
much easier in consequence.

Some recommend that the hot agar should be allowed
to cool gradually in a tall cylindical vessel placed in the

44 BACTERIOLOGY

steam steriliser, by which means the turbidities sink to the
bottom, so that the solidfied mass of agar is perfectly clear
in the upper part. This mass is removed from the glass
by slightly warming it, the clear part is separated from the
turbid by cutting it with a knife, and is then chopped up
and re-melted.

To simplify the preparation of nutrient agar it is
advisable to dissolve the finely-cut pieces of agar in boiling
water over the open fire, a sheet of asbestos being inter-
posed between the flame and the vessel ; this takes from
half to three-quarters of an hour, and the agar should not
be mixed with the broth until solution is complete. In this
case also it is better not to heat the liquid too long, as the
medium becomes dark if allowed to boil away, but from the
first to add as much more water from time to time as might
be lost through evaporation. In order to secure a clear
solution, the pieces of agar may be first of all laid in
2 per cent, hydrochloric or 5 per cent, acetic acid, which
is afterwards washed away with water. By Kichter's
method the agar is dissolved in wine by two hours' mace-
ration and subsequent boiling, and the solution is added to
bouillon.

Tischutkin allows the required quantity of agar to swell
for 15 minutes in a very dilute solution of acetic acid, washes
it in pure water, and thereupon adds it to the broth, in
which it dissolves after only 3 to 5 minutes' boiling. When
it has been neutralised and cooled, the whites of two hen's
eggs are poured in, and the mixture kept in the steam
apparatus for half to three-quarters of an hour. The sub-
sequent filtration occupies only a short time, even without
the hot-water funnel.

The agar when ready is filled into sterilised test-tubes
closed with cotton-wool, in such a way that about a third
of the test-tube is occupied by the fluid. Care must next

PEPTONE BOUILLON AGAR 45

be taken that the nutrient mass be sterilised, which is done
by heating the filled test-tubes in the steam steriliser for
twenty minutes daily on three successive days.

The tubes when filled and sterilised are laid in a slanting
position, for which purpose suitable contrivances of various
kinds are employed, the result being that the surface of the
agar after setting forms a very acute angle with the long
axis of the tube. During solidification some water, the
water of condensation, separates out and prevents the firm
adherence of the agar to the vessel, so that the medium
often turns if the test-tube is rotated, a phenomenon which
does not disappear until the water of condensation has
evaporated. Esmarch recommends the addition of some
gum arable to prevent slipping away from the surface of
the glass.

The mass of agar is clear and transparent while liquid,
bat after solidifying is somewhat cloudy and opaque.

Nutrient agar is often prepared by adding 20 grm. agar,
5 grm. extract of meat, 5 grm. grape-sugar, and 30 grm.
peptone to a litre of water. Sterilisation must be carried
out with greater care than in the case of ordinary nutrient
agar, and the prepared medium is of a brownish-yellow
colour.

The most varied modifications of nutrient agar can be
produced by the addition of solution of litmus, grape-
sugar, and other substances soluble in water. That most
commonly used is the litmus agar mass prepared by adding
40 ccm. solution of litmus to a litre of prepared agar. This
medium is par excellence of service in the carrying on of
researches on the formation of acids or alkalies during the
growth of micro-organisms.

Modifications of gelatine and agar, &c. — Glycerine agar
is often made use of, as several micro-organisms grow very
readily on this medium, which consists of nutrient agar

46 BACTERIOLOGY

modified by the admixture of about 6 per cent, of pure
neutral glycerine. It has lately been shown by Nocard
and Roux that tubercle bacilli and the bacilli of glanders
flourish very freely on a nutrient medium so made.

Kowalski prepares both nutrient gelatine and nutrient
agar in the following way : — Instead of meat, a kilogram of
calf's lung is used in the preparation of the broth. It is
cut up, two litres of water poured over it, the fluid allowed
to stand in a cool room for some time, and squeezed out
after boiling. To the filtrate so obtained are added 25
grm. peptone, 90 grm. sugar, 18 grm. common salt, 9 grm.
sodium phosphate, 9 grm. ammonium sulphate, and 25
grm. sodium sulphate, and as soon as these ingredients
are all dissolved a further addition of 10 to 15 per cent,
of gelatine or 2 per cent, of agar-agar is made, and the
whole is boiled with continual stirring. After the mass
has cooled, but before it has become viscous, the whites of
five hen's eggs are mixed in and divided up in the fluid,
and when it has been boiled once more until all the
albumen is coagulated, 8 to 10 per cent, of glycerine is
added. This culture-medium is, when clear, of a straw-
yellow colour. It is distributed into test-tubes with a
pipette, and sterilised in the steam apparatus for ten
minutes on three days in succession. Kowalski has de-
scribed very favourable results obtained with it.

The method given by Heller for the preparation of
urine gelatine can also be used to produce urine agar ; the
process already given is followed, 1 to 2 per cent, of agar
being added instead of the gelatine.

Gelatine as well as agar media may be prepared with
milk or caseine, after the method of Marie Raskin, and
peptone can be used as an addition, as also albuminate of
soda or potash.

Miquel has described a nutrient jelly which he pre-

BLOOD SERUM 47

pares from an Irish moss (Carragheen, Fucus crispus) by
boiling 300 to 400 grms. of it with 10 litres of water, and
filtering, the filtrate being then evaporated and dried at
40° to 45° C. By the addition of 1 per cent, of this extract
to broth, a solid nutrient medium is obtained, which only
begins to liquefy at 50° C.

Besides the modifications of nutrient broth, gelatine,
and agar, just mentioned, numerous other alterations and
additions have been proposed, the application of which is,
however, very limited.

Blood serum. — The use of blood- serum as a nutrient
medium was introduced into the practice of bacteriology
by Koch. The blood from a punctured or incised wound is
allowed to flow into a sterilised tall glass cylinder, which is
then placed in an ice-tank, and allowed to stand undisturbed
for forty-eight hours. In this way the serum, which should
be of a yellow or pale red colour, separates out, and it is
then poured with the aid of a sterilised pipette into sterilised
test-tubes plugged with cotton-wool, so that there are about
10 ccm. of serum in each tube. The liquid serum is exposed
to a temperature of 56° two hours daily for a week, and freed
from germs by this fractional sterilisation. In order, how-
ever, to kill those micro-organisms also which grow at a
higher temperature, it is next shaken with chloroform in
excess, and allowed to stand for a few days, the chloroform
being removed by heating before use. The test-tubes are
then laid on a slanting surface and the serum made to set
at a temperature of 70° C. When solid, it should have a
jelly-like consistence and a yellowish colour, and should
adhere in its whole extent to the test-tube as a transparent
mass. Koch has devised a special apparatus for the inspis-
sation of blood serum, in which the water is carefully heated
for about half an hour to 68°-70° C. (fig. 23). It becomes
opaque at higher temperatures. Before use it must be

48 BACTERIOLOGY

ascertained whether the serum is sterile, which is most
readily done by covering the test-tubes with india-rubber
caps and letting them stand fora few days in the incubator.
Only those test-tubes should be used in which no germs of
micro-organisms have visibly developed.

The serum of human blood is often employed, and can
be obtained sometimes at operations and sometimes from
placentae.

FIG. 23.— APPARATUS FOR THE INSPIRATION OF BLOOD SERUM.

Modifications of serum. — Those fluids which are pro-
cured from hydroceles, ovarian cysts, or dropsical effusions,
are very nearly akin to human blood-serum, and the
process for manufacturing nutrient media from them is
similar.

Loffler modified the blood-serum as follows : — Having
freed an aqueous extract of meat from albuminoid bodies,
he added 1 per cent, of peptone, 1 per cent, of grape-sugar,
and 0-5 per cent, of common salt to it. This solution,
which has an acid reaction, is neutralised with sodium
bicarbonate, then sterilised in the steam apparatus, and,
after cooling, mixed with liquid blood serum, in the proper-

BIRDS' EGGS 49

tion of one part broth to three parts serum. Test-tubes
having been filled with the mixture and sterilised by the
discontinuous method, the mass is inspissated at 70° C.

Admixture of 6 to 8 per cent, of glycerine is recom-
mended, and Hiippe also makes serum gelatine by adding a
concentrated gelatine solution, or serum agar, with a 2 per
cent, solution of agar, in the proportion of two to three
parts of either to one part of serum ; the resulting nutrient
media being freed from germs by fractional sterilisation.
These have recently been a good deal used.

Eggs of birds, — It is well known that the white of hens'
eggs, when mixed with a concentrated solution of potash
and poured from one vessel to another, coagulates to a rather
firm jelly, which is known as Lieberkuhn's_poto/i albuminate.
Taking advantage of this process, Tarchanoff and Koles-
nikoff have accordingly employed as a nutrient medium an
alkaline albuminate prepared in the following way: — Hens'
eggs are laid without being denuded of their shells in a 5 to
10 per cent, solution of potash for about fourteen days. In
this way the white becomes firm like gelatine, probably
owing to a combination between the albumen and potash
taking place through the pores of the calcareous shell and
the membrane more slowly than is the case in preparing
Lieberkuhn's potash albuminate. If this pale yellowish
jelly-like mass of albumen is cut into fine slices, and the
strong alkalinity got rid of by washing, a very useful nutrient
medium is obtained. It need hardly be said that thorough
attention must be paid to sterilisation, which can be done
in the steam steriliser.

Plovers' egg albumen. — A convenient albuminous nutrient
material, which commends itself on account of its great
transparency and colourlessness, is the white from the eggs
of insessorial birds up to the time when the embryo has
developed its vascular area. This medium was introduced

50 BACTERIOLOGY

and has been frequently used in the author's Institute.
We employ for this purpose the white of plovers' eggs, as
these are easily bought in the spring time. When a plover's
egg is opened after washing with corrosive sublimate solu-
tion, there is found round the vitelline membrane a con-
densed mass of albumen, outside of which the white is
clearer and less dense. If this outer white is distributed
into narrow test-tubes, and these laid slanting and subjected
to a temperature sufficient to coagulate the albumen, a clear
gelatinous transparent mass is obtained, which can be used
for the most widely-differing cultures : for example, even
gonococci will grow upon it, as Von Schrotter and F. Winkler
have shown in the author's Institute, and pigment -form-
ing micro-organisms develop particularly well upon this
medium.

Admixtures of various other substances — grape-sugar,
dextrine, paste, and in fact all bodies soluble in water, and
which have not an acid reaction — can be made with it, so
as to modify it in various ways. The medium can also be
prepared by diluting the concentrated albuminous mass
with water but in that case the white of egg must first be
filtered.

Although investigations show that no micro-organisms
can be detected in the normal unfecundated plover's egg, it
is nevertheless advisable to subject the test-tubes when
filled to a fractional sterilisation before use.

Plover's egg albumen is also applicable to plate cultures,
but after careful aterilisation it must be dried on a sterile
glass plate over sulphuric acid in the receiver (also steri-
lised) of an air-pump, and then kept in the moist chamber
after inoculation. The preparation of plates may, however,
be facilitated by mixing the albumen with gelatine or agar.

Hens' eggs. — According to Hiippe and Heim, hen's eggs
may themselves be used with advantage as nutrient media.

POTATOES 51

Fresh eggs are cleansed with soda solution, washed, and
laid in corrosive sublimate, which must be removed with
ammonium sulphide, spirit, and ether before they are
inoculated. The latter is done by piercing the apex with a
needle which has been sterilised at a glowing heat, and
introducing the seed material into the interior by means
of a glass capillary tube, from which it is carefully blown
out as the tube is withdrawn. Closure is then effected
with sterilised cotton- wool or collodion. This method is
particularly well adapted for the cultivation of anaerobes.

Potatoes.— An excellent culture material for the different

t/V> o>

micro-organisms, and one, moreover, which secures their
development in a quite characteristic way, is the potalbCr,
This medium is prepared as follows, according to Koch's
method : — The potatoes are carefully cleaned in water with
a brush, and after being freed from dirt are laid for half an
hour in a 1 per 1,000 solution of corrosive sublimate to
which some -J per cent, hydrochloric acid has been added,
and finally washed with water to remove the adherent sub-
limate. With the aid of a sterilised knife single specks of
dirt and ' eyes ' are removed from the surface of the potatoes,
which are next heated for about an hour in the steam
apparatus and then cut into halves with a sterilised knife,
when the free cut surface of each of the halves can be
utilised for the culture of micro-organisms. If it is desired
to ascertain whether the potatoes are perfectly sterilised,
several of those treated as above can be left in moist
chambers and watched to see whether micro-organisms
develope, in which case the process of sterilising in the
steam apparatus must be repeated.

If the potatoes after being washed in water, disinfected
in corrosive sublimate, and carefully cleaned on the surface,
are cut into several rather thick discs which fit into small
sterilised boxes of glass, each individual disc can be used as

E 2

52 BACTERIOLOGY

a medium upon which to cultivate (Esmarcli's potato discs} ;
but they must be separately sterilised on from three to
five successive days in the steam steriliser. They may
also be kept stored in several glass dishes standing one
above the other, and which mutually cover and close each
other.

In order to use potatoes in a transparent form, thin
slices can, after Wood's method, be cut from very white
potatoes, and firmly pressed upon sterilised slips of glass,
which are introduced into test-glasses and then sterilised.

Instead of discs, cylinders may be punched with the
aid of a cork-borer out of cleaned and peeled potatoes.
Each cylinder can be split into two halves in its long axis,
and each half placed in a test-tube closed with cotton-wool.
After these test-tubes have been sterilised in the steam
apparatus for three days in succession, the surface of the
piece of potato is inoculated. It is advisable to support
the potato cylinders on cotton-wool or small glass tubes,
which can absorb the water of condensation.

Potatoes are often pounded up after being peeled and
boiled, and are then pressed into little Erlenmeyer's flasks
(potato pap) ; and in this way a useful culture medium is
obtained after proper sterilisation. Eisenberg has modified
the process by using, instead of flasks, small boxes capable
of being closed with a glass lid, and by sealing these with
paraffin for permanent cultures.

Nutrient materials made from potatoes possess a
slightly acid reaction, so that the surface of the potato
mast be rendered alkaline with sodium bicarbonate solu-
tion for certain micro-organisms, whose growth demands
alkalinity.

Bice, bread, and wafers, — Milk rice is prepared by
mixing skim-milk with two and a half times its quantity of
powdered rice. This mixture is boiled until a thick pap is

MODES OF CULTIVATION 53

obtained, which is slightly cooled and introduced into a
cork-borer so that no air-spaces remain. The pap is then
pushed out with a rammer and divided with a platinum
wire into separate discs, which are placed in glass boxes,
moistened with a few drops of milk, and sterilised,
and then serve as a nutrient medium on which the most
various micro-organisms grow in a characteristic manner,
particularly those which form pigments.

The milk rice was modified by Eisenberg in the following
way:— 100 grm. rice powder, 70 grm. bouillon, and 210
grm. milk are rubbed up and introduced into glass boxes and
the mass heated on the water-bath until it solidifies. The
boxes having been closed and heated on three days in suc-
cession in the steam steriliser, a medium of the colour of
cafe au lait is obtained, on the smooth surface of which
the micro-organisms can be inoculated.

Bread pap is prepared in the following manner : — Bread
is dried until it is fairly free from water, and is then crumbled
to powder and spread over the bottom of a flask so as to
cover it. By adding water a pap is formed which after
boiling yields without being neutralised an excellent medium
for the cultivation of moulds, the Aspergillus niger developing
particularly well upon it. After neutralisation with sodium
carbonate the pap forms a nutrient medium for different
bacteria when it has been several times sterilised in the
steam apparatus.

Wafers, especially the thicker ones, are, according to
Schill, to be chosen for chromogenic bacteria. They are
moistened with bouillon, laid in glass boxes, and sterilised.

MODES OF CULTIVATION

Slide cultures. — Microscopic slides covered with a layer
of gelatine have repeatedly been used to catch the micro-

54 BACTERIOLOGY

organisms deposited from the air in different places, or have
had a linear inoculation made on the surface. These
slide cultures are less in use at the present time, since,
in order to isolate the germs, a large surface must be given
to the nutrient medium, and consequently glass plates are
employed. Moreover, Koch has devised an exceedingly
ingenious method known under the name of the plate
process.

Koch's plate process. — Gelatine is stored in sterilised
test-tubes, plugged with cotton, to the amount of about
10 c. cm. in each, and is completely sterilised by the
fractional method. A pure culture, or a mixture of many
micro-organisms in a mass of any desired size having been
obtained, the gelatine in three test-tubes is liquefied in the
water-bath at a temperature of 35° C., and a small quantity
of the mass to be examined is taken on a platinum needle
(previously sterilised at a red heat) and introduced into the
first test-tube. The platinum needle may be bent round
into a circular loop at the end or have its point somewhat
flattened out. If the seed mass is rather too coherent,
attempts must be made to separate the micro-organisms by
rubbing them with the point of the needle against the side
of the test-tube below the surface of the gelatine. The
platinum needle having been again heated, three samples
are transferred with it from the first tube to the second,
and the same procedure is repeated with the second and
third, so that we have three inoculations, of which the third
is the most diluted. When inoculating care must be taken
in opening the tube to seize the plug of cotton-wool between
the fingers, best between the third and fourth, on the back
of the hand, and thus twist it out of the tube, which must
again be carefully closed after inoculation without allowing
the cotton plug to come in contact with the surface of the
hand or with any instrument.

THE PLATE PROCESS 55

During these operations some plates should be under-
going sterilisation in the hot-air steriliser at a temperature
of 130° C. A box of sheet iron may be used with advantage
for this purpose, and is, moreover, capable of containing
a larger number of plates (fig. 24). After cooling, three
plates, which must only be seized by their corners, are next
taken out of the box and laid one after the other on Koch's
plate-making apparatus, on which they are cooled under a
bell-glass. In the absence of a hot-air steriliser the plates
may be sterilised in the interior of an oven, or in the gas
or spirit flame by holding them by the corners in the fingers
and heating both sides over the flame.

Lid

Case ~

Fu. 24.— CASE QF SHEET-IRON FOR HOLDING THE PIATES.

The plate apparatus (see p. 27) consists of a triangle with
feet formed by levelling screws, on which rests a glass vessel
covered with a thick plate of glass and filled before use with
iced water. It is rendered horizontal with the aid of a
spirit-level and covered with a bell-glass. The sterilised
plates having cooled under this bell, the first of the inocu-
lated test-tubes is then unplugged, its upper edge is heated
in order to sterilise that part over which the gelatine has to
flow, and its contents are poured from a small height upon
the plate in such a manner that the gelatine spreads out
over it in a fairly thin layer. In a short time the gelatine
sets under the bell-glass, and the plate is then brought into
a moist chamber and laid upon either little glass benches

56 BACTERIOLOGY

or pieces of glass which have been sterilised. The same
process is gone through with the second and third inocula-
tions, and the three plates can be laid on benches one above
the other in a single moist chamber, or a separate one
appropriated to each of them.

The moist chamber consists of a large glass box, which
is disinfected with corrosive sublimate solution and has a
circular piece of blotting-paper moistened with a 1 per 1,000
solution of the same substance laid upon the bottom (fig. 25).

The plates prepared as above are left at the temperature
of an ordinary room until the individual cultures show them-
selves on the surface. These appear in the form of islets

Gelatine plate

Glass bench
FIG. 25. — MOIST CHAMBER.

either lying close together or isolated. Sometimes several
run into one another, and at times a dotted mass appears
on the plate, not unusually in the form of little clouds, all
of which vary in figure according to the kind of micro-
organism. Under a moderately high power of the microscope
the colonies are seen to be sharply defined, and sometimes
granular, sometimes fibrous, according to the manner in
which the micro-organisms are arranged in relation to one
another. If the microbes under observation are pigmented,
the individual colonies will appear of various tints, or the
colour may be diffused through the gelatine, and phosphores-
cence or fluorescence may be seen in single spots. By
comparison of all these peculiarities it is possible to isolate

ISOLATION OF MICEO-OEGAHISMS

57

microorganisms and to identify them, and a further point
is that some microbes liquefy the solid gelatine, while
others leave its consistence unchanged (fig. 26) .

The colonies of micro-organisms are best isolated in
the most diluted of the three cultures.

If it is wished to obtain further cultivations from such
a plate, the following is the course adopted : — A sample is
taken from a colony with the point of a platinum needle
fused into a glass rod (the needle being first sterilised at

FIG. 26. — GELATINE PLATE, SHOWING COLONIES OF VARIOUS FORMS.

red heat), or the whole colony is lifted on the point. In
either case a thrust is made into the sterilised gelatine in a
test-tube, or a streak is drawn over the oblique surface
of the solid agar-mass, or sterilised potatoes are infected.
Such a transference to different nutrient media enables
us to note all peculiarities in growth, and hence to gain ah
inkling of the class in which the micro-organism under con-
sideration is to be included. This procedure is carried
out under a low power, and is designated ' fishing.' It

58 BACTERIOLOGY

requires a certain amount of skill, and therefore Unna,
Fodor, and others have quite recently described contri-
vances for facilitating the operation.

Roll cultures, — A modification of the plate process which
is known as roll culture has been invented by Von Esmarch,
in which the gelatine, after being liquefied and inoculated,
is kept rolling round the sides of a wide test-tube until it
sets.

Eoll cultures are prepared by inoculating the tube in
the usual way, closing it with a cotton-wool plug which has
first been singed in the flame, and drawing over the cotton
an india-rubber cap sterilised in solution of corrosive subli-
mate. The tube is then seized by its upper end with three
fingers of the right hand and by its lower with three fingers
of the left, laid horizontally in a vessel of iced water, and
kept turning on its long axis until the gelatine has set in a
layer of even thickness. The roll cultures when finished
must at once be conveyed into a cool place.

Modifications of the plate process. — A quicker and more
convenient procedure is the introduction of portions of the
gelatine into Petri's capsules. In using Soyka's plates a
small quantity of liquefied gelatine is deposited in each
hollow, and prepared with the seed-material by means of a
platinum needle. By transferring the material from one
hollow to another, it is possible to have all degrees of
attenuation on the same plate.

In order to economise gelatine Giinther places on a
sterilised surface of glass a few drops of sterilised water or
bouillon, lying isolated from one another. A sample of the
material to be examined is mixed with the first drop by
means of the platinum wire, and by the same means the
inoculating matter is transferred to the remaining drops one
after the other, the needle being continually sterilised at
a red heat after each inoculation. From the last drop a

MODIFICATIONS OF THE PLATE PROCESS 59

loopful is conveyed into a test-tube of liquefied gelatine,
which is then poured out into a Petri's capsule.

Agar can be used instead of gelatine for the plate pro-
cess, but this medium requires greater care in preparing
the cultivations than is the case with gelatine. Agar be-
comes fluid at 90° C., and passes into the solid state at 40° C.,
hence the liquefied mass must be cooled down to 40° before
it is inoculated, since at a higher temperature the micro-
organisms might be destroyed. The mass when inocu-
lated is poured out upon sterilised plates with the precau-
tions mentioned above, and as the water of condensation
which separates out renders the film of agar liable to slip
from the plate, it is prevented from doing so by dropping
some sealing-wax on the edge of the medium. But a more
convenient plan is to use Petri's capsules or Soyka's plates
to contain the mass. Agar plates have the special
advantage that they can be kept for a considerable time
at incubation temperature, and that they do not undergo
liquefaction.

The roll process can also be applied to cultures on agar.

To facilitate the isolation of micro-organisms, Dahmen
has devised an apparatus consisting of a double capsule, of
which the upper part extends beyond the lower. The latter
is placed on a glass plate and surrounded with an india-
rubber ring, on which the upper capsule rests securely.
The lower capsule is prepared with the inoculated nutrient
agar, placed in the centre of the rubber ring, and covered
over with the larger capsule ; the whole is then surrounded
with an india-rubber band and deposited in the incubator.

The individual colonies form on the agar plate in the
same way as on the gelatine (except that they do not liquefy
it), appearing coloured or glossy, and showing characteristic
outlines.

Plate cultures on serum and plover's egg albumen. — Blood

60 BACTERIOLOGY

seruni is only used in the solid state, and is principally
adapted for surface or streak cultures (Strichciilturen) . In
order, however, to render it available for plate cultivation,
Hiippe mixes it with an equal quantity of a warm solution of
agar ; and it may also be suspended in gelatine. Unna in-
creased the coagulability of blood serum by' rendering it
strongly alkaline, in order to add it to gelatine or agar.
This medium has special excellences for a number of micro-
organisms.

The albumen taken from plovers' eggs which have been
previously sterilised with 1 per 1,000 corrosive sublimate
may be inoculated and then dried over sulphuric acid under
the receiver of an air-pump. Plates so inoculated can be
kept for a considerable time if preserved in a sterile place,
and when laid in a moist chamber there appear on the thin
dry transparent film of albumen variously shaped areas of
different sizes, which may be transferred to other culture
media. Plover's egg albumen may also, like blood serum, be
mixed with gelatine or agar, and so used for plate cultures.

The micro-organisms can be transferred to other media
from agar and serum plates in the same way as from gela-
tine, and the appearances presented by them in their growth
observed.

Cultivation of anaerobic micro-organisms. — For the culti-
vation of anaerobic microbes, that is, those which grow with
a scanty supply of oxygen, or when it is totally excluded,
an entire series of methods have been devised, of which the
following are a few.

The most direct plan is to introduce at once into the
nutrient medium such substances as will extract the oxygen
present, a result which is attained by adding to nutrient
gelatine 2 per cent, of grape sugar or O'l per cent, of
resorcine, or to liquefied nutrient agar ^ per cent, of formic
acid or of sodium sulphindigotate.

CULTIVATION OF ANAEEOBES 61

In order to prepare plate cultures of anaerobic micro-
organisms the oxygen can be excluded after Koch's method!
by laying upon the gelatine or agar before it has fully set
a thin sterilised plate of mica or selenite, which adheres
closely to the surface of thu nutrient mass. The exclusion of
oxygen is rendered complete if melted paraffin be run round
the border of the mica plate.

The removal of oxygen is effected by Buchner in a very
simple way by means of an alkaline solution of pyrogallol,.
prepared by dissolving a gram of pyrogallol in 10 c.cm.
water, and adding 1 c.cm. concentrated solution of caustic
potash.

Another mode of cultivating anaerobic microbes on
plates consists in bringing the plate prepared with the
micro-organism under the receiver of an air-pump and
expelling the oxygen by pumping.

Bliicher and Botkin secure the removal of oxygen by
displacing the air in the receiver with another gas, viz.
hydrogen, by means of an india-rubber tube, the lower
opening of the receiver being closed with paraffin or with
glycerine and water.

Complete removal of the oxygen from a test-tube by pump-
ing has been effected by Gruber in the following way :—
A test-tube of more than the usual length is drawn out afc
about 15 cm. from the bottom to a narrow neck. It is
filled with 10 c.cm. of nutrient material by the aid of a
funnel, closed with cotton- wool and sterilised. After inocu-
lation, the wool plug is pressed down as low as the narrowed
part and a tight-fitting rubber cork introduced into the
mouth, through a hole in which passes a right-angled tube of
glass connected with an air-pump. The air is then pumped
out (the culture medium in the rarefied space being
meanwhile kept in a water-bath at 30°-40° C.), after which
the tube is sealed at the constricted part over the flame of

62 BACTERIOLOGY

a Bunsen burner, and the gelatine rolled out by Esmarch's
method.

In order to displace the air in a test-tube by means of
hydrogen, Fuchs recommends that the inoculated tube
should be inverted and hydrogen conducted into it from
below through a glass pipe, after which the test-tube is
closed with a rubber cork.

For test-tube cultivation, however, Liborius' method of
preparing high cultures is specially adapted. A tube is filled
high up with gelatine or agar, which is then freed from air
and oxygen by thorough boiling and is cooled to 40° ; the
matter to be inoculated is distributed in it as evenly as pos-
sible with a platinum needle, and it is made to set rapidly in
iced water. By this means the deeper layers of the nutrient
mass are protected from the air by those higher up, while
the superficial ones are exposed to the action of oxygen.
When several varieties of bacteria develope, a means is
hereby afforded of distinguishing the aerobes which grow
on the surface, from the anaerobes growing in the deeper
parts.

High cultures are also used for obtaining thrust culti-
vations (Stichculturen} of anaerobic micro-organisms, the
platinum needle charged with infecting matter being thrust
as deeply as possible into the stiff nutrient mass. Although
at first* development only occurs in the deeper parts, the
growth gradually mounts upwards as the gaseous products
of its metabolism displace the air from the higher layers of
the medium.

Nikiforoff cultivates the anaerobes in the ' hanging drop .'
A cover-glass is prepared with an inoculated drop of bouillon
and sealed to a hollowed slide with a layer of vaseline.
Between the edge of the cover-glass and that of the well in
the slide, the contents of a platinum loop of strong solu-
tion of pyrogallol are allowed to flow in on one side, and

PERMANENT CULTURES 63

on the opposite, after pushing aside the cover-glass, a
similar quantity of caustic potash, so that the two fluids
mix when the object has been brought into the right
position.

Hens' eggs seem to afford a suitable medium for anae-
robic cultures. This kind of cultivation, which is recom-
mended by Hiippe and Heim, has been described more in
detail above (see p. 50).

In cultivating obligate anaerobes the materials used in
the investigation must, according to Kitisato, be previously
heated, in order to remove by this means the facultative
anaerobes.

Permanent cultures. — To preserve cultivations of bacteria
so that they can be examined at any time, evaporation of
the moisture contained in the nutrient medium must be
prevented, as well as all possibility of contamination, and
consequently the vessels must be hermetically closed. Krai
punches cylinders out of boiled potatoes, cuts them into
discs, and places them in round glass boxes, the covers
of which are tightly ground on and the channels in them
filled with glycerine. After inoculation they are closed
germ-tight with paraffin and spirit varnish. Cultures may
also be preserved in test-tubes hermetically sealed by
melting the glass.

Prausnitz pours an aqueous solution of gelatine con-
taining 1 per cent, of carbolic or 5 per cent, of acetic acid
over thrust cultures placed in iced water, and then closes
them with corks and seals them.

By Duclaux's method the cultures of bacteria are en-
closed in the small tubes used to contain lymph.

Jacobi first exposes the gelatine which contains the
colonies, and which has been spread out in the thinnest
possible layer, to the action .of 1 per cent, bichromate of
potash for from one to three days in the presence of light,

64 BACTERIOLOGY

hardens it in alcohol, and cuts it into pieces which can
then be strained like sections of tissue, and thus rendered
permanent.

To preserve small portions from agar plates, Giinther
lays them in a drop of glycerine placed on a slide, deposits
thereon a second drop, and finally the cover-glass. The
superfluous glycerine having been absorbed out the prepara-
tion is closed with cement.

65

CHAPTEE IV

EXAMINATION OF MICRO-ORGANISMS UNDER THE MICROSCOPE,
AND BY EXPERIMENTS ON LIVING ANIMALS

Examination in the fresh state. — In making microscopic
examinations we begin with the simplest mode of procedure,
which consists in taking minute samples from the individual
colonies on the plate with the help of the platinum needle,
floating them in water, and subjecting them to observation.
It must be seen to that too much fluid is not taken, but only
enough to fill the interspace between cover-glass and slide.
The former ought not to float about loosely, nor should
the fluid extend beyond its edges. When examining with
high powers it must be noted which form the particular
micro-organism takes — that is, whether rods or cocci are to
be dealt with, whether they are connected with one another
in chains, whether, if so, the chains run straight or spirally,
and, in the case of cocci, whether they lie scattered or in
rows. Size is measured in micromillimeters (micra, com-
monly written p, = the thousandth part of a millimeter),
or by comparison with other similar forms, especially red
corpuscles.

Examination in the hanging drop. — A very useful method
of observing freshly -obtained micro-organisms is the
examination in the ' hanging drop.' For this purpose the
end of a platinum wire is bent with the aid of a pliers into
a little loop. When this is dipped into a liquid containing
bacteria, enough of the liquid remains adhering to it to

66 BACTERIOLOGY

form a small drop, which is transferred to a cover-glass.
A ' hollow ' slide is then taken, that is, one with an exca-
vation or well ground in the centre ; this well is surrounded
with vaseline by means of a fine hair pencil, and the cover-
glass with the drop turned downwards is laid on the slide
in such a way as to adhere firmly to the vaseline. If the
substance, the- microbic contents of which it is desired to
examine, is not a liquid containing bacteria, but animal
tissue or solid culture medium, a drop of sterile water or
sterile salt solution is conveyed on to the cover-glass with
the loop of the platinum needle, and a minute sample of
the mass to be examined is transferred into the drop.

In observing the hanging drop the edge must first be
sought for with a low power, and then focussed with
a higher ; since, as it appears bounded by a sharply-
drawn line, the micro-organisms in the drop can in this
way be more easily focussed, which very much facili-
tates the examination for beginners ; and, morever, the
elements are met with in a thinner layer at the border than
in the centre. As the elements under observation are not
stained, the narrowest possible aperture of the diaphragm
must be used in the examination.

With the hanging drop attention must in like manner
be paid to the peculiarities which can be observed in bacteria
examined in the fresh state, as detailed above, and their
rnotility is more distinctly brought out in this mode of
investigation. The closure of the space prevents the fluid
from evaporating, but if the examination is too prolonged
the micro-organisms sink into the concavity of the drop,
and so sometimes elude observation.

When it has been ascertained by means of fresh pre-
parations that micro-organisms are present, and their form,
mode of propagation, and power of movement have been
observed, the next step is that of staining. So many pro-

STAINING OF MICRO-ORGANISMS 67

cesses have been established in the course of the researches
which have been made up to the present that we must
describe them here more in detail, especially as they afford
marks by * which bacteria can be distinguished, and
consequently staining is of the highest importance in
determining the individual varieties.

Staining of micro-organisms. — Staining constitutes an
indispensable aid to the study of the finer structure
of the micro-organisms, and of their relation to the cells
of the body. In carrying it out a series of colouring
matters are used which have been already detailed (p. 30),
and solutions are prepared from these in different ways,
which are employed both for isolated micro-organisms
and also for those in the tissues. The basic aniline
colours are for the most part kept in stock in alcoholic
solutions which are mixed with water before use, so that
we really employ a dilute alcoholic solution in staining.
The dilution, however, must not be carried too far.

Giinther has pointed out that absolute alcohol is not
suitable for use in staining with basic aniline colours, just
as it is incapable of extracting the dye from cells when
once they have been stained.

Simple staining of cover-glass preparations, — In this, the
simplest kind of staining, the mode of procedure is as
follows : A sample of the matter to be examined is con-
veyed on to a cover-glass with the point of the sterilised
platinum needle and is diluted, if needful, with water, after
which the organisms suspended in the water are spread
out over the surface of the glass with the flattened end of
the needle (smear preparation) ; or a better way of managing
this is to press another cover-glass upon the prepared one,
and then slide it off, so that the mass under examination
appears equally distributed on both cover-glasses. The
mass which contains the micro-organisms is frequently

F 2

68 BACTERIOLOGY

found to have so much moisture as to render the addition
of water superfluous. If it is desired to examine the juice
of organs, a piece of the organ is seized in a forceps and
the cover-glass smeared with it, dried in the air, and passed
three times through a flame to fix the micro-organisms to
its surface, after which the staining is done by depositing a
few drops of dye on the infected surface of the cover-
glass, or by pouring some into a watch-glass and floating
the cover-glass upon the solution with the prepared side
downwards. After from one to five minutes it is freed from
superfluous stain by washing in water, is dried in the air
— a process facilitated by soaking up the drops with blotting-
paper — and is mounted in fairly fluid Canada balsam ; or,
if it is not wished to preserve the preparation, it may be
examined in water, or in a very dilute solution of potassium
acetate. Such objects are examined with ordinary or
homogeneous immersion objectives by the aid of Abbe's
illuminating apparatus without a diaphragm. Coloured
preparations admit of being seen with distinctness, and
their outlines can be accurately determined, such figures
being spoken of as ' coloured images,' to distinguish them
from the unstained ' structural images,' which should
only be examined with a diaphragm of narrow aperture.
When Abbe's apparatus is used without a diaphragm, all
the rays which enter the lower lens, and which form
a very obtuse-angled pencil, are enabled to reach the
object.

Bacteria are difficult to observe in fluids and tissues,
being only visible through the shadows caused by the
differences in refractive power of the several structures.
Hence but little light must be allowed to reach the prepara-
tion, and consequently as small a diaphragm as possible
used, and the result is an impairment of distinctness. If,
however, the bacteria be stained, it becomes possible to

PREPARATION OF STAINING SOLUTION'S 69

remove the diaphragms, and to examine with the full power
of the Abbe's illuminator.

The coloured image is best if the structural image be
effaced by rendering the shadows of the unstained parts
invisible in the broad cone of light.

It must further be remarked regarding the structural
image, that the diaphragm should have the narrowest
possible aperture with a low power, but should increase in
size as higher powers are employed.

Preparation of staining solutions. — For the simplest kind
of staining of bacteria solutions of fuchsine, methyl blue,
gentian violet, Bismarck brown, vesuvine (in equal parts of
water and glycerine), and methyl violet are used. Gentian
violet and fuchsine stain quicker and more intensely than
the others. In order to increase the staining power with
the various micro-organisms, certain mordants are employed,
as in other histological methods of staining. Aniline oil and
phenol are the mordants most used in bacteriological re-
search. The former, which is not a true oil, is obtained
from coal tar, and it is used for preparing an aniline water
in which the dyes, especially gentian violet or fuchsine, are
dissolved. The aniline water should be prepared freshly
each time, or in any case should not be allowed to stand
long, as it rapidly decomposes. To make it a test-tube
is filled with water which is shaken up with 1 to 2 c.cm.
of aniline oil until an emulsion is formed, which is
filtered. The clear filtrate is aniline water ready for use, and
enough of the alcoholic solution of the dye is then added
to render the liquid of a dark colour. The concentration
of aniline water amounts to 5 to 100.

Trenkmann prepares his aniline water solution of gen-
tian violet in the following way : — A drop of a concentrated
alcoholic solution of gentian violet is let fall into a test-
glass and 10 c.cm. of water are added. Half of this is then

70 BACTERIOLOGY

poured away, and the glass filled with aniline water ; a
solution is thus obtained which remains clear and stains the
bacteria themselves deeply, but the ground very slightly. The
cover-glasses should remain about half an hour in the stain-
ing fluid.

Instead of the aniline water a 5 per cent, aqueous solution
of carbolic acid (phenol) may be used, to which an alcoholic
solution of fuchsine is added, until the mixture becomes of
a dark colour. This mixture is known as ZieliUs solution,
and its composition is as follows :

Crystallised carbolic acid 5'0

Water lOO'O

Alcohol 10-0

Fuchsine . . . ... . 1-0

According to Kiihne's formula, methyl blue instead of
fuchsine is mixed with 5 per cent, carbolic acid, water, and
alcohol, and a solution of strong staining power so obtained .

Instead of carbolic solution a 1 per cent, solution of
ammonium carbonate can be used as a mordant.

Koch employs a solution of caustic potash as an ingre-
dient, adding to 1 c.cm. of a concentrated alcoholic solution
of methyl blue 200 c.cm. of water and 0-2 c.cm. of a 10
per cent, potash solution, and Loftier also adds 100 c.cm. of
0*01 per cent, solution of caustic potash to 30 c.cm. concen-
trated alcoholic solution of methyl blue.

In certain staining processes, particularly that for tubercle
bacilli, a sulphuric acid solution of methyl blue is employed,
prepared by mixing 100 parts of a 25 per cent, solution of
sulphuric acid with 2 parts methyl blue ; or a nitric acid
solution consisting of a saturated solution of methyl blue
in 20 parts nitric acid, 30 parts alcohol, and 50 parts
distilled water.

The different methods of staining are in many cases
assisted by heat, the colouring solutions being kept warm

STAINING OF FLAGELLA 71

on the cover-glass or in watch glasses while the process is
going on. When, however, sections of tissue containing
bacteria are to be warmed, precautions must be taken to
avoid spoiling the tissue, especially as staining takes
longer in the case of sections.

Staining of flagella. — For the purpose of rendering
visible the flagella of motile micro-organisms, Loffler uses
a mixture of 10 c.cm. of a 20 per cent, solution of tannin
and a few drops of saturated ferrous sulphate solution,
with fuchsine or 4 or 5 c.cm. of extract of logwood. Stain-
ing is effected with fuchsine in aniline water, to which a 1
per 1,000 solution of caustic potash has been added until it
becomes turbid owing to a floating precipitate. For bacteria
which form alkalies, the mordant must be rendered corre-
spondingly acid; for those which form acids, alkaline.

According to Trenkmann the cilia are brought into view
if the preparations are treated before staining with tannin
and hydrochloric acid or catechu tannic acid to which
carbolic acid has been added, or extract of logwood treated
with acid ; and they become still more distinct if the pre-
parations, after being treated with the mordant and stained,
are examined in a drop of iodine water. Two or three
drops of boiled water are allowed to fall upon a slide, and a
small drop of the culture to be examined is added and
mixed well in. A minute droplet is conveyed from this to
a cover-glass, spread out, dried in air, and laid without
previous heating in a solution containing 2 per cent, of
tannin and J per cent, of hydrochloric acid. The cover-
glass remains for from 6 to 12 hours in this solution, and
is then washed in water, laid for an hour in iodine water,
washed again, and deposited for half an hour in a weak
solution of gentian violet in aniline water.

Staining of spores. — It is possible to effect a staining of
the spores, in those micro-organisms which form them, by

72 BACTERIOLOGY

warming the staining fluids. Carbolic acid fuchsine (Zielil's
solution) is used for staining, and the infected cover-glass is
left for an hour in the boiling dye, when the spores in the
bacilli will remain of a red colour after washing with water
and decolorising with alcohol ; or if double staining with
methyl blue is carried out the bacilli appear blue, and the
spores dark red. The hay bacillus, and especially the Bacillus
megatherium, should be selected for the study of these methods
of staining. In many of the microbes hitherto known the
discovery of spores has not as yet been made.

Spores are also brought out distinctly as little granules
by staining with dilute alkaline methyl blue, in which case,
after double staining with aqueous solution of Bismarck
brown, they appear blue on a brown ground.

According to Moeller, spores are most conveniently
stained by the following method : — The cover-glass prepara-
tion is brought for two minutes into absolute alcohol and for
two more into chloroform, washed in water, plunged for from
a half to two minutes into a 5 per cent, chromic acid solu-
tion and rinsed again with water, after which some aqueous
carbolic fuchsine solution is dropped on, and it is warmed
for one minute in the flame, being brought once to the
boil. The carbolic fuchsine is poured off, the cover-glass
dipped into 5 per cent, sulphuric acid until decolorised, and
once more thoroughly washed with water. Finally, an
aqueous solution of methyl blue or malachite green is
allowed to act on it for half a minute, arid then washed off.
The spores are dark red in the interior of blue or green
bacteria.

DECOLOKISING AGENTS

In staining with the various aniline dyes a phenomenon
of practical importance has been observed, viz. that stained
micro-organisms part with their colouring matter to certain

THE KOCH-EHKLICII METHOD OF STAINING 73

reagents. These are known as decolorising agents, and
include amongst them water, alcoliol, acetic, hydrochloric,
sulphuric, and nitric acids, iodine, &c.

Upon decolorising with one or other of the above-named
fluids depend various methods which have acquired an
extraordinary significance for the diagnosis of micro-organ-
isms, and for the practical work of staining them in sec-
tions.

Koch and Ehrlich method of staining. — The Koch-
Ehrlich method of staining tubercle-bacilli stands in the
first rank of these, and brings into action both the mordant
and bleaching processes. Aniline water is prepared as
described above, and alcoholic solution of fuchsine, gen-
tian violet, or methyl violet is added to it until a fairly
saturated solution in dilute alcohol is obtained. Small
masses of sputum, or of the wall of a pulmonary cavity, are
then conveyed on to a cover-glass and spread out by rub-
bing with a second, so that both glasses become coated
with a fine film of the mass under examination. The
cover-glasses, having been dried in air, are passed three
times through the flame with the prepared side up by
means of a forceps, and then deposited in the staining fluid
and either left for twenty-four hours at the temperature
of an ordinary room, or heated for fifteen minutes until
bubbles rise. The cover-glass is next lifted from the dye
with a forceps and plunged for a few seconds into a solu-
tion of about 33 per cent, of nitric acid until the prepara-
tion, previously of a red colour, becomes yellowish green, and
is then washed in 70 per cent, alcohol. If fuchsine has been
used for the staining, the after-staining may be done with
methyl blue, malachite green, or picric acid ; but if the
first staining has been done with gentian or methyl violet,
Bismarck brown must be used for the second. The secondary
staining lasts from one to five minutes, until the particular

74 BACTERIOLOGY

colour used is plainly visible on the cover-glass, after which
this is washed in water, dried, and mounted in Canada
balsam. After washing in water the cover-glasses may
also be brought into alcohol and then treated with oil of
cloves and Canada balsam.

In this staining process the nitric acid acts as a bleaching
agent on the different micro-organisms contained in the
mass under examination. The tubercle-bacilli alone refuse
to yield up their stain to the acid unless it has acted for
a long time.

Ziehl and Neelsen's method of staining. — The Koch-Ehrlich
method was modified by Ziehl and Neelsen by using car-
bolic fuchsine instead of aniline water fuchsine. Here, also,
either the stain is applied on the cover-glass, or this is laid
prepared side downwards in the warm dye. It is after-
wards washed with water and decolorised in 33 per cent,
nitric or 5 per cent, sulphuric acid. In all other particulars
the process resembles the Koch-Ehrlich method, and
secondary staining is effected by means of malachite green,
picric acid, or methyl blue.

Ehrlich's method of staining, — For demonstrating tubercle-
bacilli in pus, Ehrlich recommends that it be spread out
very thinly, and the preparation placed for one to two hours
in cold aniline fuchsine and decolorised with sulphanil-nitric
acid (1 part nitric acid to 3 to 6 parts saturated solution of
sulphanilic acid). The double staining is done with methyl
blue.

Gunther's method of staining. — In this process the stain-
ing is effected with warm aniline water fuchsine, from which
the cover-glass is conveyed with the prepared side upper-
most into alcohol containing 3 to 100 of hydrochloric acid,
in which it is moved about for a minute and then rinsed in
water. By means of a pipette a few drops of dilute alco-
holic solution of methyl blue are now allowed to fall upon

VARIOUS STAINING PROCESSES 75

the cover-glass, which is then washed in water, dried, again
passed three times through the flame, and mounted in
Canada balsam and xylol.

Weichselbaum's method. — This is a modification of the
Ziehl-Neelsen method, in which the red-stained cover-glass
preparations are transferred directly to an alcoholic methyl
blue solution, in which they remain until they show an
even blue colour. They are then rinsed in water, dried,
and mounted in Canada balsam. The alcohol is here the
only bleaching agent.

Fraenkel's method, — The cover-glasses are stained with
aniline water fuchsine and transferred to a fluid consisting of
a saturated solution of methyl blue in 50 parts water, 30
parts alcohol, and 20 parts nitric acid. When the preparation
appears blue it is washed in alcohol and acetic acid or in
pure water, and is examined in water.

Gabbet's method. — After staining in carbolic acid fuchsine
the preparation is brought into a sulphuric acid methyl
blue (2 grms. methyl blue to 100 grms. of a 25 per cent,
solution of sulphuric acid), and double stained with mala-
chite green.

Method of Pfuhl and Petri. — The preparations are stained
in a mixture of 10 c.cm. alcoholic solution of fuchsine to
100 c.cm. of water, and subsequently decolorised in glacial
acetic acid. They are then washed in water and double
stained with malachite green, again washed in water, dried,
and mounted in Canada balsam. In this case the glacial
acetic acid is the decolorising agent.

Method of Pittion. — The cover-glass preparation is dipped
for a minute into a mixture of 1 part alcoholic fuchsine
solution with 10 parts of a 3 per cent, solution of ammonia,
and transferred after rinsing in water to a concentrated
solution of aniline green in 50 grms. alcohol, 30 grms. water,
and 20 grms. nitric acid, in which it remains J minute.

76 BACTERIOLOGY

Arens' chloroform method. — In order to avoid heating, as
well as the preparation of a complicated staining fluid, an
alcoholic solution of fuch sine mixed with chloroform is used
as the dye, and alcohol with hydrochloric acid for decolorising.
The fuchsine solution is prepared by pouring 3 drops of
absolute alcohol on a crystal of fuchsine the size of a millet-
seed in a watch-glass and adding 2 to 3 c.cm. chloroform.
The solution becomes turbid and then begins to clear,
flocculent particles of fuchsine being separated. When
the clearing is complete the cover-glass preparation is laid
in it for from 4 to 6 minutes, until the chloroform is evapo-
rated, and is then decolorised in concentrated alcohol, to
which hydrochloric acid has been added in the proportion
of 3 drops to a watch-glass full. It is next rinsed in water,
and finally double stained with dilute methyl blue.

Gram's decolorising method. — This appears to be the
most extensively used of all the bleaching methods, and
depends upon the employment of iodine in aqueous solu-
tion combined with potassium iodide (1 part iodine, 2 parts
potassium iodide, and 250 parts water) after the prepara-
tion has been stained in aniline water solution of gentian
violet. The iodine forms with the colouring matter a
precipitate which adheres to the micro-organisms, but can
be easily washed out of the tissues, and if this is properly
done the bacilli or cocci appear isolated by the stain. The
following is the mode of procedure in carrying out Gram's
method : — The prepared cover-glass or section containing
bacteria is warmed in aniline water gentian violet ; too
strong heating, however, has an injurious effect. The best
mode of warming the solution is to place it for 15 minutes
on the water-bath ; or to hold it over the flame for 1 minute,
let the watch-glass cool for 3 minutes, then heat it again for
a minute more and let it cool again for two or three, and so
on, until the process has been repeated four or five times.

GRAM'S DECOLORISING METHOD 77

The preparation is next laid for 1 to 2 minutes in the
iodine and potassium iodide solution and transferred from
that into absolute alcohol, in which it remains until the
colour is discharged. The bacteria come out stained with
gentian violet, and the tissue may be double-stained red
with picrocarmine, Magdala red, or other dyes.

Gram's method can be used as an aid to the diagnosis
of the vast majority of micro-organisms. For example,
the Pneumococcus Friedldnder shows no staining after going
through the process, and similarly the bacilli of Cholera
Asiatica, typhoid fever and glanders, gonococci, the spirilla
of recurrent fever, &c., cannot retain the colouring matter,
but give it up, as do also the nuclei of cells, when iodine
solution is applied.

It is strongly to be recommended that the preparation
should not be brought directly from the staining fluid into
the iodine and potassium iodide, but be first rinsed free
of superfluous stain in plain aniline water before being
transferred to the iodine solution (Botkin) .

In staining sections of tissue it is advisable to carry
out the ground staining before that of the bacteria, which
is done by immersing the sections in picrocarmine for one
or two minutes, washing in water, transferring to alcohol,
and then subjecting to Gram's process.

Every pigment is not, however, suitable for this method,
since Unna has shown that it gives no results if fuchsine,
methyl blue, or Bismarck brown are used. The process
can only be carried out with the pararosanilines (methyl
violet, gentian violet, and Victoria blue).

Gunther's modification of Grain's process. — Not only pure
alcohol, but also alcohol to which 3 per cent, of hydrochloric
acid has been added, is used for decolorising. The cover-
glass or section of tissue is left for about two minutes in
aniline water gentian violet, but in the case of tubercle

78 BACTERIOLOGY

bacilli the dye is allowed to act for twelve hours, and in
that of lepra bacilli for half a day. Superfluous stain is
removed with blotting-paper, and the preparation is brought
for two minutes into solution of iodine and potassium
iodide, then for half a minute into pure alcohol, for
exactly ten seconds into 3 per cent, hydrochloric acid in
alcohol, and immediately afresh into plain alcohol for
several minutes, changing the spirit as long as any colour
is extracted from the preparation. The cover-glass is now
dried and mounted in balsam, and sections of tissue are
laid in xylol (which renders them transparent in half a
minute), and then mounted on slides with Canada balsam
dissolved in the same liquid.

Weigert's modification of Grain's process. — The sections
stained with gentian or methyl violet are not transferred
to alcohol from the iodine solution, but laid upon slides and
covered with aniline oil, which dehydrates and differentiates
them. The aniline oil is then removed with blotting-paper,
xylol is poured upon the preparation, and it is put up in
Canada balsam in xylol.

Impression preparations. — These are made for the purpose
of rapidly gaining an idea, when examining plates, regarding
the arrangement of the colonies and the microscopic
peculiarities of the organism under investigation. A cover-
glass is laid on the plate, pressed gently down, lifted care-
fully with a forceps, and laid aside to. dry. It can then be
stained like an ordinary cover-glass preparation.

Examination of micro-organisms in sections of tissues.—
The examination of micro-organisms in the tissues, whether
in the interior of the individual cells, or in the structures
which are formed by them, is of pre-eminent importance in
research directed to medical ends. Not only have the nature
of the micro-organisms and their mode of entrance into
the body to be discovered, but attempts must be made to

EXAMINATION OF MICRO-ORGANISMS IN TISSUE 79

ascertain their bearing towards the elements of the tissues,
and their exact situation in and between them. In par-
ticular, their physiological action, in spite of very advanced
methods of investigation, has not up to the present been
fully explained. When tlit, microbes are present in masses
of considerable size, but only then, their position in the
tissues can be recognised in the simplest way in pieces of
the fresh organ by examining some of its tissue on a slide in
a drop of sterile water or salt solution. In the case of
fluids the addition of water may be omitted. The minute
elements are then examined with a high power (an oil-
immersion with Abbe's condenser and a diaphragm). We
cannot, however, ascertain by this method how the microbes
are related to the tissues, nor their exact situation in the
tissue and its elements. A small piece of it should there-
fore be torn up with needles and treated with a drop of
acid or caustic potash, so as to cause the connective tissue
which forms the bulk of organs to swell up. In this way
the bacteria, which exhibit greater power of resisting re-
agents, are brought out distinctly. Through the introduc-
tion of staining processes, however, methods have been
discovered which render it possible to demonstrate the
micro-organisms in uninjured tissue.

Examination by the freezing method. — In order to be able
rapidly to examine pieces of organs, recourse is had to the
freezing microtome. The substance in the fresh state is laid
upon a roughened metal plate and frozen by means of an
ether spray apparatus. It is then cut into sections with a
cooled knife, and these are laid on slides, allowed to thaw,
and subjected to staining processes which will be described
later on ; after which they are most conveniently examined
in dilute glycerine.

Hardening. — Owing to the frequent destruction of the
tissue in using the freezing microtome, caused by the

80 BACTERIOLOGY

crystals of ice which form, the organs are usually not cut
until they have undergone a hardening process.

In Histology an entire series of reagents is employed for
hardening, the use of which is, however, impracticable in
Bacteriology, because they deprive bacteria of their property
of taking up aniline colours easily. The most convenient way
is to harden the pieces, which should each be a cubic centi-
meter in size, singly in absolute alcohol, which must be
changed several times. The alcohol may be obtained as
free from water as possible in the following way : Crystals
of copper sulphate (blue vitriol) are heated in an iron
capsule with frequent stirring until they have completely
parted with their water of crystallisation and subsided to a
white powder, which, after cooling, is introduced into a bottle
and the alcohol is poured over it, when it greedily extracts
the water therefrom, becoming again blue. As the piece of
tissue contains water, it sinks to the bottom if thrown into
absolute alcohol, and the hardening process goes on more
slowly in the lower than in the upper half of the vessel,
the alcohol above being less rich in water. Hence it is
advisable to keep the organ in the upper part of the alcohol,
either by means of a layer of cotton wool, or by suspending
it with a thread fastened outside.

A half per cent, chromic acid solution, with or without the
addition of platinum chloride and acetic acid, has also been
recommended, as in it the bacteria are well preserved. After
eight days the pieces are rinsed in water until that which
flows away shows no yellow coloration, and the hardening
is then completed in alcohol.

Instead of chromic acid, a concentrated aqueous solution
of picric acid renders good service. The pieces are left in
this for two days, washed for twenty-four hours in water,
and transferred first to dilute, and from that to absolute,
alcohol.

IMBEDDING 81

Portions of tissue so fresh as to be still warm are best
hardened in corrosive sublimate. They are left for from ten to
thirty minutes in a 5 per cent, solution prepared at 70° C., and
are then transferred directly into moderately dilute alcohol,
in which they remain for a day, and hardening is then
completed in absolute alcohol.

Imbedding. — The hardened specimens are prepared for
section-cutting in many different ways, to enable them to be
fixed in the microtome.

Imbedding in gum arabic. — One of the simplest method
consists in fastening them with gum arabic to cork or elder
pith, or to little bits of wood, when, after drying, sections
are cut from them, great care being taken to prevent the
hardened gum from injuring the knife. The process con-
sists in immersing the pieces to be cut in a concentrated
solution of gum arabic of a syrupy consistence, after
imbedding in which they are deposited in concentrated
alcohol. This extracts the water from the gum, so rendering
the mass sufficiently firm to be cut.

Imbedding in glycerine jelly, — The most useful method
is that of attaching the pieces to little bits of cork or wood
by means of a concentrated glycerine jelly prepared with
the aid of heat ; Frankel recommends boiling together one
part gelatine, two parts water, and four parts glycerine.
The portion of organ having been made to adhere by
means of this glycerine glue, nothing further is done until
the gelatine sets, when the piece is laid in alcohol and
becomes after some time so firmly adherent that the
cork can be clamped in the microtome and sections made.
It is necessary to bring the knife to the preparation
obliquely, and to keep everything constantly wet with
alcohol while cutting. Before staining, the sections must
always be brought into absolute alcohol. To enable gly-
cerine jelly to be kept in stock, a drop of corrosive subli-

82 BACTERIOLOGY

mate must be added, in order to prevent the growth of
micro-organisms.

Imbedding in celloidine. — The celloidine method is a very
convenient one. It consists in fastening the portions of
organs to bits of cork or wood by means of celloidine
dissolved in alcohol and ether, and then, after the celloidine
has set, immersing them in alcohol, in which they gain a
consistence suitable for cutting. The pieces are placed in
absolute alcohol and left there for twenty-four hours, after
which they are transferred to a mixture of equal parts of
alcohol and ether, and finally to a celloidine solution of
medium consistence, in which they remain for at least twenty-
four hours, in order that the tissue may become thoroughly
saturated with that substance. The pieces are now taken
out one by one and fixed to corks by means of celloidine, and
as soon as it has set in the air, which requires only a few
minutes, the pieces fastened to the corks are immersed in
very dilute (30 per cent.) alcohol. In this the celloidine
becomes cloudy after a short time, until after several days it
is changed into an opaque white mass of such firmness that
the piece of organ to be cut is securely adherent to its cork
support, and if this be now fixed in the clamp of the micro-
tome it is possible to obtain the finest sections. These
sections are enveloped, so to speak, in a mantle of celloidine,
which is capable of taking the aniline dyes.

This method gives good results with Gram's process.
When it is wished to stain in this way several sections follow-
ing one another in series, the section stainer l in use at the
author's Institute is well adapted for this purpose. Several
sections having been laid in serial succession upon a
slide of larger size than usual, are covered with a nickel-
plated grating and clamped in the section stainer. The
whole is then passed through the various fluids and stains

1 Sold by Sieberi in Vienna.

IMBEDDING IN PARAFFINE 83

one after another, the delicate grating preventing the
sections from slipping off, without in any way injuring
them ; and when finally it is raised after full completion of
the treatment, the sections remain lying in their original
order, and the result is a serial preparation (fig. 27) .

Imbedding in paraffine. — This method serves for the pre-
paration of finer sections, but is only rarely used in bacte-
riology. It is employed for making single sections as well
as for series. The pieces of organ are brought into absolute
alcohol for twenty-four hours, then into a mixture of chloro-
form and alcohol for twenty-four more, and finally for the
same length of time into pure chloroform. Xylol, oil of
cloves, and oil of turpentine do not yield such good results.
If the pieces are saturated with chloroform they should sink

Nickel-plated grating

FIG. 27.— SECTION STAINER FOR PREPARATIONS IMBEDDED i\ CELLOIDIXE.

to the bottom in that liquid. After this they are laid in
paraffine dissolved by heat in chloroform, and remain in this
solution for two or three hours at a temperature of 30°-40° C.
Finally they are imbedded in paraffine. Little boxes of paper
having been made ready and floated on cold water, fluid
paraffine is poured into them, and after it has solidified the
pieces of organs are laid upon it and covered with more
melted paraffine, which liquefies again the surface of the layer
already solidified, so that the specimen seems enclosed in a
block of the substance. After a few hours this is trimmed
to a suitable form with a knife, clamped in the microtome,
and sections are cut with the knife transverse or slightly
oblique, and without using any moistening fluid. The micro-
tome can be arranged to cut sections of any desired thickness.

G 2

84 BACTERIOLOGY

The sections are transferred one by one to xylol, in order
to extract the paraffine from them, and are thence brought
into alcohol and then into water. If they do not sink in
the water the paraffine has not been completely removed,
and in this case they must be returned to the alcohol and
from that into xylol, and then transferred afresh to alcohol
and water. After removal from the water they are sub-
jected to suitable staining processes, cleared in xylol, and
mounted in xylol Canada balsam. Oil of cloves should
not be used for clearing the tissue, as it decolorises the
micro-organisms.

In the preparation of serial sections a softer paraffine is
used for imbedding; the imbedding-block is otherwise
prepared in the same way as before and cut to a square, and
the microtome knife is fixed transversely. The sections,
which adhere to each other, forming bands which resemble
a tape-worm in outline, are laid one beside the other in
corresponding order and fixed to the slide, usually with the
white of a hen's egg diluted with water and glycerine. At
ordinary temperatures the white of egg takes a long time
to dry, but this may be expedited by gentle heating. A
drop of creosote or carbolic acid should be added to the
fluid to make it keep. Other fixing media are collodion,
glycerine agar, or glycerine gelatine in a dilute condition.

The adherent pieces are freed from paraffine with xylol,
which is extracted in turn with alcohol ; they are then
washed in water, subjected to staining processes, rendered
transparent with xylol in the same way as single sections,
and put up in Canada balsam.

ON THE STAINING OF SECTIONS

The staining of sections is carried out after various
m3thods,but a certain order of procedure is common to all.

ON THE STAINING OF SECTIONS 85

The sections, whether single or in series, are transferred
from the alcohol to water, and remain in it until thoroughly
saturated, which serves as proof that they are freed from
alcohol and from any other fluid that may by chance
adhere to them ; after which they are subjected to the action
of the selected stain for from two to five minutes to
twenty-four hours. The time during which the stain must
be allowed to act may, however, be shortened by warming,
so far as this can be done without spoiling the tissue.
The preparation must now be washed in water as long as
any colour comes away from it. The various bleaching
agents are next used, and from them the preparation is
transferred to water, then to alcohol in order to dehydrate
it, and is finally cleared with xylol. It is advisable several
times to change the alcohol used for dehydrating. Xylol
is employed because it behaves in a completely indifferent
manner towards basic aniline colours, whether in nuclei
or bacteria, which is not the case with other clearing
reagents ; and moreover it evaporates without deposit,
never becomes resinous, and consequently does not 'soil
articles with which it comes in contact so much as does
oil of cloves. Besides xylol, oil of turpentine, aniline oil,
phenol, oil of bergamot, oil of cedar, oil of origanum, oil of
cinnamon, &c., are used. When the preparation has been
rendered sufficiently transparent by means of the xylol, it
is transferred to a slide and dabbed with blotting-paper,
a drop of Canada balsam in xylol is placed on it, and a
cover-glass applied.

Tlnna's drymg-on process (dry method). — Sections cut with
the freezing microtome are stained in a dilute alcoholic
solution of fuchsine, washed in water, laid for a short time
in alcohol, double-stained in methyl blue, dabbed with
blotting-paper, dried on a slide over the flame, and put up
in Canada balsam and xylol.

86 BACTERIOLOGY

Combination of staining methods. — The dyes are selected
in the same manner as when staining the bacteria from a
plate-culture or from a mixed mass of them ; and the com-
bination of several colours is indicated, because then that
of the bacteria stands out distinctly from the ground tint
of the tissue.

Kiihne's methyl blue method, — Kiihne, to whom the most
marked advances in the technique of staining are due, re-
commends as the most reliable method the staining of the
sections with methyl blue dissolved in a 5 per cent, carbolic
acid or a 1 per cent, ammonium carbonate solution. In
order to differentiate the preparations, they jare brought after
staining into a weak aqueous solution of lithium carbonate
or into slightly acidulated water, then dehydrated in abso-
lute alcohol to which some methyl blue has been added,
and transferred to aniline oil, similarly mixed with methyl
blue. Each section is then cleared by immersion in pure
aniline oil, next in a light fluid etherial oil, such as that
of thyme or terebene, and finally in xylol, and is mounted
in balsam.

For staining the bacilli of tuberculosis, leprosy, and
mouse septicaemia a method may be used which differs
from the foregoing only in the substitution of fuchsine for
the methyl blue.

Koch's method, — The sections after staining are trans-
ferred to a saturated solution of potassium bicarbonate
which has been diluted with an equal volume of water, and
thence to alcohol, cedar oil, and Canada balsam.

Lbffler's method. — Loffler stains the sections in an alka-
line solution of methyl blue, decolorises in half per cent,
acetic acid, and thence brings them into absolute alcohol,
cedar oil, and Canada balsam.

Chenzynsky's Method. — The sections are immersed in a
methyl blue and eosine solution containing forty parts con-

GRAM'S METHOD 87

centrated alcoholic solution of methyl blue, twenty parts of a
^ per cent, eosine solution in 70 per cent, alcohol, and forty
parts water, and after staining are rinsed in water, and the
remainder of the treatment carried out in the usual way.
Plehn recommends the addition of twelve drops of a 20 per
cent, caustic potash solution to the water.

Grain's method, — This method is in a high degree suited
for sections. They are stained in aniline water gentian
violet, to the action of which they are exposed for from ten to
thirty minutes ; but the time of staining may be shortened
by heating. After staining they are rinsed in water and
immersed for two to three minutes in a solution of iodine and
potassium iodide, and are then kept moving to and fro in
90 per cent, alcohol until no more colouring matter comes
away. The sections, which now appear of a slate-grey
colour, are next transferred to alcohol, cedar oil, and
Canada balsam. The bacteria are seen in violet on a
yellowish ground. Double-staining with picrocarmine or
Magdala red causes the violet tint of the micro-organisms to
stand out distinctly against the red colour of the tissue.

The method of Gram may also be reversed, and the
sections first stained for fifteen minutes in picrocarmine or
Magdala red, rinsed in 50 per cent, alcohol, and then laid
in aniline water gentian violet. After decolorising in
iodine solution, the preparation is treated with alcohol, oil,
and Canada balsam.

Giinther's modification, which is characterised by the
exposure of the sections, after decolorising in alcohol, to
the action of a 3 per cent, solution of hydrochloric acid,
yields brilliant results (compare p. 54).

Kiihne's modification of Gram's process, — Gram's method
has undergone many further modifications in its use for
sections, and Kiihne in particular has devised a number of
processes, of which the following are the most important.

88 BACTERIOLOGY

A solution is prepared of 1 grm. Victoria blue in 50
c.cm. of alcohol diluted to half its strength, and this is again
diluted to the same exten with a half per cent, aqueous
solution of ammonium carbonate. Staining lasts from one to
five minutes, and the sections are decolorised in iodine and
potassium iodide, and further treated as directed by Gram,
except that instead of alcohol a solution of fluoresceine
(1 grm. fluoresceine to 50 c.cm. absolute alcohol) is used for
extracting the colouring matter.

A further process consists in adding some hydrochloric
acid (1 drop to 50 grms. water) to a concentrated aqueous
solution of violet and using this to stain the sections, which
are otherwise treated as in Gram's method.

In using carbolic methyl blue the sections are stained
for from half an hour to two hours, rinsed in water mixed
with hydrochloric acid, passed through a weak aqueous
solution of lithium carbonate, and transferred from that
to absolute alcohol and to aniline oil, in both of which a
little methyl blue has been dissolved. After rinsing in
pure aniline oil they are cleared in an etherial oil which is
then removed with xylol, and are mounted in Canada
balsam.

Pragl recommends a modification of the carbolic methyl
blue method, which consists in staining the sections, fixed
to a slide or cover-glass, for from half to one minute in
carbolic methyl blue, after which they are rinsed in water
for a short time, decolorised in 50 per cent, alcohol, de-
hydrated in absolute alcohol, cleared in xylol, and mounted
in resin.

Another method which is easily applied consists in stain-
ing the sections for three to five minutes in carbolic fuchsine,
rinsing in water, and passing through alcohol. They are
then laid for a quarter of an hour to two hours in aniline
oil containing some methyl green, in order to decolorise and

KUHNE'S AND AVEIGERT'S METHODS 89

differentiate them, and after clearing with an etherial oil and
removal of this with xylol, are put up in Canada balsam.

In i}\Q fluoresceine and oil of cloves method the sections are
immersed for five to ten minutes in a concentrated aqueous
solution of oxalic acid, which acts as a mordant, rinsed in
water, and dehydrated in alcohol. The staining which
follows is done with fuchsine in aniline water, or methyl
blue dissolved in £ to 1 per cent, aqueous solution of
ammonium carbonate. To dehydrate, the sections are left
for five to ten minutes in absolute alcohol, to which is added
a little fuchsine or methyl blue as the case may be, and
differentiation is effected with oil of cloves containing
fluoresceine. They are thereupon cleared in etherial oil, the
.oil is extracted with xylol, and they are mounted in Canada
balsam. Sections stained in methyl blue are transferred
from the fluoresceine and oil of cloves to eosine and oil of
cloves before being brought into etherial oil.

Kiihne's dry method. — A one per cent, solution of ammo-
nium carbonate is mixed with a concentrated aqueous
solution of methyl blue, and this is allowed to act on the
sections for ten to fifteen minutes. They are then washed
in water, decolorised in an aqueous solution of hydrochloric
acid, again washed in water, dried upon slides, cleared in
xylol, and mounted in Canada balsam.

Weigert's iodine method, —By Weigert's method the
sections are stained in aniline water gentian violet, rinsed
in a solution of common salt, laid upon slides, and dried,
and solution of iodine is dropped on them. After they
have been again dried, aniline oil is poured over them
and renewed several times. It is then removed with xylol,
and the sections are mounted in Canada balsam.

A combination of Weigert's with Kiihne's violet method
(see p. 88) consists in staining the sections in a concen-
trated aqueous solution of violet to which some hydrochloric

90 BACTERIOLOGY

acid has been added (1 drop to 50 grms. water). After
staining, the sections are rinsed in water, decolorised in
iodine and potassium iodide solution, transferred to absolute
alcohol, treated with aniline oil and with xylol, and preserved
in Canada balsam.

Unna's borax methyl blue method, — A process which is
particularly to be recommended for tubercle and lepra
bacilli is the treatment of the sections for 5 minutes with
aqueous borax methyl blue, from which they are transferred
for 5 minutes more to a 5 per cent, solution of potassium
iodide to which a crystal of iodine has been added.
Rinsing in alcohol follows until a blue cloud forms, and
then differentiation in creosote, lasting from a few seconds
to half an hour, according to the intensity of the staining.
The sections are afterwards transferred to rectified oil of
turpentine, in which the bluish colour immediately changes
to red or brown, and are put up in a solution of colopho-
nium in oil of turpentine.,

Unna's method of demonstrating the organisms of the skin.
— Unna has devised several methods for staining micro-
organisms in furuncles and abscesses of the skin, which
can also be used to show the micro-organisms of pus. In
all these methods the sections are previously stained in
carmine and treated for two minutes with borax methyl
blue (1 part each of borax and methyl blue in 100 of water),
after which [in the first method^] they are rinsed in water
and placed for a few seconds in a 1 per cent, aqueous
solution of arsenic acid, then in alcohol, bergamot oil, and
balsam.

According to a second method the sections, after a slight
preliminary staining with carmine and methyl blue, are
brought for five to ten seconds into a 20 per cent, solution of
ferrous sulphate, then into alcohol so long as any colour
comes away, then for some seconds into a 1 per cent, solu-

EXPERIMENTS ON ANIMALS 91

tion of potassium binoxalate, and finally direct into absolute
alcohol, oil of bergamot, and balsam.

In the soap method the previously stained sections are
immersed in alcohol to which a few drops of Spiritus Sapo-
natus Kalinus l have been added, and then into pure alcohol,
bergamot oil, and balsam.

The chromic method consists in immersing the sections,
after previous staining, in a 1 per cent, solution of potas-
sium bichromate, washing them in water, and then trans-
ferring them for a considerable time into aniline oil, and
finally from that into bergamot oil and balsam.

Noniewicz's metliod. — Noniewicz combined Lofiier's and
Unna's methods of staining in order to show the bacilli of
glanders. The sections are transferred from alcohol to
methyl blue for two to five minutes, rinsed in water and
decolorised in a mixture of 75 parts of half per cent, acetic
acid and 25 parts of half per cent, aqueous solution of
tropseoline. Thin sections are only dipped quickly into the
solution ; thicker may remain in it for two to five seconds
or longer. After being washed in water they are spread
out upon a slide, dried in the air or over a flame, laid in
xylol to clear, and mounted in Canada balsam.

EXPEEIMENTS ON LlVING ANIMALS

Transmission of micro-organisms to animals. — So far a

series of methods of research has been described which are
necessary for the diagnosis of bacteria ; the observation of
micro-organisms in the recent state, of their growth on
different nutrient media, and of their behaviour in relation to
staining materials forms, when taken together, the methods
by which it is possible to demonstrate the micro-organisms

1 [The Spiritus Saponatus Kalinus of the Austrian pharmacopoeia con-
sists of 200 parts of potash soap and 100 parts of spirit of lavender, prepared
from lavender flowers by maceration and distillation.]— TR.

29 BACTERIOLOGY

in the tissues and fluids of the human body, as well as ex-
ternal to it. It still remains for us to give a brief account of
those methods which are employed to ascertain the special
significance of the different micro-organisms for the human
body, that is to say, to recognise by means of experiment
their pathogenic powers.

Amongst micro-organisms a distinction is drawn, as
we have learnt, between those which exercise a specific
injurious influence upon the bodies of men and animals, and
those which do not possess this property, although they may
perhaps occasion disturbances of various kinds by their
numbers ; the former being known as parasites, the latter
as saprophytes.

In order, then, to investigate micro-organisms with
reference to their power of causing disease, experiments
must be made by transmitting them to animals, for which
purpose monkeys, dogs, cats, hedgehogs, rabbits, guinea-
pigs, white mice, rats, marmots, poultry, pigeons, or even
frogs (kept at abnormally high temperatures) are used.
[To prove that a particular micro-organism is the specific
cause of a given disease it should be shown — 1

1st. That its presence can be detected with the micro-
scope in all cases of that disease.

2nd. That it is never found in any other disease.

3rd. That when isolated and cultivated through many
generations a culture inoculated on a susceptible animal
invariably produces a disease identical with that in the
animal from which the virus was taken, and

4th. That the same bacteria are found to be present in
the animal so inoculated.]

Transmission can easily be effected on the cutaneous
surface, or on the mucous membranes of readily accessible

1 [See on this subject Giinther's Einfilhr. in das Stud, der BakterioL,
pp. 139 et seq.t 2nd ed.]— Tn.

INFECTION BY THE DIGESTIVE CANAL 93

cavities, an experiment made in the latter way being often,
indeed, attended by better results than follow transmission
into small wounds of the skin ; but care must be taken that it
does not become possible for the animals to remove the micro-
organisms which have been introduced. Whether infection
can take place through the epithelial structures of the skin,
if unbroken, has not yet been finally decided.

Infection by the air ' passages. — Entrance can readily
be effected through the respiratory tract ; indeed, infection
seems to be able to gain admission with particular ease by
the internal surface of the lungs, especially sore ; the degree
of moisture all over the surface assists by fixing the micro-
organisms and enabling them to develop. For artificial
infection by the respiratory passages a spray apparatus is
used, by means of which the micro-organisms, suspended
in bouillon, reach their destination in the form of a fine
shower; but it is not easy to prevent the simultaneous
occurrence of a second infection, since during the process the
infectious matter may reach the intestinal canal by being
swallowed, or may be deposited on the skin. To render this
less easy of occurrence the excessively fine mist must be con-
ducted by means of a tube into a closed chest in which the
animal to be experimented on has been placed, so that it
can thus freely breathe in the micro-organisms suspended
in the air of the interior space.

Infection by the digestive canal. — Infection is communi-
cated through the intestinal tract either in the food or
directly by means of an cesophageal bougie, or the micro-
organisms may be introduced by establishing a gastric or
intestinal fistula. The best mode is, however, to hollow
out pieces of potato, fill them with the bacterial culture,
and push them so far back into the animal's pharynx that
they must be swallowed. Fluid infecting material is ad-
ministered to animals by means of cesophageal bougies

94 BACTERIOLOGY

introduced into the gullet — in the case of rabbits, through
the gap between their teeth, in that of guinea-pigs, through
a small perforated gag clamped between the incisors — the
bougie used being a soft elastic catheter. When it is
wished to infect an animal artificially the micro-organisms
must be introduced into the intestine, as the acid gastric
juice frequently impairs their vitality.

Nicati and Eietsch in their experiments on cholera
injected the infecting liquid directly into the duodenum,
which they had laid bare by a laparotomy performed with
the strictest antiseptic precautions. Koch recommended
the following mode of procedure for the purpose of excluding
the injurious effect of the gastric juice on the micro-
organisms : — A wooden gag perforated in the centre having
been introduced into the mouth of the animal, a sound is
inserted through it, and 5 c.crn. of a saturated solution
of sodium carbonate is injected to neutralise the acid gastric
juice. One grm. of tincture of opium for every 200 grms. of
body-weight is then injected subcutaneously, in order to keep
the animal in a state of narcosis, after which cholera-bacilli
suspended in bouillon are injected by means of an oesopha-
geal tube, and the experiment of introducing infection by
the intestinal canal is complete.

Subcutaneous infection. — Inoculation can also be per-
formed subcutaneously by introducing the infecting matter
beneath the skin with a Koch's syringe. In the case of small
animals, such as white mice, the hair of the back in the
neighbourhood of the tail is carefully removed, a minute
incision is made into the skin with disinfected instruments
(forceps and scissors), and the infecting matter introduced
subcutaneously with the help of a sterilised platinum loop.

Experiments of transmission into the peritoneal and
pleural cavities, or into the organs themselves, are con-
ducted after a similar fashion.

INFECTION INTO THE EYE 95

Intravenous infection, — This is most conveniently done
into one of the superficial veins of the neck, or by puncture
of an aural vessel.

Infection into the anterior chamber of the eye. — One of
the most elegant modes of inoculation is the introduction
of micro-organisms into the anterior chamber of the eye.
This is done by opening the chamber with a lancet entered
at the junction of the cornea and sclerotic, and introducing
the infecting material through the wound so made. The
aqueous humour which flows away is soon restored after
cicatrisation of the wound, while the multiplication and
visible peculiarities of the micro-organisms can be observed
through the transparent cornea.

96 BACTERIOLOGY

CHAPTER V

THE BACTERIOLOGICAL ANALYSIS OF AIR

Micro-organisms in the air, — Floating in the air are
particles of dust consisting of organic substances, amongst
which are also to be included, as a rule, dried-up colonies
of micro-organisms. Such may either sink downwards
of themselves under the influence of gravity, and so be
caught, or they may be obtained by calling in the aid of
currents of air, but in all cases they must be transmitted
to a suitable nutrient medium before they can develop.
As a rule we find in the air moulds, yeasts, and the spores
of bacteria. On the open sea, far out from shore, the
number of micro-organisms is considerably smaller, and in
like manner the air on high mountains is almost entirely
free from germs, or at least there are but few, whereas on
the plains 100 to 500 germs capable of living have been
counted in each cubic centimeter. The air of dwelling-
rooms contains them in considerable numbers only when
they have been whirled up from between the flooring and
from the coatings of the walls, and this detachment of
bacteria by draughts of air can only take place when the
surfaces are dry.

Simple methods of examining air. — The simplest way of
examining air consists in letting a plate prepared with agar
or gelatine stand in any locality for a definite time, and
afterwards placing it in a moist chamber, when colonies of
micro-organisms will form in a few days. Agar plates

POUCHET'S METHOD OF ANALYSIS

97

may also be placed in the incubator, in order to observe the
micro-organisms which develop at a higher temperature.

The method can be simplified by pouring the gelatine
into capsules, which, after catching the germs from the air,
are closed and kept. Such capsules may be exposed in a
glass vessel of cylindrical form, the volume of air in which
is known ; after a fixed time the process can be stopped,
and the capsules with the gelatine set aside for the organisms
to develop. Knowing the volume and the time of exposure,

Connecting tube

Aspirator

FIG. 28. — POUCHET'S AEROSCOPE.

it is possible to gain an approximate idea of the number of
micro-organisms contained in the air.

Pouchet's method. — Pouchet employed for the examina-
tion of the dust of the air an aeroscope consisting of a glass
cylinder, capable of being closed air-tight by means of a
screw and clamps ; it is placed vertically upon a stand and
perforated above and below. In the upper aperture is a
glass tube with a very narrow exit, the lower one communi-
cates with an aspirator through an indiarubber pipe, and
in the centre of the cylinder is a little table supporting a

n

98 BACTERIOLOGY

small glass plate, which is smeared with glycerine. The
aspirator being put in action, air streams in through the
upper aperture and deposits the greater part of the dust it
contains upon the glycerine, and the preparation is removed
from the cylinder and examined as soon as sufficient air has
been drawn through. The dunt is distributed as evenly as
possible through the glycerine by stirring with a sterilised
steel needle, and the glass plate is covered with a second and
brought under the microscope. To calculate the amount of
dust in a litre of air, the particles in several microscopic fields
are counted, so as to ascertain the average number in each ;

Glass head closed with
cotton wool

Tube connected with
the aspirator

Glass flask

FIG. 29. — MIQUEL'S APPARATUS FOR EXAMINING AIR.

from this the number spread over the whole plate is calcu-
lated, and thence the amount contained in a litre.

Instead of the glycerine plate one of gelatine or agar
may be laid on the little table, and an attempt thus made
to isolate the micro-organisms (fig. 28) .

Miquel's method. — Miquel constructed a flask with two
lateral tubes (fig. 29) and another fitting by a ground joint
into the aperture at the top, and supporting a cap or head of
glass closed with a cotton-wool plug. One of the lateral
tubes is connected with an aspirator, the other (by means of
a piece of rubber piping) with a narrow glass tube sealed at
one end. The flask is filled with 30 to 40 c.cm. water, and
sterilised in the steam current ; the glass cap is then taken
off and a given volume of air aspirated through, after which

EMMERICH'S METHOD 99

the cap is again put on, and, by blowing air through the
lateral tube which was connected with the aspirator, the
fluid is driven up into the vertical one, so as to wash it out.
Finally the point of the glass tube on the opposite side is
broken off, and the fluid contained in the flask distributed
into tubes of bouillon.

Emmerich's method. — In the apparatus devised by Em-
merich for bacteriological research of this nature, the air
is drawn slowly through a coiled tube filled with nutrient
bouillon, and the germs are in this manner retained (fig 30) .

Aspirator tube

Plug of cotton wool —

Coiled tube containing
nutrient bouillon

FIG. 30.— EMMERICH'S APPARATUS FOR EXAMINING Am.

Welz's method. — Two small flasks, one as a receiver,
and the other as a control flask, are prepared with 20 c.cm.
each of a neutral liquid composed of equal parts of glycerine,
bouillon, and water, and are connected together by means
of a glass tube bent twice at right angles, the longer limb of
which reaches to the bottom of the control flask, the shorter
to just below the stopper of the receiving flask. Two large
flasks connected by means of a rubber tube are used for
aspirating, one being filled with water and united to the
controlling flask. The other, which is empty, stands at a
lower level than that containing the water, so that this

H 2

100 BACTERIOLOGY

may be able to flow into it ; and in this way a volume of
air, corresponding to the quantity of water used, is aspirated
into the receiving bottle. For the purpose of regulating
the flow, two little glass tubes, drawn out to fine points,
are fixed in the india-rubber tube which connects the two
aspirating bottles. Cultivation is effected by conveying 1
c.cm. of the fluid in the receiving flask (after it has been
thoroughly mixed) by means of a sterile pipette into 10
c.cm. gelatine, and pouring this out on plates.

Hesse's method. — In this method the air is caused to
pass by means of a small slowly-acting aspirator through a
disinfected tube, the walls of which are coated with gelatine
after the manner of Esmarch's roll cultures. This tube is
70 cm. long, and has a diameter of 3 to 4 cm. ; it is placed
horizontally, and covered at one end with a tightly- stretched
rubber cap having a round piece cut out of the centre, and
over which a second cap, not perforated, can be drawn ;
while the other end is closed with a caoutchouc cork, bored,
and fitted with a small glass tube about 1 cm. wide and
10 cm, long, connected with the aspirator. While the air
is being aspirated, the unperforated cap must be removed.
Two bottles are used by way of aspirator, as in Welz's
method, one filled with water, the other empty (fig. 31).
The bacteria develop chiefly in the fore part of the tube,
while the spores of moulds, being isolated and therefore
lighter, are carried further and develop further on in the in-
terior. When air is examined which presumably contains
but few germs — for example, air out of doors during a calm—
10 to 20 litres are drawn through, but if it is probable that
large numbers are present only 1 to 5 litres are aspirated.
The process is concluded by replacing the unperforated
rubber cap. In a few days the gelatine is seen to be
covered with colonies which can be distinguished from one
another by their form, their colour, and their action on the

METHOD OF STRAUSS AND WURZ

101

gelatine (liquefaction). The germs may then be isolated
by further transference to culture plates, and submitted to
microscopic examination.

Glass tube

J lubber cork

Aspirator

FIG. 31. — HESSK'S APPARATUS FOR EXAMINING AIR.

Method of Strauss and Wiirz. — Air is drawn into a glass
vessel full of liquid gelatine by means of a tube affixed to

Aperture for the admission of air

Vessel filled with gelatine

Lateral tubuluie

FIG. 32.— Aiu-TKSTiNG APPARATUS OF S'IRAT ss AND WURZ.

the side and connected with an aspirator (fig. 32). A large
volume of air, 100 to 200 litres, is thus tested, and when
the aspiration is concluded the gelatine is poured out on

102 BACTERIOLOGY

plates, or a roll-culture is made after Esmarch's method in
the vessel itself.

Petri's Method. — A sand-filter is prepared and carefully
sterilised. This consists of a tube 8 or 9 cm. long and 1'5
cm. in diameter, into which sand is introduced after one
end has heen closed with wire netting. When the layer of
sand has reached a depth of 3 cm. another wire netting is
laid on it, [another layer of sand introduced, and a third
netting last of all], so that the tube is now provided with two
sand-filters kept together by wire gauze. Quartz-sand, each
grain of which is £ to ^ mm. in size, is the best. About 50
to 100 litres of air are drawn through with a water aspirator

Sand-filter -=^ 3=s=*Win; netting

Aspirator tube

Receiving bottle _"~"

FIG. 33. — PETRI'S SAND-FILTERING APPARATUS.

at the rate of some 10 litres per minute, the quantity being
determined by means of a gas meter. When aspiration of
the air is concluded, each sand-filter is partitioned out
separately into several glass capsules prepared with nutrient
gelatine or some other solid culture medium. The second
layer of the filter should be free from germs.

Miquel uses powdered sodium sulphate to absorb the
microbes instead of sand.

Tyndall's method, &c. — Sterilised cotton wool is used
for absorbing the micro-organisms, instead of air-filters con-
sisting of substances in the form of powder, and is then
transferred to gelatine, and plate cultures made therefrom.

PENICILLIUM GLAUCUM 103

Percy Frankland ^ises glass wool instead of cotton.

With the aid of these methods, moulds, yeasts, micro-
cocci, bacilli, and spirilla are found, all of which are con-
tained in greater or less quantity in the air, though their
distribution is not the same in all parts of the earth's sur-
face, nor at all times, either as regards quantity or quality.
For example, the author found that the Micrococcus pro-
digiosus grew in abundance on a paste medium in the Alps
(Hollenegg, Styria) in the month of September, 1891,
whereas in the months of July and August in the same
year no perceptible trace could be found.

Penicillium Glaucum. — The Penicillium glaucum, or pen-
cil fungus, grows in the form of locks of cotton-wool, and
during sporulation forms a green fur of a peculiar musty
odour. Its mycelium consists of horizontally-arranged,
straight, or slightly undulating jointed filaments, from
which the spore-bearing hyphce (Frucliihypheri) stand
vertically up, dividing at their upper ends into forks
(basidia) from which fine processes branch off (sterig-
mata) in the shape of a hair pencil, and are segmented at
their ends into rows of fine globular bodies (spores or
conidia), which in the mass give the fur its green colour (see
fig. 4). Sterilised bread-pap is particularly well adapted
for the growth of the pencil fungus, which forms a fur upon
it, white at first, but afterwards taking on a fine green
colour ; but besides this it grows in all sorts of places where
as a rule only mould can develop. Gelatine is liquefied by
it. The growth of mycelium takes place very well accord-
ing to Wiesner at a temperature of 26° C. ; sporulation
progresses best at 22° C.

The fungi appear on plate cultures first as threads
diverging from a point, and do not form sharply-defined
dark-coloured colonies upon the gelatine, but radiate out
over a considerable extent of surface. The spore-bearing

104 BACTERIOLOGY

hyphae which rise free above the level of the gelatine are
put in motion by currents of air (such as lightly blowing
upon the plate culture), and when this occurs the shedding
of the spores can be readily observed. The earliest forma-
tion of spores occurs in the centre of the colonies, and is
indicated by a green coloration.

Loffler's methyl blue stains the filaments of mycelium
and the hyphse, the spores on the other hand remaining
unstained. Moulds cannot easily be moistened with water,
as their surface has no affinity for it, owing to the presence
of a thin coating of fat ; hence the first proceeding is to
treat the unstained moulds with alcohol to which a little
ammonia has been added, after which they are examined in
glycerine and water or plain glycerine. For making per-
manent preparations, glycerine or glycerine jelly is suitable,
the cover-glass being cemented with asphalt lac dissolved
in turpentine over a water-bath.

Hansen recommends the addition of 0*1 to 0*2 per cent,
of hydrochloric acid when growing moulds upon gelatine, in
order to keep away bacteria.

Brown mould. — The fur formed by the brown mould de-
scribed by Hesse is brownish yellow, and is further distin-
guished from penicilium by its closely felted mycelium, the
hyphse being scanty, ramified, and segmented. Gelatine
is very rapidly liquefied, and in thrust-cultures becomes
softened with the mycelium of the fungus into a brown,
viscid, stringy mass. Growth takes place best at 15° to 20° C.
According to Trelease the mould is identical with an alga,
the Cladothrix dichotoma, very frequently found in water.

Yeast. — This micro-organism consists of cells and
masses of cells of which the individual elements possess an
oval figure and multiply by gemmation. They have a
thin limiting membrane and a granular protoplasm con-
taining vacuoles (see fig. 6). They are obtained from the

YEASTS IN THE AIR 105

air upon gelatine or agar plates, or upon sterilised potatoes,
and grow on the first-named in the form of round colonies
raised into knobs of a drop-like appearance, which do not
liquefy the medium.

Pink yeast is distinguished by the rose-pink colour of
the mass, and shows on gelatine-plates small round, rather
coarsely-granulated, rose-coloured colonies. In thrust cul-
tures there appears after eight days a coating with a dull
surface like a drop of wax, which slowly increases in cir-
cumference and shows raised edges, while a row of little
dots forms along the track of the thrust. The gelatine is
not liquefied. On agar there grows an irregular thin slimy
coating of a pink colour, and on potato a deposit of a beau-
tiful rose-red tint.

Black yeast and white yeast only differ in the colour of
the coatings formed by them.

Yeast grows at the temperature of an ordinary room.
When stained with aniline colours the cells shrivel some-
what, and do not show the fine figures seen if they are
examined in the unstained condition. They can easily be
distinguished from cocci by their remarkable size (1*5 ^ to
3 //, long, 2 yu, broad).

Micrococcus radiatus. — The Microcpccm radiatus, iso-
lated by Fliigge, forms small cocci in short chains or
clumps. On gelatine plates little yellowish-brown colonies
first appear, from which outgrowths push forth in a radial
direction, and in thrust-cultures rays are seen running out
horizontally from the centre of the track, so that it ac-
quires an almost feathery appearance. The gelatine is
slowly liquefied. Colonies of a yellowish colour form on
potatoes.

Micrococcus versicolor. — This micro-organism, which
was also found by Fliigge, is distinguished by the mother-
of-pearl gloss seen on its colonies. It forms minute

106 BACTERIOLOGY

clumps of cocci, which appear on the very first day as
round white points on the gelatine plate, and these after
several days become yellowish-brown and in certain posi-
tions of the plate shimmer like mother-of-pearl. The latter
appearance is also seen on the surface of a thrust-culture
as well as on superficial cultures upon agar. The shimmer
sometimes strikes into a yellowish -green. Gelatine is not
liquefied.

Micrococcus cinabareus. — The microbe so named by
Fliigge, and which is noticeable on account of its cin-
nabar colour, forms diplococci. On gelatine plates the
colonies do not appear for four to six days, and are reddish-
brown ; but the colour gradually changes to vermilion. A
thrust- culture also shows the colour on the surface in the
form of a red knob, from which a white stripe extends into
the gelatine, which is not liquefied.

Micrococcus flavus tardigradus of Fliigge appears in
large single elements, and forms colonies which become
yellow in six to eight days. In the thrust-canal isolated
and disconnected yellowish balls develop. It is distin-
guished by its yellowish colour, and does not liquefy gela-
tine.

Micrococcus candicans (Fliigge) often appears as a con-
tamination on gelatine plates, forming white colonies which
are darker in the centre and lighter towards the margin.
Thrust-cultures are nail-shaped with a nodular elevation.
Gelatine is not liquefied.

Micrococcus viticulosus. — This micro-organism, described
by Katz, consists of oval elements, and is particularly cha-
racterised by the tendril-like shapes of its colonies. These
appear both on and below the surface of the gelatine plates,
and send out lateral outgrowths in the form of fine pro-
cesses resembling tendrils. In thrust-cultures it grows
along the track of the puncture, from which a delicate net-

M1CROCOCC1 IN THE AIR 107

work of fibres likewise runs into the substance of the gela-
tine without liquefying it.

Micrococcus urese. — This is a micro-organism which
does not liquefy gelatine, and which, although it occurs in
the air, can also be obtained from decomposed ammoniacal
urine. It was described by Pasteur and Van Tieghem.

The cocci are for the most part arranged in pairs, and
when they have grown for a considerable time upon gelatine,
show a rather large-sized colony
which is raised above the surface
like a drop of solidified stearine,
and diffuses a smell like that of
paste. It sets up fermentative Fll.t 34._ MICKOCOCCUS

. , (After Jaksch.")

processes in urine, owing to its
property of converting urea into ammonium carbonate.
Growth takes place at room-temperature, or best at 30° C.,
but it does not lose the power of development even at
temperatures below zero (fig. 34).

Micrococcus roseus. — This is a micrococcus occurring in
gonorrhceal disease of mucous membranes, and was found
in the air by Bumm. The cocci are immotile, and are
arranged in pairs, each half of the diploccocus so formed
being hemispherical, and separated from the other by a
fissure. The small colonies formed by it on the gelatine
plate are rose-red and raised above the surface. Thrust-
cultures show liquefaction of the gelatine, but only after a
considerable time, and a rose-red colour then forms at the
bottom of the needle-track.

Diplococcus citreus conglomeratus. — This micro-organism
was found by Bumm in the dust of the atmosphere, with
which it probably enters the human organism. Its elements
are not motile, and are sometimes arranged in pairs, at
other times in fours. They form small oblong colonies on
the gelatine plate, which soon become fissured, and possess

108 BACTERIOLOGY

a lemon-yellow colour ; the thrust-culture slowly liquefies
the gelatine, a yellow mass being found in the deeper part.
Surface cultures on agar exhibit a lemon-yellow coating,
which later becomes brownish.

Micrococcus flavus liquefaciens and Micrococcus desidens.
— Both have been described by Fliigge. The former has
larger, the latter smaller elements frequently arranged as
diplococci, and both form yellowish-coloured collections on
discs of potato. On gelatine plates small round yellowish
colonies occur, which begin to liquefy the gelatine in from
one to two days. Thrust-cultures liquefy in a few days,
earlier in the case of Micrococcus liquefaciens than with
Micrococcus desidens, and when the elements have sunk to
the bottom of the funnel-shaped fluid area, a slight yellow
coloration forms below.

Sarcina alba. — The Sarcina alba grows slowly on gelatine
plates in little round white colonies, and in the same manner
along the track of the thrust in the test-tube, forming in
the latter a small white head on the surface. It also grows
very slowly on potatoes, in the form of a whitish-yellow
deposit round the site of inoculation. Gelatine is very
slowly and only very slightly liquefied,

Sarcina Candida. — The microbe of this name, found by
Reinke in breweries, shows shining white colonies on
gelatine, which later become yellowish and very soon
liquefy. On agar there appears a white deposit with smooth
edges.

Sarcina aurantiaca. — Small smooth-edged colonies appear
on the gelatine plate, having a dotted granular aspect when
seen under a low power, and an orange-yellow colour.
Thrust-cultures liquefy slowly along the entire track, and
excrete an orange-yellow pigment at the surface ; but when
they have stood for a longer time, the principal mass sinks
to the bottom and the superficial part of the medium

S ARCING IN THE AIU 109

becomes clear. Agar cultures also show a fine golden-
yellow glossy coat. The sarcina grows slowly on potato.
Gelatine is but little liquefied. Sulphuric acid turns the
golden-yellow pigment bluish-green, and caustic potash,
red.

Sarcina rosea. — This micro-organism, which was dis-
covered by Schroter, grows very rapidly on gelatine, slowly
on agar, forming minute cartilaginoid clumps ; while a
vigorous, intensely red deposit forms on potato. Multi-
plication takes place in broth with extraordinary rapidity,
and with development of a red sediment. Gelatine is very
speedily liquefied. The red pigment exhibits the same
chemical reactions as the colouring matter of Sarcina
aurantiaca.

Sarcina lutea. — The sarcina of this name, described by
Schroter, grows very slowly on the gelatine plate, forming
small round colonies. A scanty coating appears diffused
over the surface, and advances into the deeper parts over
a narrow area in the form of yellow7 granules. A thickish
deposit of a fine yellow colour appears upon agar. Cultures
on potato are sulphur-yellow, and confined to the place of
inoculation. Gelatine is sometimes liquefied slowly.

Staphylococci. — The Stapliylococcus pyogenes was fully
described by Eosenbach, by Ogston, and by Passet. Its
distinctive characteristic is its power of causing suppuration,
and it may consequently be described as a specific pus-
coccus, being constantly found in suppurative processes.
There are distinguished, according to colour, a Staphylo-
coccus pyogenes aureus, a Stapliylococcus pyogenes albus, and a
Stapylococcus pyogenes citreus. It is but seldom in the
analysis of air that a plate culture destitute of staphylo-
cocci is obtained.

According to E. Ullmann, these micro-organisms are
found in considerably greater numbers in the air of rooms

110 BACTERIOLOGY

which are much used than in localities but little frequented
by human beings. Ullmann also found them very widely
diffused elsewhere in nature, not only in the air but in
river-water and rain-water, though not in spring-water ;
also in ice, in the earth, and on walls.

They are small globular immotile cells, always tend-
ing to form closely-packed clusters, particularly in the

interior of tissue (fig. 35). Those
cells, however, which are not in-
cluded in the clusters possess the
power of moving with tolerable
activity. The individual cells take
up all the different aniline dyes,
FIG. 35.— STAPHYLOCOC grow even at ordinary tempera-

PYOGENES ATJKEU8. . at f n

tures, though more energetically at

degrees of heat approaching that of the human body, and,
if added to sterilised milk, precipitate the casein e.

The Staphylococcus pyogenes aureus grows very quickly
on the gelatine plate at the temperature of an ordinary

Smallest islets

Larger
islets

FIG. 36. FIG. 37.

ISLETS op STAPHYLOCOCCTTS PYOGENES AUREUS ON A GELATINE PLATE.

room, so that even as early as the second day small puncti-
form colonies are to be seen, which are round and possess
a sharply- defined circumference, and these soon approach
the surface and liquefy. The liquefaction extends out at
the periphery, and soon shows a yellowish colour in the
centre (figs. 36 and 37). In the thrust-culture the gelatine
begins to undergo liquefaction on the second or third day,

STAPHYLOCOCCUS PYOGENES AUKEUS

111

and this gradually advances deeper along the thrust-canal,
while, as the funnel-shaped liquid area enlarges, the cocci

Liquefied part
Collection of bacteria

Needle-track in the
non-liquefied part

Liquefied part of the

gelatine

Mass of bacteria collected
at the bottom of the
liquid funnel

tFiG. 38.— THRUST-CULTURES IN GELATINE OP STAPHYLOCOCCUS TYOGENES AUREUS ;

— TWO DIFFERENT FORMS OF GROWTH.

sink to the bottom and begin to take on colour (fig. 38).
When, however, the culture is exposed, not to ordinary tern-

1 1 2 BACTERIOLOGY

perature, but to a higher degree of heat, although, indeed,
the growth is more luxuriant, there is not so fine a forma-
tion of colour as at the former temperature. This is true
particularly of surface cultures on agar, in which a thick
column forms at first along the streak, and then gradually
spreads out further so as to cover the surface of the agar
with a complete coating of culture displaying the charac-
teristic colour. On potatoes there occurs a deposit which is
at first whitish but afterwards takes on a yellow or orange hue.

Staphylococci also grow excellently on serum and the
white of plovers' or pigeons' eggs. All cultures of them
very soon develop a strong smell of paste, which, as the
age of the cultures advances, is modified to an odour re-
sembling that of sour milk.

Successful infections have repeatedly been made with
staphylococci. When brought upon the surface of wounds,
they set up a progressive suppuration, while subcutaneous
injections originate abscesses, and injections into the circu-
lation cause inflammation of joints and abscesses in the
kidneys and myocardium. According to Orth, Wyssoko-
witsch and Eibbert, they set up an ulcer ative endocarditis
on diseased or perforated cardiac valves. All these phe-
nomena are dependent either on the occurrence of a
mechanical derangement of the vascular areas by the
micro-organisms, or on the development of metabolic pro-
ducts having a toxic action on the tissues. In addition to
entering by wounds, the staphylococci can find their way
into the cutaneous and subcutaneous tissue from the hair
follicles and the ducts of the cutaneous glands.

The Staphylococcus pyogenes albus is distinguished from
the last only by the absence of pigment ; it appears to be
less energetic in its action. Staphylococcus pyogenes citreus
differs also in colour, and liquefies gelatine more slowly than
either of the other two.

STREPTOCOCCUS EEYSIPELATIS 113

Leber isolated the active principle from the cultures in
the form of a crystalline body, to which he gave the name
ofphhgosin, and which, if injected in small quantity, leads
to suppuration without the presence of micro-organisms.
Christmas obtained a pyogeiiic body from the cultures in the
shape of a substance of the nature of a ferment, which set
up suppuration when introduced into the anterior chamber
of a rabbit's eye. E. Ullmann caused osteomyelitis by intra-
venous injection of dead cultures after a previous fracture.

Streptococci. — Emmerich and Hartmann succeeded in
isolating a streptococcus from the air, which, when inocu-
lated on rabbits, set up a typical erysipelas, and is there-
fore described as Streptococcus erysipelatis. A pure culti-
vation was first obtained by Fehleisen. Gelatine is not
liquefied. On the plate small colonies appear in the sub-
stance of the gelatine on the third or fourth day, and
gradually assume a brownish colour. In thrust cultures
the superficial growth is very scanty, but along the needle-
track very minute white globular colonie£ appear, forming a
white stripe. Small round isolated colonies develop upon
agar, resembling drops of dew. No growth takes place on
potatoes.

According to Jordan, Erankel, and Von Eiselsberg, it is
identical with the Streptococcus pyogenes (see p. 201).

Bacillus subtilis. — This bacillus, also called the hay
bacillus, was described by Ehrenberg, and is most easily
obtained from an infusion of hay made by chopping up the
hay, pouring water on it in a flask, and bringing it once to the
boil. In this way all the other different micro-organisms are
easily killed, the hay bacillus alone suffering no impairment
of vitality. After two or three days a thick whitish pellicle
forms on the surface, and consists of a pure culture of the
Bacillus subtilis. The bacillus takes the form of very long, fine
thin rods, possessing marked power of movement by means

i

114 BACTERIOLOGY

of flagella, and a disposition to unite into groups. Owing to
their motility, the bacilli, or the threads formed by them,
are seen to dart with an undulating motion across the field
of the microscope. On the gelatine plate little white dots
occur, which soon extend and liquefy the gelatine over a
still wider surrounding area, while around the liquefied
mass fibres of bacilli are moreover seen growing into the
gelatine in the form of a halo. Thrust-cultures likewise
show an energetic liquefaction (fig. 39), and as soon as the
gelatine in the test-tube has become completely fluid a coat-
ing or pellicle forms on the surface. An extensive growth
develops upon agar, and on potato there appears a creamy
deposit, which in a few days takes the colour of wine.
Serum and the coagulated albumen from plovers' and
pigeons' eggs are liquefied, and on these also the superficial
formation of membrane is very marked. According to
Wyssokowitsch, if the spores are introduced into the circu-
lation they expand into,, rods, and remain lying in the liver
and spleen without exercising any influence on the organ-
ism. According to Vandervelde, the Bacillus subtilis sett
up active fermentation of sugar.

Bacillus prodigiosus. — The Bacillus prodigiosus, which is
especially remarkable on account of the development of a red
pigment, falls from the air at certain times upon substances
containing starch, on which it grows with tolerable rapidity .
and it has thus given origin to the legends of showers of blood.
The rods are so very short that their long diameter scarcely
exceeds their breadth, and for this reason the bacillus wa^
formerly classed with the rnicrococci. The individual rod:-,
are motile. On acid nutrient media, however, they expand,
according to Rubier, into larger bacilli, which also possess
the power of motion. They form spores. On gelatine platen
they show even after ten or twelve hours small round
granular colonies, which soon liquefy from the surface

BACILLUS PRODIGIOSUS

115

downwards and coalesce with one another if they lie closely,
the diagnosis being established by the early appearance of a

Funnel-shaped
area of lique-
faction

Funnel-shaped
area of lique-
faction

FIG. 39.— THRUST-CULTURE IN GELATINE
OF BACILLUS SUBTILIS (THIRD DAY).

Needle-track in
the unliquefied
part

FIG. 40.— THRUST-CULTURE IN GELATINE
OP BACILLUS PRODIGIOSUS (FOURTH DAY).

red colour. The thrust-culture liquefies from the surface
down, and soon a funnel-shaped area of liquefaction is

i 2

116 BACTERIOLOGY

formed, upon the surface of which the pigmentation takes
place, owing to contact with the air, and then sinks gradually
downwards (fig. 40) . A beautiful purple-red colour develops
on the surface of streak-cultures on agar, but the finest
growth takes place upon slices of potato or wafers, on which,
moreover, it progresses very rapidly at the temperature of the
room, being less luxuriant at higher degrees of heat. The
bacillus liquefies serum, and soon appears on plovers' egg
albumen with a beautiful rose-red colour, which extends only
as far as the coagulated mass has become liquid. The
coating shows a punctiform appearance under a low power.
The spectrum of the pigment, which is readily soluble in
water, alcohol, and ether, has three absorption bands, one to-
wards the violet end of the spectrum at [Fraunhofer's line]
D, one at E, and another at r. The red colour becomes
brown in the air, owing to the action of ammonia, but
recovers its raspberry-red colour if acetic acid be applied.

Wyssokowitsch, and quite recently E. Ullmann, have
proved that dead cultures of this bacillus are capable of
exciting suppuration, and Grawitz and De Bary found that
its pathogenesis is connected with the pigment.

Potato bacillus. — Three varieties are distinguished : Bacil-
lus mesentericus fuscus (Fliigge), Bacillus mesentericus ruber
(Globig), and Bacillus mesentericus vulgatus. They show
short filaments which are often connected together into
chains, and have the power of active movement. ,pThey
liquefy gelatine very quickly during their growth, whether
on the plate or in thrust-cultures, and form round colonies
which soon become yellowish, and in the case of the brown
bacillus (Bacillus mesentericus fuscus) assume a dark brown
colour. The liquefied gelatine, which swarms with bacilli,
also darkens (fig. 41). Upon discs of potato they grow
very luxuriantly, and soon spread from the upper to the lower
surface. The Bacillus mesentericus ruber shows at a higher

BACILLLUS MESENTERICUS 117

temperature (of about 37° C.) a reddish-yellow or rose-red
colour. The individuals of all the varieties included under
the name of potato bacillus adhere together and form an ex-
tensive wrinkled membrane, which can easily be detached
from the slice of potato. The Bacillus mesentericus vulgatus
has the property of curdling milk, as rennet does, and render-
ing it stringy, the substance to which it owes its viscidity
being probably metamorphosed cellulose. It displays upon
the whole the same behaviour towards gelatine and agar that
the two other potato bacilli do, but whereas the cultures of
Bacillus mesentericus fuscus have a yellowish colour, and
those of the ruber variety a reddish, the membrane on the

Peripheral radiating

processes

Liquefied part
FIG. 41. — ISLET OP BACILLUS MESENTERICUS VULGATUS ox A GELATIXE PLATE.

potato shows no pigmentation at all in the case of Bacillus
mesentericus vulgatus.

The potato bacillus develops with particular readiness
on pieces of potato which are not completely sterilised, often
destroying the cultures of other micro-organisms.

Bacillus liodermos. — Fliigge found very widely distributed
in the atmosphere, and often as a guest upon our nutrient
materials, short, exceedingly motile rods, the growth of
which on gelatine causes it to liquefy with great rapidity, a
white pellicle floating on the surface. In thrust-cultures
dirty grey flakes swim about in the fluid mass. A smooth,
glossy coat resembling thin mucilage develops on potato,
changing as the spores form into a thick and much-
wrinkled membrane. The mucilaginous mass is soluble in

118 BACTERIOLOGY

water. The Bacillus liodermos grows with especial luxuri-
ance in milk.

Bacillus melochloros. — The Bacillus melochloros was origi-
nally discovered in the author's Institute by F. Winkler
and Yon Schrotter, in the caterpillars' excreta found in
worm-eaten apples. It is at times a constant inhabitant
of the air of the author's laboratory, and often appears as
a guest upon cultures of other micro-organisms ; and it is
possibly identical with the Bacillus butyri fluorescens, found
by Lafar in butter. It consists of slender, fairly long rods,
with smoothly rounded ends and actively motile, and is
distinguished by its unusually rapid growth, so rapid that
even -in four hours there appear on the plate greyish-white
colonies, in which darker and more closely-packed masses
are to be seen ; while as early as the second day the gela-
tine is liquefied with development of a greenish-yellow
colour. In thrust-cultures also, on the second * day, an
hourglass- shaped depression shows itself, around which
there is very rapid liquefaction (fig. 42). The speedy
growth and greenish-yellow colour are also seen in super-
ficial cultures on agar, the surface of which very soon
becomes overspread with a thick yellowish coating, while
all the rest of the medium acquires a green tinge. On
plovers' egg albumen it grows with a splendid emerald
green colour, and on potato it forms a dirty reddish-yellow
layer. The pigment developed by the Bacillus melochloros
is very readily soluble in water, but not at all in alcohol or
chloroform. It is destroyed by acids, but restored again by
alkalies. Older cultures acquire an exceedingly unpleasant
odour. When the pure culture is injected into the veins or
peritoneal cavity of rabbits the animals perish in a week at
furthest.

Bacillus multipediculosus, which was discovered by Fliigge,
shows small thin immotile rods. The colonies on a gela-

BACILLUS MULTIPEDICULOSUS

119

tine plate appear under a low power as circular, sharply -
denned discs with radiating processes, resembling insects

Hour-glass
depression

• Liquefied
portion

. Superficial
coating

Xeedle-track

.Needle-track

FIG. 42.— THUUST-CULTURE IN GELATINE
OF BACILLUS MELOCHLOROS (SECOND

DAY).

FIG. 43.— THRUST-CULTURE IN GELATINE
OF EMMERICH'S BACILLI'S (Bac. Nea-
politanus).

with numerous radially arranged feet. In thrust -cultures
also the processes appear extending from the needle-track

120 BACTERIOLOGY

in all possible directions, and these peculiar projections
have procured for this micro-organism its name of ' multi-
pediculosus.' Gelatine is not liquefied.

Bacillus neapolitanus. — This microbe was first discovered
by Emmerich in the blood and in evacuations from the
corpses of cholera patients in Naples, and it was subsequently
ascertained to be present in normal faeces. Pathogenic
powers were ascribed to it, because a disease resembling the
cholera in human beings develops after the introduction of
considerable quantities of it into the bodies of guinea-pigs,
dogs, cats, and monkeys ; the introduction may be effected
subcutaneously, or into the abdominal cavity or the lungs.
Microscopic examination demonstrates the bacilli in all
the organs. There have, however, been objections made
by Weiser to ascribing pathogenic properties to it, and he
has shown that it is present in the air also.

The bacillus appears as a short rodlet with rounded
ends and destitute of motile power, which forms on the
gelatine plate colonies resembling porcelain and lying at a
greater or less depth, of which the superficial ones spread
as a coating over the surface of the gelatine, and the deep
have a figure like that of a whetstone. In thrust-cultures
the more vigorous growth takes place on the surface (fig.
43). Gelatine is not liquefied, but loses its alkalinity, which
causes a clouding of the transparent jelly and a simultaneous
separation out of crystals of salt. If tincture of litmus is
added to the gelatine the blue colour disappears and becomes
changed to a red. A dirty white mass forms on agar
and potatoes. With regard to staining processes, it is a
special characteristic of this micro-organism that it does
not colour by Gram's method. Its resistance to external
influences is so great that it retains its vitality after being
frozen for twelve days and then thawed again.

ATMOSPHERIC SPIRILLA 121

Atmospheric spirilla. — The spirilla occurring in the air.
have been described by Weibel. They usually generate
yellow pigment, according to the degree of intensity of
which there have been distinguished a Vibrio aureus with a
colour varying from golden to orange-yellow, a Vibrio flavus
of an ochre-yellow tint, and a yellowish-green Vibrio flaves-
cens. The individual spirilla frequently appear remarkably
thin, generally S-shaped, and without power of automatic
movement. Islets of an oval or whetstone form, or some-
times circular, develop on the gelatine plate; they are
sharp-edged and granular, and generate pigment in a few
days. There is no liquefaction. Thrust-cultures in gelatine
and superficial cultures on agar display also a copious
development of colour, which takes place only on the surface
of the former ; while on discs of potato there appears
a luxuriant pap-like deposit of a very pronounced tint.
Spirilla are decolorised by Gram's method.

122 BACTERIOLOGY

CHAPTEE YI

THE BACTERIOLOGICAL ANALYSIS OF WATER

Micro-organisms of water. — Water, both in its liquid
and solid state, almost always contains micro-organisms,
although in variable quantity, and these have been named
water bacteria by Percy Frankland. They are for the most
part bacilli — in general such as do not liquefy gelatine — and
they do not grow at the higher degrees of temperature.
Some of them have the property of setting up ammoniacal
fermentation. But pathogenic varieties are also found, in
the foremost rank of wKich stand the cholera bacillus de-
scribed by Koch, which was discovered in drinking water in
the neighbourhood of Calcutta, and the bacillus of typhoid
fever ; but besides these, others also occur as a contamination
of water. Some micro-organisms cannot grow in water
alone, as it does not afford sufficient pabulum for their
development, but large numbers also perish from being
overwhelmed by the growth of the water bacteria.

Very many of the micro-organisms met with in water
generate pigment, often in such quantity that considerable
volumes appear coloured or fluorescent owing to it, and a few
exhibit a brilliant phosphorescence.

Filtration and filters. — Microbes are removed from water
bj filtration, for which purpose use is made of sand filters
constructed with sand and gravel, charcoal filters of plastic
carbon, filters of asbestos, of unglazed porcelain, of earthen-
ware made from burnt diatomaceoiis clay, &c. Forster's filter

THE KAOLIN FILTER

123

allows the water to trickle through sandstone, and in that of
David it is forced in turn through layers of wool treated
with iron tannate, sandstone, animal charcoal, and gravel.

The kaolin niters on the Chamberland-Pasteur principle
consist of porous tubes of porcelain (the so-called * candles')
about 20 cm. long and 2 cm. thick, closed at one end and
provided at the other with enamelled points for the outflow.

Supply-pipe connected with
the metal case

Water

Porcelain tube

Enamelled discharge-pipe
FIG. 44.— KAOLIN FILTER, ox THE CHAMBERLAND-PASTEUR SYSTEM.

These are placed in the water to be filtered or fixed in metal
cases and screwed on to the supply-pipes ; several may
also be connected to form a battery (fig. 44).

The micro-membrane filter of Breier consists of a fine
netting of metal covered with densely-packed asbestos,
which thus forms a thin filtering layer having excessively
fine pores.

124 BACTERIOLOGY

Variations in water depending on source.— The bacteria
which live in water multiply considerably when it has been
stagnant for some time, and observations made in flooded
districts show that the bacilli of anthrax, typhoid fever, and
cholera are capable of growth on dead portions of plants
when moist. Koch has also found the bacillus of mouse
septicaemia in water, and the Staphylococcus pyogenes aureus
is not seldom encountered. This is explained by the fact
that fresh water contains carbon dioxide ; and, moreover,
large numbers of germs are often found also in artificial
aerated waters, such as seltzer, which contain the gas.

In fresh spring-water the germs are said to sink to the
bottom, and hence in some wells which have been disused
for a considerable time a larger proportion of germs is
demonstrable after the first pumping than later. The
micro-organisms are not, as a rule, carried to the well by the
ground water, but come from the surface and the superficial
layers of soil, and the more ground-water is caused to flow
in by constant pumping, the fewer will become the bacteria
contained in the water of the spring. When, however, the
distance of the spring from the surface is small, or when
the well has been made artificially by damming up the
earth, or when sewers extend down into the ground-water,
then this water in which the spring stands will be very rich
in bacteria (Arnold).

A drinking-water which can be termed good from a bac-
teriological point of view must be poor in fission-fungi, and
consequently must not have stagnated in the water-pipes,
and there must be security that no communication can take
place by crevices and fissures between the reservoir or the
mains on the one hand, and drains or sinks on the other.
According to Eubner, the natural filtration through the
soil under which the spring water lies purifies it so
thoroughly that it comes to the light of day in an almost

WOLFFIIUGEL'S APPARATUS 125

sterile condition, only containing two or three germs per
cubic centimeter.

Examination of water. — For purposes of examination
i to 1 c.cm. is taken with a sterilised pipette and mixed
with sterile melted gelatine, which is then poured upon a
plate, and the development of the colonies carried on at the
temperature of the room. The number of islets formed is
then ascertained with the aid of a counting apparatus, and
in this way the relative value in micro-organisms of various
samples of water is determined.

Supporting slab

Glass plate engraved

in divisions
Frame

FIG. 45.— WOLFFHUGEL'S COUNTIXG-PLATE.

The counting apparatus (Wolffhiigel's counting-plate,
fig. 45) consists of a black slab upon which the plate with
the gelatine culture is laid, and over this is arranged a pane
of glass on which squares of uniform size have been
engraved. The islets in the individual squares are then
counted with the help of a lens, and an average struck, when
the number so obtained multiplied by the total number of
squares on the plate gives approximately the total number
of colonies for a certain area, a number which varies with
different kinds of water. The water to be used in this
experiment must not be kept, but must be examined imme-
diately after collection. In examining water presumably
rich in germs — for example, that from rivers or ponds — the

L 2 6 BACTERIOLOGY

volume of water used for observation must be diluted with
sterilised distilled water (generally in equal parts or in the
proportion of one to nine), as otherwise the colonies lie so
close together that they cannot be counted, or else they
liquefy the gelatine too speedily.

The counting apparatus can be rendered complete by
cutting a piece of some size from the upper part of a square
box, and placing a plane mirror obliquely in the interior.
If the gelatine plate be now laid upon the box, the number
of islets on each square can easily be ascertained by the
transmitted light.

Pfuhl's method, — If the examination can be carried out
immediately at the spring, the water to be analysed is poured
into sterilised vessels, which are at once closed with a
sterilised plug of cotton-wool. To obtain the water without
catching extraneous germs, Pfuhl uses flat-bottomed
glass tubes partially emptied of air, and having the
ends drawn out into capillaries, bent at a right angle, and
sealed. The points are broken off actually at the spring,
and the tubes filled with water and again sealed. For the
purpose of transport small cylindrical glass bottles, provided
with ground-glass stoppers, are used, which have been
sterilised and covered with india-rubber caps. To collect
the water from a delivery-pipe the cap and glass stopper
are removed, the bottle completely filled, and carefully closed
again. The water which first flows away, however, must
not be used for examination. To obtain water from a spring
the rubber cap is taken off, but the stopper is only removed
under the surface of the water, which is allowed to flow into
the flask for about a minute, and then the bottle is closed
again and lifted out and the rubber cap drawn over the
glass stopper.

Kirchner's method, — About 36 cm. length of glass tube,
of the diameter commonly used for making connections

METHODS OF EXAMINATION 127

between apparatus, is bent to a U form in the flame, and both
ends are drawn out to points, after which it is sterilised and
sealed hermetically at both extremities while still hot. At
the place where water is to be collected both ends are broken
off, and one held in the water while suction is exerted at the
other until the tube is full, when both points are sealed on
the spot. The tubes are sent packed in ice.

Other methods. — To gain some preliminary information
as to what micro-organisms are present, a few drops of the
water to be examined are evaporated on a cover-glass, which
is drawn several times through the gas flame to fix the dry
residue and covered with one or two drops of a staining
solution. In washing off the stain, the stream from the
wash-bottle should not be directed on the actual deposit
from the water.

It is advisable that every examination by culture should
be preceded by sedimentation, for which purpose Finkelnburg
has contrived an apparatus consisting of a cylindrical vessel
with a bottom capable of being lifted out. Water being
allowed to drop in through an opening in the bottom which
can be closed by a glass tap, the floating particles gather
on the movable bottom of the glass, and in this way a de-
position of the organised impurities can rapidly be obtained.
Csokor's or Gartner's centrifugal machine serves this pur-
pose still better. In order to isolate the bacilli of cholera
and typhoid and other bacteria endowed with motility, AH
Cohen has brought forward a peculiar method depending
on the chemotactic action of certain stimulating substances,
especially of the juice of raw potatoes. A small glass
capillary tube is filled with the fluid found on the cut sur-
face of the raw potato, and is sealed at one end. A ridge
of paraffin is now made on a microscope slide, enclosing a
space into which the fluid to be examined is introduced,
and the sealed end of the capillary tube is fixed in the

128 BACTERIOLOGY

paraffin ridge while the open end extends into the fluid.
The whole is now protected with a cover-glass, when it can
be seen under the microscope that none but the motile
micro-organisms make their way into the interior of the
tube. They can easily be isolated afterwards by cultivation
on plates.

Micrococcus aquatilis. — According to Bolton, this microbe
is one of the commonest inhabitants of water. The cocci
are very minute, and are usually grouped in irregular clumps.
Their growth does not liquefy gelatine, on the surface of
which there develop circular deposits with a gloss like that
of porcelain, from the- centre of which furrows radiate out,
so as to give the colony the figure of a liver acinus. In
thrust-cultures growth takes place both on the surface and
along the needle -track. A white coating develops on agar.

Micrococcus agilis, found by All Cohen in drinking-water,
is met with in the form either of diplococci or streptococci,
which possess the power,, of lively automatic movement. It
liquefies gelatine very slowly, so that an evaporation of the
fluid along the thrust canal often takes place within three
weeks, leaving a dry funnel-shaped cavity. It forms a rose-
red deposit both on agar and potato.

Micrococcus fuscus, described by Maschek, consists of
immotile cocci which frequently have an elliptical form.
Eound light- or dark-brown colonies appear on the gelatine
plate and speedily liquefy, and in the canal of a thrust -
culture liquefaction also progresses with tolerable rapidity,
a sepia-brown pellicle forming on the surface of the fluid.
The slimy deposit on potatoes is also distinguished by a
brown colour.

Micrococcus luteus. — This, which was described by Cohen,
consists of small immotile elements, forming a rather
flocculent zoogloea. It appears in irregular colonies on the
gelatine plate, while in thrust-cultures a yellow deposit is

Jft

MICROCOCCI IN WATER 129

seen on the surface, and grannies form along the track.
The gelatine is not liquefied. A slimy coating develops on
potatoes and agar, and prominences and hollows appear on
old cultures. The yellow pigment shows itself capable of
resisting the action both of acids and alkalies.

Micrococcus aurantiacus was also discovered by Cohen,
and consists of small immotile elements, which sometimes
occur in the form of diplococci. The colonies on plates as
well as thrust-cultures in gelatine show a fine orange-yellow
colour, and the growths on agar and potatoes are also
beautifully tinted. Gelatine is not liquefied.

Micrococcus fervidosus. — Adametz has described tfte
Micrococcus fervidosus, which consists of small elements'-
whose colonies are first seen on the gelatine plate as dots of
a pale yellow colour, becoming brown later. In gelatine
thrust-cultures a granular growth appears along the canal
and a thin coating on the surface. Superficial cultures on
agar show a gloss like that of mother-of-pearl, and a dirty
white deposit occurs on potato. Abundant bubbles of gas
are disengaged on glycerine jelly, but there is no liquefaction
of the gelatine.

Micrococcus carneus. — This micro-organism, described by
Zimmermann, is distinguished by the cluster-like arrange-
ment of its elements, which are immotile. Bound reddish-
coloured colonies appear on the gelatine plate, but in older
cultures the red tint fades towards the circumference. In
thrust-cultures the colour only appears on the surface. A
flesh-red, or sometimes violet layer develops on agar and
potato.

Micrococcus concentricus. — This, like the preceding, was
found by Zimmermann in the Chemnitz water-supply.
The cocci are arranged in clumps, and occur on gelatine
plates in the form of blue-grey dots, while thrust -
cultures in gelatine show on the surface a greyish-brown

K

130 BACTERIOLOGY

disc notched in a radiating manner at the margin, round
which a light brownish circle runs, and this again is
surrounded by a second circle of a brightly-shining appear-
ance, so that the surface of the gelatine appears marked
with concentric rings. A dirty grey deposit forms on
potato.

Diplococcus luteus. — This diplococcus, found by Adarnetz,
forms rather long chains, whole pieces of which move about
in a lively manner in the hanging drop. It liquefies gela-
tine slowly and forms round brownish-yellow colonies which
spread with tolerable rapidity. In thrust-cultures the
growth is more active on the surface, showing lemon -
yellow deposits in concentric layers. It appears on agar
as a yellow or brownish-red coating, while that on potato is
dirty yellow and exhales a mouldy odour. Caseine is precipi-
tated from milk by cultures of the micro-organism.

Bacillus fluorescens liquefaciens and Bacillus nivalis
(glacier-bacillus). — These both display short, thick rods
possessing the power of easy movement. Fick found the
Bacillus fluorescens liquefaciens also in the conjunctival
secretion, and Schmolk found the Bacillus nivalis in glacier
ice. Both form on the gelatine plate colonies which have
a funnel-shaped hollow in the centre, and exhibit a greenish-
yellow fluorescence. Thrust-cultures grow slowly in the
deeper part, but somewhat more rapidly in the case of
Bacillus fluorescens liquefaciens than in that of Bacillus
nivalis, and the former shows on the surface a depression
resembling an air-bubble, owing to evaporation, whereas
with the latter the liquefaction extends over the gelatine.
The non-liquefied portion of the medium shows a greenish -
yellow fluorescence. A whitish layer appears on agar along
the inoculated streak, and the mass becomes fluorescent.
The deposit on potato is yellowish-brown.

Bacillus fluorescens non-liquefaciens, — A bacillus closely

BACILLI IN WATER 131

related to the above-described fluorescent but liquefactive
bacilli has been discovered in the Bacillus fluorescent
non-liquefacicns, small rods destitute of motility which form
on the gelatine plate shimmering colonies with indented
edges, having a darker spot in the centre, and a lighter
coloured leaf-like figure all round. In thrust-cultures there
is a superficial growth of considerable vigour, but nothing
can be observed along the needle-track ; the shimmer, how-
ever, pervades the whole of the gelatine. This bacterium
is distinguished from the Bacillus erytlirosporus by the fact
that the latter shows red-coloured spores.

Bacillus erythrosporus was found by Eidam in drinking-
water and in various putrefying albuminous fluids. The
rods are slender, have rounded corners, and are actively
motile, and the cultures are characterised by the develop-
ment of a dichromatic pigment, appearing orange-yellow by
direct, green by transmitted light. The colonies on the
gelatine plate are circular, and show in the centre a darker
spot around which spreads a wider light zone. Round
every colony fluorescence appears, and soon spreads over
the gelatine, so that the entire plate exhibits the phenome-
non. Thrust-cultures show a growth upon the surface from
which the fluorescence advances deeper until it extends
along the whole track of inoculation. A reddish colour,
becoming later nut-brown, develops on potato. The spores
are characterised by a red gleam, which gives them the ap-
pearance of being stained with fuchsine.

Bacillus arborescens has been frequently detected by
P. Frankland in the water-supply of London. Its rodlets
are thin and motile, and it appears on gelatine plates in
iridescent colonies resembling a trunk with its branches,
the latter being arranged in sheaves. A superficial irides-
cence is visible likewise in the thrust-cultures, and slow
liquefaction soon sets in ; while on agar and potato a yellow

K 2

132 BACTERIOLOGY

or orange-coloured growth develops, with an iridescent
margin.

Bacillus violaceus, — In water of a violet tint, in that from
rivers and water-works — in the Thames and the Spree, for in-
stance— there are found motile rods with rounded ends, whose
colonies on the gelatine plate are at first small, resembling
air-bubbles enclosed in the gelatine, but later on liquefy
it and form granular islets of a bluish-violet colour, which
are distinctly visible as early as the fourth or fifth day. In
the thrust-culture a funnel-shaped area is formed by the
liquefaction, to the bottom of which the blue-coloured
masses of bacilli sink. A dark blue coating develops on
agar, and the bacillus grows very well on potato, spreading
out radially from the place of inoculation until the disc is
nearly covered. Blood serum and plovers' egg albumen
are liquefied with the formation of a violet colour.

Bacillus gasoformans, — The gas- forming bacillus consists
of small highly motile rods, and forms islets on the gelatine
plate, which, though at first small, rapidly liquefy the
medium and spread into its substance as well as over the
surface. In this wray a capsule-like figure is formed, in
which bubbles of gas are visible ; but the formation of these
bubbles is especially characteristic in the clear gelatine along
a thrust-canal. The gelatine is speedily liquefied. Growth
only takes place at the temperature of an ordinary room.

Bacillus phosphorescens. — Fischer found the Bacillus
phosphorescens indigeuus, or native luminous bacillus, in
phosphorescent water from Kiel harbour. Its rods are
short, rounded at the ends, and actively motile, and will
not grow on serum nor on potato. Small round colonies form
on the gelatine plate, which liquefy rapidly, and in about
eight days present the appearance of holes cut with a
punch in the gelatine, and evidently containing air. The
young colonies are sea-green, the older of a dirty greyish-

BACILLUS PHOSPHORESCENS 133

yellow, forming variously shaped flakes. The thrust-culture
is of a slightly conical or sand-glass form, with a thin de-
posit on the edge of the gelatine ; but in old cultivations
the colonies become heaped on the bottom without fluid.
The light given off by them is of a bluish-white colour, and if
traces of the culture be added to sea-water, it acquires the
property of phosphorescence like that observed at sea.

Closely allied to the foregoing is the Bacillus phos-
phorescens indicus, or Indian luminous bacillus, which was
also found in sea-water by Fischer, and which forms small,
thick, energetically motile rods. Bound, sharply-defined
colonies develop on the gelatine plate about the third day,
and are also of a sea-green colour with a rosy shimmer, but
later turn dirty yellow. The thrust-
culture shows on the fourth day a
cavity filled with air on the site of
the puncture, and later the loss of
substance increases, the liquefied
gelatine being covered by a dirty
yellow pellicle. A white layer de-
velops on potato. Boiled fish and

FIG. 46.— ISLET OF BACILLUS RA-

mpQ-J- fnvm'csli ft o-nnrl rnprlinm fnr MOSUS ON A GELATINE PLATE,
1 a 8U(J IN PROCESS OF LIQUEFACTION.

the growth of the micro-organism,

which is checked at temperatures below 10° C. The fact of
its growing on potato distinguishes it from the indigenous
species. In the presence of air and moisture it shows a
luminosity in the dark, best at a temperature of 25° to
30° C. Both bacilli admit of being photographed by their
own light. They grow best on herring gelatine, which is
prepared in a manner similar to ordinary gelatine.

Bacillus ramosus, or root-bacillus (Wurzelbacillus) , shows
short rods with rounded ends and possessing slight power
of movement, and is found in the water both of wells and
rivers, and also on the surface of the ground. Small

134 BACTERIOLOGY

colonies with ill-defined margins develop on the gelatine
plate, and later on send out shoots resembling the filaments

ft.

Liquefied portion

Processes extending radially
from the needle-track

FIG. 47.— THRUST-CULTURE IN GELATINE OF BACILLUS RAMOS us (THIRD DAY).

of mould. When, after growing for some time, the fibres
have become entangled with one another, they form an
interlacing root-like network (fig. 46), an appearance which

BACILLUS RAMOSUS 135

is seen still more characteristically in the thrust-culture, pro-
cesses springing out radially from the needle -track, after the
manner of fibres from a tap-root. The aspect of the culture
has been aptly compared to that of an inverted pine-tree
(fig. 47) . Older cultures are completely liquefied, and carry
a pellicle on the surface. The ramification is equally
distinct upon agar, and spores are also formed on potato.
On plovers' egg albumen there soon develop whitey-grey
colonies growing in root form, which become continually
more and more interlaced and entangled. The liquefaction
of the albumen is very tardy.

Bacillus aurantiacus. — This bacillus, found by P. Frank-
land in deep wells, shows only a feeble motility. The rods
are often arranged in pairs, and grow out into threads.
Gelatine is not liquefied. Small raised colonies appear on
the plate, which are at first dark-coloured but subsequently
become bright orange-yellow, and this orange colour is also
very distinct on the surface of thrust-cultures, whereas the
track of the thrust shows no growth. On agar the deposit
is also of an orange colour. On potato the growth is re-
stricted to the spot inoculated.

Bacillus aureus. — Adametz found the Bacillus aureus
in water, and Unna upon the skin of persons suffering from
seborrhoeic eczema. The rodlets are slender, have but slight
motility, and do not liquefy gelatine. The colonies on the
gelatine plate are irregular, coalesce with each other, and
develop a golden-yellow pigment, which also occurs in the
form of hemispheres on the surface of the streak-culture.
On potato the golden-yellow colour soon changes to a brownish
red.

Bacillus bruneus. — The brown bacillus described by
Adametz and Wichmann shows small irregular rods. The
colonies, which do not liquefy the gelatine in their growth,
are rounded and whitish at first, later brown. The brown

136 BACTERIOLOGY

colour is also developed along the whole length of the thrust -
canal in the gelatine.

Bacillus aquatilis, which was described by P. Frankland,
exhibits short, feebly motile rods. On gelatine plates
the colonies develop in the deeper parts, and spread thence
to the surface, liquefying the gelatine. From the surface
bundles of filaments extend to the periphery. In thrust-
cultures a weak yellowish coloration first shows on the
surface, and liquefaction takes place comparatively late.
The growth on agar is restricted to the line of inoculation,
and on potato the colonies show a similarly meagre de-
velopment. The bacillus was found in the water of deep
wells sunk in a chalky soil.

Bacillus aquatilis sulcatus, — Weichselbaum found four
micro-organisms in the water of the high-level water-works
of Vienna at a time when water from the Schwarza was
laid on,1 and these he designates as Bacillus aquatilis
sulcatus 1-4. The first three forms have motile elements,
those of the fourth variety are shorter, and do not move.
On the gelatine plates colonies develop which are thicker
in the centre than at the periphery, and possess a distinctly
coloured border, while on their surface furrows can be seen
with a low power crossing one another at the most varying
angles, and this network of wrinkles is denser in the centre
than at the periphery. The points of distinction of these
varieties are limited to variations of temperature and
differences of odour, which is like that of whey in the first
form, resembles urine in the second, and is exceedingly
unpleasant in the third, while the cultures of the fourth
variety give off no smell. A superficial growth appears

1 The drinking-water supplied to Vienna is brought from the Schneeberg,
a distance of 100 kilometers. In summer, however, sufficient cannot be
obtained from this source, so that other water must be laid on in addition,
and this is derived from the Schwarza, a little river in the vicinity of the
So3mmering.

BACILLUS MEMBKANACEUS AMETHYSTINUS 137

in thrust-cultures. The fourth form does not grow on
potato.

Bacillus aquatilis radiatus. — Zimmermann found this
bacillus in the water of the Chemnitz water-works. The
rods are motile and form irregular colonies on the gelatine
plate, with root-like processes arranged in a radiating
manner. The gelatine is liquefied with tolerable rapidity,
and the mass of bacteria is collected in the centre of the
fluid colony, while all round it extends a yellowish-coloured
ring which is environed by a cloudy yellowish mass. Thrust-
cultures show a circular pellicle on the surface when lique-
faction has taken place. A layer forms on agar which
appears bluish by direct, yellowish by transmitted light.

Bacterium Ziirnianum, described by Adametz, displays
short, immotile rods. The colonies, which do not liquefy
gelatine, form masses like clusters of grapes, and the surface
of thrust-cultures has also a similar appearance. It grows
on potatoes at 25° to 30° C.

Bacillus membranaceus amethystinus. — Eisenberg and
Jolles have discovered this bacillus in the spring- water of
Spalato. It has the power of liquefying gelatine with de-
velopment of a dark violet pigment, and its rods are short
and immotile. Small, dark violet colonies develop on
gelatine plates and liquefy slowly, while a violet pellicle
appears on the surface of the liquefied mass, resembling a
membrane stained with gentian violet. A deposit with
serrated edges develops on thrust-cultures and acquires a
violet colour in about a fortnight, slowly liquefying the
gelatine. The liquid mass is covered with a violet pellicle
in this case also. A whitish layer, which later becomes
violet, forms upon agar ; the deposit on potato is of a
dirty olive-green, while a violet sediment and a violet pellicle
develop in bouillon.

Bacillus indigoferus. — The bacillus of this name, detected

138 BACTERIOLOGY

by Claessen in the water of the Spree, has slender rods with
rounded ends and showing vigorous motility, but which
often adhere together and form entire clumps, and seem to
possess a jelly-like envelope. The colonies, which do not
develop on the gelatine plate until the third day, contain
an indigo-blue pigment as early as the day following. In
thrust-cultures a punctiform mass of a deep indigo blue
appears round the site of inoculation even in twenty-four
hours, and the gelatine is not liquefied. On agar an indigo-
blue layer develops over the extent of the line of inocula-
tion, its surface being covered with a shining pellicle. On
potato an intensely deep indigo blue develops in three or four
days, but only when the reaction is acid. The formation of
pigment does not seem to depend on the presence of light.

Bacillus ianthinus was found by Zopf in the Chemnitz
water-supply. It consists of short rods with active motile
power, which liquefy gelatine, and form a bluish-violet
pigment.

Bacillus ochraceus. — Zimmermann obtained from the
same source as the foregoing a bacillus with ochre -yellow
pigment, which has been described as the Bacillus ochraceus.
Gelatine is slowly liquefied. The rods show little motility.

Bacillus gracilis. — Zimmermann described a micro-or-
ganism to which this name has been given on account of
the form of the rods, which are slender and fine and
possess an oscillating rotatory motion. Gelatine is liquefied.
On agar a bluish-white pigment appears. There is but little
growth on potato. It has the property of thriving better
in the substance of the solid media than on the surface ;
hence it will also develop under the mica plate (see p. 61).

Bacillus sulphydrogenus. — Miquel found not only in
drinking-water, but in rain and canal water, an anaerobic
bacillus possessing the peculiar property of decomposing
albumen with the formation of sulphuretted hydrogen. Its

BACILLUS OF CHOLERA 139

cells are very short motile rods, which increase in length
when grown artificially. Carbon dioxide and hydrogen are
liberated from a nutrient medium which contains no sulphur.
1 grm. of sulphur is decomposed in forty-eight hours in a
bouillon mixed with ammonium tartrate and with sulphur
in excess.

Other bacteria, however, also generate sulphuretted hy-
drogen, to prove the presence of which it is only necessary
to hang strips of paper saturated with lead acetate in the
test-tubes containing the cultures, when a blackening of
the paper will be found with most water and air bacilli.
According to Mace this reaction becomes more distinct if
some flowers of sulphur be
added to the culture medium.

Bacillus of Asiatic cholera,
and allied micro-organisms.—
Koch discovered a comma-
bacillus in the evacuations
and the intestines of cholera

patients, and adduced evi- FIG. 48.— CHOLERA BACILLI, FROM A PURE

CULTURE. (After Jaksch.)

dence proving that this micro-
organism is the cause of the disease. He found it also in
the water of a tank in the neighbourhood of Calcutta. Ex-
periments have shown that the comma-bacilli can multiply
very well in sterilised water, whereas in ordinary water they
are soon overgrown by the water-bacteria ; and they can
tolerate just as little the concurrence of other bacteria, arid
consequently perish in decomposing liquids, although Gruber
has obtained pure cultures from alvine evacuations which
had been allowed to putrefy for a week.

The pathogenesis of this bacillus has, however, recently
been rendered doubtful by the experiments (performed on
themselves) of Pettenkofer and Emmerich at Munich,
and Hasterlik in Vienna ; and of Dr. A. J. Wall, of H.M.

140 BACTERIOLOGY

Indian Army, in London, who swallowed cultures of the
bacilli.

Cholera bacilli appear as plump rods curved in the
direction of their long axis so as to resemble a comma in
figure, hence the name 'comma-bacillus' (fig. 48). They
have a twist in addition to this curve, so that they represent
a kind of spiral bacteria, and on this account have been
described as Vibrio or Spirillum cholera. The bacteria lie
connected in chains forming a tolerably steep spiral, and
when examined in the hanging drop show an exceedingly
lively motion, so that they might not inaptly be likened to
a swarm of midges dancing all over the field. Loffler found
a flagellum at one end of the cell. Koch was unable to
detect spores in them, but Fliigge, on the other hand, has
succeeded in demonstrating the formation of arthrospores.
They offer but small resistance to the influence of chemical
substances, and are destroyed by the acid of the gastric
juices ; and they refuse to grow upon feebly acid gela-
tine. They also perish at temperatures above 50° C., but
grow at room temperature as well as in the incubator.
Drying causes speedy loss of the power of development.
They stain in about ten minutes in diluted alcoholic solu-
tions of the aniline dyes, but the process, is accelerated by
heating the solution, which also increases the intensity of
the stain. Special importance is to be attached to the fact
that they are decolorised if Gram's process be employed.

Staining is done as follows : An alcoholic solution of
fuchsine or methyl violet is diluted with water sufficiently to
form a fairly strong aqueous staining fluid, which is poured
into a watch-glass. A trace of the pure culture or a minute
flake from the intestinal contents is next placed upon a cover-
glass and spread out by rubbing lightly with another laid over
it, after which the two glasses are drawn apart, dried in air,
grasped with a forceps and passed several times through a

STAINING OF CHOLERA BACILLI 141

flame to fix the micro-organisms, and then laid on the
surface of the staining solution with the infected side down-
wards. The solution is allowed to act for ten minutes on
the preparations, with, or even without, slight warming, and
it need hardly be said that care must be taken to exclude all
substances liable to decompose the stain. The cover-glasses
are next taken from the solution with the forceps, washed
in water, and dried with the prepared surface uppermost,
after which a drop of Canada balsam is laid upon the
prepared face of the glass, which is in this way cemented
to the slide. Microscopic examination is carried out with
the help of the oil-immersion lens and Abbe's condenser.

When a sample of the material to be examined for
cholera bacilli is diffused through gelatine, and a plate -
culture made, colonies are soon obtained possessing in-

Non-liquefied marginal portion

FIG. 49. — ISLET OF BACILLUS CHOLER^E ASIATICS ON A GELATINE PLATE,
IN PROCESS OF LIQUEFACTION.

dented and bowed margins, and presenting, when magnified
100 diameters, an appearance as if strewn with bits of
glass. After a time, small round excavations appear,
corresponding to the colonies, and soon give the entire
gelatine the aspect of pock-marked skin, the funnel-shaped
cavities being due to evaporation of water from the slowly-
liquefying gelatine. The liquefaction does not extend very
far at first (fig. 49).

In thrust-cultures also the gelatine liquefies very slowly,
the liquefaction being chiefly seen on the surface, and in
this case too evaporation of the fluid occurs, so that a
bubble communicating with the external air appears in the
upper part of the funnel-shaped excavation. From this
bubble a thin prolongation runs down along the track of

142

BACTERIOLOGY

inoculation, and comes to resemble a capillary tube as the
liquefaction advances downwards and the evaporation pro-

>uperficial air-bubble

Needle-track

FIG. 50.— THRUST-CULTURE IN GELATIXE OP THE CHOLERA BACILLUS (THIRD DAY).

gresses. When liquefaction has gone still further the
bacilli sink in the needle-track, and the coherent mass of
bacteria forms a loose thread which is sometimes thicker

GROWTH OF CHOLERA BACILLI 143

and sometimes thinner, and which extends to the bottom
of the funnel-shaped liquid cavity (fig. 50). In four weeks
liquefaction has made such progress that the entire mass
of gelatine is fluid.

Cholera bacilli grow with a fair degree of luxuriance
upon other media also, forming on culture bouillon a wrinkled,
much-folded membrane, while the bouillon itself remains
tolerably clear. Only on shaking do the masses rise from
the bottom and render it cloudy.

They grow also in sterilised milk, but in milk which has
not been freed from germs they undergo speedy destruction,
owing to the occurrence of acid fermentation.

In sterilised water they grow rather actively and main-
tain themselves for a considerable time, so that cholera
bacilli find in many places the conditions required for their
increase.

On agar the bacilli grow in the form of a whitish layer
spreading out from the line of inoculation.

On potato they thrive even when the surface shows a
slightly acid reaction, but only at 30° to 40° C. In the region
surrounding the site of inoculation there develops a greyish-
brown layer, which gradually spreads and presents an
appearance almost identical with that exhibited by cultiva-
tions of the glanders bacillus.

Blood serum is slowly liquefied by the growth of cholera
bacilli.

The inoculated streak in the case of plovers' egg albumen
(Von Hovorka and F. Winkler) becomes covered with a
coating which reflects more light than the surrounding mass
of transparent white of egg, and consequently looks lighter.
If examined with a lens the streak, which slowly widens,
exhibits closely-packed, distorted colonies with a grey sheen,
partly united with one another. There is no liquefaction
of the nutrient medium.

144 BACTERIOLOGY

During the progress of the disease the bacilli are found
principally in the mucous membrane and contents of the
intestinal canal, but not in the blood.

To examine cholera stools for bacilli, the evacuation is
mixed with an equal bulk of alkaline meat -bouillon and let
stand in an open glass for twelve hours at a temperature of
30° to 40° C. An abundant development on the surface
results, and preparations are obtained by this method of in-
vestigation which consist of cholera bacilli only. To re-
cognise the presence of the bacilli even without the micro-
scope, a good way is to add 10 per cent, hydrochloric acid,
which after only a few minutes stains the cholera cultures

a rose-violet colour, forming
the cholera reaction.

The introduction of pure
cultures in bouillon into the
stomach of guinea-pigs re-
sulted in Koch's hands in an
unmistakable infection, when

FIG. 51.— FIN-KLER-PRIOR BACILLI PROM A Combined with the
PURE CULTURE. (After Jaksch.)

tration of sodium bicarbonate

and opium. To avoid the detrimental action of the gastric
juice, and at the same time to diminish intestinal peri-
stalsis, Nicati and Eietsch ligatured the ductus choledochus
and made the injection directly into the duodenum. Intro-
duction of cholera cultures into the circulation also causes
the death of the animals, and in that case the bacilli can
be detected in all the organs.

The problem frequently arises, to determine with cer-
tainty whether in a given case it is the bacilli of cholera
Asiatica that have to be dealt with, or other micro-organ-
isms resembling them in form. One of these is the Vibrio
proteus, which was discovered by Finkler and Prior in the
evacuations of persons suffering from cholera nostras, and

FINKLER-PRIOR BACILLUS 145

which may possibly be identical with the comma-bacillus
found by W. D. Miller in carious teeth.

The Finkler-Prior bacillus, or Vibrio proteus, is somewhat
larger and thicker than the bacillus of Koch, but the spi-
rilla formed by it are never so long as the cholera spirilla
(fig. 51). The culture on a gelatine plate liquefies so
rapidly and extensively that the difference between it and a
culture of cholera bacillus must at once become apparent.
If two gelatine tubes are inoculated by making in them two
thrusts parallel with one another, the one with the bacillus
of cholera Asiatica, and the other in the same way with Vibrio
proteus, speedy liquefaction will be observed in the latter,
whereas with cholera Asiatica it occurs very slowly and
shows at the surface an excavation occupied by a bubble of
air. Furthermore the liquefaction of Vibrio proteus ex-
tends widely into the surrounding parts, the masses of
bacilli sink to the bottom, and there is obtained in the
liquefied area corresponding to both thrusts the form of
an empty stocking-leg (the so-called ' trouser-leg culture,'
fig. 52). Superficially the liquefaction of the gelatine soon
causes the two thrust-cultures to coalesce, and a skin then
forms upon the surface.

The Vibrio proteus grows on potato even at ordinary
temperatures, whereas the cholera bacillus only grows at
that of the incubator.

On plovers' egg albumen a very distinct difference
quickly becomes apparent, liquefaction beginning in the
case of Vibrio proteus as early as the second day, and an
intense yellow coloration of the entire nutrient mass soon
setting in, whereas the culture of cholera Asiatica shows
neither the one nor the other. The nutrient mass runs
round from the sides of the test-tube towards the bottom,
where it gathers in a comparatively thick stratum (Von
Hovorka and F. Winkler).

146

BACTERIOLOGY

Deneke's comma-bacillus is more difficult to distinguish
from that of cholera. It was grown by Deneke from old

-Expanded part
of the funnel-
shaped area of
liquefaction

Air-bubble

Liquefied
gelatine

'Constricted, part
of the liquid
funnel

FIG. 52.— THHUST-CTJLTURE IN G-ELATINE
OP THE FINKLER-PRIOR BACILLUS
(THIRD DAY).

j Mass of bacilli

at the bottom
of the liquid
funnel

FIG. 53.— THRUST-CDLTURE IN GELATINE
OP DENEKE'S COMMA-BACILLUS. BEGIN-
NING OF THE THIRD DAY. (After Baum-
garten.)

cheese, and scarcely differs from the cholera bacillus in its
morphological relations. It is, however, distinguished by

VIBRIO METSCHNIKOFFI 147

its speedier liquefaction on the gelatine plate, and the
yellowish colour of the colonies, which appear irregular
under the microscope, and are surrounded by a thick ram-
part. Like the cholera bacillus, it shows an air-bubble in
thrust-cultures at the uppermost part of the funnel-shaped
excavation, but the bubble is larger than in the former case
(fig. 53). A delicate yellowish coat, composed of beautifully-
formed spirilla, appears on potatoes at incubation tempera-
ture. Like the Vibrio proteus, this bacterium possesses
pathogenic properties.

The differential diagnosis of the Bacillus neapolitanus
(p. 120), regarded by Emmerich as the originator of
cholera, presents no difficulties, as its elements are short
immotile rods and the gelatine is not liquefied by their growth,
which takes place more chiefly upon its surface.

The Vibrio Metschnikoffi, which was found by Gamaleia
in the intestinal contents in a Kussian disease of poultry, is
a curved bacterium forming screw- shaped spirilla of con-
siderable length, but which is shorter and thicker than the
cholera bacillus. Its accurate identification is often difficult,
as, although the gelatine of plate-cultures becomes fluid
at times as rapidly as with Vibrio proteus, the liquefaction is
sometimes greatly protracted. The thrust-culture shows a
distinct air-bubble, which gradually enlarges, and disappears
when liquefaction is complete. It grows on potato as a
brown deposit, but only at the temperature of the incubator.

A peculiarly characteristic property of the cholera
bacillus lies in the cholera reaction already alluded to, which
was discovered by Bujwid and Dunham. Cultures of the
bacilli in media containing peptone (bouillon or gelatine)
give a reddish- violet or pur pie -red colour in a short time
when treated with pure hydrochloric or sulphuric acid, in
which process a definite pigment, cholera red, is developed.
Salkowski explains this as an indol reaction, since that sub-

L 2

148

BACTERIOLOGY

stance gives a red coloration with nitrous acid, the theory
being that the cholera bacilli split off indol from the peptone
of the nutrient medium, and at the same time develop
nitrites which are decomposed by the addition of a strong
acid. Of all the other morphologically and biologically
similar micro-organisms, Vibrio Metschnikoffi alone gives
this cholera reaction.

According to Gruber and Schottelius, a still more trust-
worthy point of difference is the fact that the cholera bacilli
grow with particular luxuriance in a very dilute bouillon, and
develop a wrinkled membrane on the surface in a few hours,

Flagella

FIG. 54.— TYPHOID BACILLI FROM A PURE
CULTURE. (After Jaksch.)

FIG. 55. — TYPHOID BACILLI ('SPIDER-
CELLS'). Magnified 1,100 times.
(After Loffler.)

whereas the remaining micro-organisms show no such rapid
growth. But the most certain diagnostic of all assuredly
lies in the manner of growth on plover's egg albumen
characterised above.

Brieger was able to obtain some alkaloids and toxalbu-
mins from cholera cultures, amongst others cadaveririe,
putrescine, and choline. Pouchet also extracted toxines
from the actual cholera stools.1

Bacillus of typhoid fever. — The bacilli of typhoid or en-
teric fever (the Typhus abdominalis of the Germans) are met

1 [For an account of recent researches on cholera vaccination, see
Appendix]— TR.

BACILLUS OF TYPHOID FEVEE 149

with in water as well as in the faeces and organs of patients
suffering from the disease. They were described by E berth
and Gaffky, and have been more thoroughly studied by
Klebs and Eppinger. The bacilli are short plump rods with
rounded ends, the length of which is three times as great as
their breadth, and which sometimes unite to form what are
apparently threads of considerable length (fig. 54). Accord-
ing to Gaffky and Birch-Hirschfeld they develop spores.
The rodlets are distinguished by a high degree of motility,
dependent, as Loftier has found, on the possession of flagella,
which are present in such abundance that the bacilli, when
subjected to the proper staining processes, take on the
appearance of spiders (fig. 55).

They thrive whether oxygen is excluded or has free
access, although in the latter case the growth is more
vigorous, and they stain in watery solutions of the aniline
dyes, yielding up their colour, however, very easily on
application of different bleaching fluids, so that the demon-
stration of them in tissues is beset with difficulties ; and
under this head it is to be observed that they lose their
colour completely when treated by Gram's method.

On the gelatine plate there form whitish colonies lying
superficially, and at first mere dots, which have an un-
evenly indented margin. Sometimes the growths lie deeper,
and take the form of a whetstone. They soon become
yellowish-brown, particularly in the centre of the islets.
The gelatine is not liquefied. Thrust-cultures show on the
surface a thin growth, which also takes place along the entire
inoculated track (fig. 56). A white layer covering the whole
surface develops on agar, blood-serum, and plover's egg
albumen.

According to Petruschky, the typhoid bacillus is one of
those which form acids.

When it is desirable to come to a positive decision

150 BACTERIOLOGY

regarding fthe presence of the typhoid bacillus, the experi-
ment of growing it on potato is indispensable. In three or

Superficial coating upon the
gelatine, which is not liquefied

Xeedle-track

FIG. 56.— THRUST-CULTURE IN GELATINE OP THE BACILLUS OF TYPHOID FEVER.
XIXTH DAY. (After Baumgarten )

four days at room temperature, and in as little as two at
that of the incubator, there appears on the surface a moist,
even gloss, although no deposit can be seen even in the part

RECOGNITION OF TYPHOID BACILLI 151

immediately surrounding the site of inoculation. Particles
of the surface of a potato showing this appearance exhibit
under the microscope micro-organisms shooting hither and
thither with the most extreme velocity. These phenomena
of growth are of particular importance in order to avoid
mistaking them for other species of bacteria which
resemble them in all remaining particulars, such as the
bacillus of Emmerich, which forms a greasy yellowish layer
upon the discs of potato. E. Frankel and AH Cohen have
given prominence to the fact that this growth only occurs
upon slices of potato having an acid reaction, so that the
reaction of the potatoes must always be previously tested.
Frankel himself, however, as well as others, most recently
Kamen, have drawn attention to an atypical growth of the
typhoid bacilli upon potato, in which a yellowish layer,
which later becomes brown, develops slowly out from the
area of inoculation and spreads in a tongue-shaped figure,
the potato assuming a violet tint after some days.

As a further means of recognition, Chantemesse and
Widal have mentioned a peculiarity of typhoid bacilli,
namely, that they thrive on a nutrient gelatine which has
been mixed with 2 per 1,000 of carbolic acid, whereas all
other micro-organisms perish on this mass.

Eodet has proposed to heat the gelatine, after inoculation
with the water to be examined, for from half an hour to
two hours in the water-bath at 45° C., by which means
at least the troublesome liquefactive germs should be
eliminated.

Vincent recommended that a sample of the water under
investigation should be transferred to bouillon, kept at 42° C.,
with which five drops of a five per cent, solution of carbolic
acid have been mixed.

Holz prepared an acid gelatine by the addition of the
juice of raw potatoes to ordinary nutrient gelatine. Only

152 BACTERIOLOGY

a small number of indifferent varieties develop on this
medium, while the typhoid bacilli grow characteristically.

Parietti adds to several test-tubes, each containing
10 c.cm. of neutral bouillon, from three to nine drops of a
hydrochloric acid phenol solution (4 grms. hydrochloric and
5 grms. carbolic acid to 100 grms. water), deposits them for
twenty-four hours in the incubator, and then treats them
with one to ten drops of the water under examination. If
turbidity is visible after another twenty-four hours' standing
in the incubator, it may be concluded with certainty that
typhoid bacilli are present.

Intravenous injections kill rabbits in about twenty-four
hours, when the bacilli may be detected in the urine, blood,
and excreta.

Infection takes place principally by means of water
contaminated with the bacilli, but also by contaminated
milk and linen. The microbes are capable of effectually
resisting the action of the gastric juice, and as soon as they
reach the intestinal canal they penetrate into the lymphatic
canals and are carried by the stream of lymph into the
other organs, particularly the [mesenteric glands] spleen and
liver. Eberth has shown that they may penetrate into the
placenta, and in this way reach the foetus.

[The typhoid bacilli have also been found in the
blood, not only in the rose spots (Neuhauss) but in the
general circulation, having been detected in blood from the
finger.]

Bacterium coli commune. — Kodet and Eoux found this
bacterium in the water of localities where typhoid was
prevalent, and it has been constantly met with by Escherich
in the intestinal canal of suckling infants. It consists of
short, slender rods possessing a sluggish motility, which
occur sometimes singly and sometimes in pairs, and which
are decolorised if treated by Gram's method. Gelatine is

SPIEILLA IN WATER 153

not liquefied, the colonies having a tendency to spread out
over the medium in a thin superficial film. They show a
dull white colour and an irregularly indented border. In
thrust-cultures white buttons develop along the needle-
track and a delicate film on the surface. The colonies
on potato are yellow and juicy, and a white layer appears
on serum. The micro-organism is also capable of growth
in the absence of oxygen upon nutrient media containing
grape-sugar, and then generates a gas consisting of hydrogen
and carbon dioxide. Eabbits succumb to a subcutaneous
injection in from one to three days, with the symptoms of
diarrhoea and collapse.

According to Gasser, an agar medium tinted with
fuchsine is decolorised only by this bacterium and the bacillus
of typhoid fever, whilst a sufficient point of distinction
between these two is, that the growth of Bacterium coli

FlageUa
FIG. 57. — SPIRILLUM UNDTTLA, WITH FLAGELLA. Magnified 800 times. (After L'dffler.)

commune remains restricted to the strip inoculated, where-
as that of the typhoid bacillus forms a tolerably broad
streak with very bowed and irregular edges.

Spirilla in water. — Spirilla are found in copious numbers
in stagnant water, and are marked by an exceedingly active
motility, darting across the field with manifold twists and
turns. They often lie together in clumps, which look to
the naked eye like flakes of mucus. The individual spirilla
possess from one and a half to four turns, or sometimes as
many as six, and have some flagella on their ends. They
are described as Spirillum undula (fig. 57).

154 BACTERIOLOGY

Other micro-organisms in water. — A considerable number
of the micro-organisms met with in the air find a congenial
pabulum in water also, and hence are constantly met with
in the various examinations of that medium. Amongst
these are found the Micrococcus radiatus, Micrococcus cina-
bareus, Micrococcus flavus liquefaciens, Micrococcus desidens,
Micrococcus flavus tardigradus, Micrococcus candicans, Micro-
coccus viticidosus, Staphylococcus pyogenes aureus, Staphylo-
coccus pyogenes albus, Staphylococcus cereus albus, Sarcina
alba, Sarcina Candida, Bacillus subtilis, Bacillus multipedicu-
losus, &c., as well as some which decompose milk and will
be described under that head.

155

CHAPTER VII

BACTERIOLOGICAL ANALYSIS OF EARTH AND OF PUTREFYING
SUBSTANCES

Micro-organisms in the soil. — The examination of soil
proves that very many micro-organisms which thrive in
the air and in water can also grow in earth. Moreover, in
every putrefactive process on the surface of the ground
there occurs an oxidation, or resolution of organic matter
with the aid of atmospheric oxygen ; consequently, in all
these processes a considerable number of micro-organisms
are afforded the possibility of maintaining themselves and
multiplying. In agriculture the land is manured with the
view of enabling highly complex organic substances to
undergo decomposition on the surface of the ground into
simple combinations, capable of serving as nutriment for
plants and of being assimilated by them in order that they
may be once more converted into higher combinations. In
these putrefactive processes the action of bacteria takes a
very prominent part. That it is the surface of the soil which
is so very rich in varieties of germs becomes evident from
the fact that at so short a distance as one to two meters
beneath the surface the amount of bacteria present rather
suddenly decreases, and that further down, at a depth of
three or four meters, the earth is found to be completely
free from germs. The ground- water level of the soil is
tolerably pure in this respect, and hence also only very few
micro-organisms are found in the water of springs. For
the same reason fountains fed by pipes, if properly con-

156 BACTERIOLOGY

structed and kept clean, deliver water which is poor in
germs, or entirely free from them (C. Frankel).

According to Kirchner, the freedom of ground-water
from germs is due to the filtering action of the soil, and
therefore, where this is too coarse and porous, the filtering
power fails, and the whole or a part of the germs met with
in the upper stratum of earth pass unhindered into the
ground- water. According to Keimers, the germs contained
in the deeper part grow more slowly than those derived
from superficial layers.

Method of examination. — If it is desired to examine soil,
or the dust of windows and rooms, for micro-organisms, a
small sample is taken — freshly, if possible — and introduced
into sterilised nutrient gelatine, which has been melted but
is not too hot, in order to prepare a roll- culture by Von
Esmarch's method. Cultures of this form are preferable
to plates, because the small particles of earth do not sink
to the bottom of the tube and get missed, as is liable to
happen in pouring out the contents on the plate. Special
instruments are employed for obtaining earth from different
depths, of which a borer constructed by C. Frankel is that
principally in use.

When the individual islets have been isolated by means
of the roll-culture, they are transferred to different plates,
in order to obtain pure cultivations of the particular
organisms. The examination of anaerobic micro-organisms
is carried on by the methods detailed above.

The broivn mould is very widespread in the earth as
well as in air (p. 104), and some micrococci are found which
liquefy gelatine. Generally speaking, the cocci are more
numerous than the bacilli.

Bacillus mycoides (earth bacillus). — Fliigge found, as a
very frequent guest in the soil of fields and gardens, a
micro-organism whose rods strongly resemble those of

BACILLUS MYCOIDES

157

anthrax, but are distinguished from them by a lively
motility. Gelatine is liquefied. On plate-cultures there

Liquefied portion

Processes from the needle-track

FIG. 58.— THRUST-CULTURE i\ GELATINE OF BACILLUS MYCOIDES (FOURTH DAY).

appear colonies which ramify like mycelium, so that the
plate looks as if overgrown with moulds. In thrust-cultures
liquefaction sets in on the surface as early as the second

158 BACTERIOLOGY

day, while very delicate fine branching filaments run out
from the track of inoculation on all sides, and have a
tolerably even length, thus differing from Bacillus ramosus,
the processes of which dimmish in size from above down-
wards (fig. 58). Kamifications resembling mycelium
develop in like manner upon agar, and have at first some
likeness to the barb of a feather ; but the surface gradually
becomes covered with a thick coating. On serum an irregu-
larly-outlined granular layer appears even in twenty-four
hours, and spreads in a fern-like manner over the surface.
Potatoes are seen after two days to be invested in a fine close
mycelium -like texture of fibres.

Bacterium mycoides roseum, — This microbe, described by
Scholl and Holschewnikoff, exhibits tolerably large rods
which are destitute of motility. A red colour develops early
in the colonies upon the gelatine plate, which coalesce with
one another and very soon liquefy. Thrust-cultures in
like manner show rapid liquefaction, with a red-coloured
superficial skin and a red sediment, but without any stain-
ing of the gelatine itself. Development takes place at
room temperature. Surface-cultures on agar display a
beautiful rose colour if grown in the dark, whereas the
growth is white if cultivated in daylight. Solutions of the
red pigment show an absorption band in the green when
placed before the slit of a spectroscope.

Bacillus radiatus, — Liideritz found the Bacillus radiatus
in the ground, and in the juice from the subcutaneous tissue
of white mice which had been inoculated with garden mould.
Its rodlets possess a ready motility and grow anaerobically,
liquefying gelatine. Upon the gelatine plate, in 'high'
thrust-cultures, and on agar, there appears a tangle of
anastomosing fibres, recalling the radiating forms of moulds.
A very unpleasant -smelling gas is generated in cultures on
serum or sugared gelatine.

VARIOUS BACILLI IN THE SOIL 159

Bacillus spinosus was also found by Liideritz in the juice
from the tissues of white mice inoculated with garden mould.
Like the last, it can only develop anaerobically, and liquefies
gelatine. In high cultures there are visible in two days
little punctiform fluid spots, from which radiating processes
soon push out ; in later stages an expansion appears at the
layers of gelatine above and below the track of inocula-
tion, giving the slimy, liquefied mass the form of a sand-
glass. This bacillus also generates a gas, which has an
odour resembling cheese in growths on gelatine containing
sugar.

Bacillus liquefaciens magnus. — In the juice of the sub-
cutaneous tissue of animals inoculated with garden mould,
Liideritz also found the bacillus of this name, which consists
of large rods rounded at the ends, and endowed with an
active motility. It is also an anaerobe, and its growth
resembles that of the moulds and liquefies gelatine, the
liquefaction in thrust-cultures not taking an hour-glass-,
but a more cylindrical sausage-like, form. Mossy colonies
develop on agar. The gas generated by it smells very
unpleasantly, recalling the odour of onions.

Bacillus scissus. — This bacillus was discovered in the
earth by Percy Frankland. It displays short, thick, immotile
rods, and does not liquefy gelatine. The colonies on the
gelatine plate develop superficially, and in thrust-cultures
no growth takes place along the puncture canal, but an ir-
regular deposit with smooth edges forms on the surface. A
slight greenish colour is imparted to the gelatine.

Besides the above there exists in the earth an entire
series of different micro-organisms, amongst which especially
the Bacillus ramosus and Bacillus subtilis are met with.
They possess, however, no significance, so far as research
has shown up to the present.

Clostridium foetidum, — By the name Clostri-dia are under-

160 BACTERIOLOGY

stood those forms of bacillus which develop spores in the
centre, so that, owing to the bulging there and tapering of the
ends, figures of a distinctly spindle shape are formed (see

fig. i).

The Clostridium foetidum is, according to Liborius, an
uncompromising anaerobe, and displays rods of various
lengths endowed with active motility. It admits of being
easily cultivated artificially, if the care is taken to fulfil the
conditions necessary for the growth of anaerobes. The
gelatine is liquefied in the form of roundish globular
cloudinesses occurring in its substance. Surface-cultures
on agar show little collections with short processes, and
colonies with irregular ramifications form in like manner
beneath the surface of serum. Development of a gas takes
place in the cultures, the evil odour of which has given the
micro-organism its name.

Bacillus oedematis maligni. — As long ago as the year
1872, Coze and Feltz found in their researches on septicaemia
a micro-organism which was more fully described by Pasteur,
and which obtained the name of vibrion septiqne, or Bacillus
septicus. Koch called it the Bacillus wdematis maligni.
The bacilli are found in the superficial layers of garden soil
and in dust from the packing of the floors of rooms, as well
as in various putrid matters during the process of decom-
position. Van Cott found them also in unprepared musk-
sacs, and from this explained the circumstance that patients
are occasionally attacked by malignant oedema after injection
of tincture of musk.

The best mode of carrying out an investigation is to
introduce as much earth as will lie on the point of a knife
beneath the skin of the abdomen of a rabbit or guinea-pig.
The animal dies in from twenty-four to twenty-eight hours,
and the bacilli are found in the oedematous fluid and on the
surface of the organs, but not in the blood-vessels, whereas

BACILLUS OF MALIGNANT (EDEMA 161

the bacilli of anthrax can be detected in the blood. They
also differ essentially from the latter in appearance, being
thinner and ending in rounded points. They unite to form
curved threads, possess the power of automatic movement,
depending, as E. Pfeiffer has found, upon flagella, and form
central spores.

The bacilli soon lose their motility in the hanging drop,
access of oxygen being fatal to them, as they are strictly
anaerobic. Growth takes place either at the temperature
of the room or at that of the incubator. They stain quickly
in aniline dyes, but the colour is easily discharged by ap-
plying Gram's method. Consequently, the points on which
reliance is to be placed in distinguishing between the bacilli
of malignant oedema and those of anthrax are the form,
motility, distribution in the organs, manner of staining,
and relation to oxygen.

In gelatine cultures, which must be made with a regard
to their strict anaerobiosis, small colonies occur, the contents
of which soon liquefy, so that each forms a liquid globule
in the interior of the gelatine. In the high cultures there
is soon seen, as Liborius pointed out, an extensive decom-
position of the nutrient medium, which is changed into a
turbid fluid with simultaneous disengagement of abundant
bubbles of gas (fig. 59). The addition of grape sugar to the
gelatine may prove advantageous. Dull, cloudy, indistinctly-
defined colonies of a shaggy appearance show themselves
on agar after eight hours. They consist of a closely- woven
network of finely granulated fibres, and develop lenticular
gas-bubbles ; indeed, this development of gas is so abundant
that thick layers of agar are thrown towards the upper part
of the test-tube, while a considerable quantity of a cloudy,
whitish liquid condenses and gathers at the bottom. A
network of bacilli forms on potato at incubating heat. The
temperature most suitable for their growth lies between 37°

M

162

BACTERIOLOGY

and 39° C. ; under 16° C. no development takes place. The
spores lie at one end of the rods, and are very resistent.

Gas bubbles in the colonies

Fluid globules (colonies)

FIG. 59.— ANAEROBIC CULTURE OP THE BACILLUS OF MALIGXAXT (EDEMA ix
GLYCERINE AGAR. (After Fraenkel and Pfeiffer.)

The Bacillus spinosus is often found in conjunction with
the above, being obtained in like manner from garden

BACILLUS OF TETANUS 163

mould, but, although anaerobic, it may be distinguished by
the fact that its rods are not endowed with motility, and
are of a different shape.

An oedema containing a reddish fluid which swarms
with bacilli, and is charged with bubbles of gas, is found
on making post-mortem examinations of the infected
animals, and bacilli are also encountered in great numbers
in the peritoneal fluid.

Cultures in bouillon retain their power of infection for
a long time, even for several months.

In the case of the mouse the bacilli effect an entrance
from the tissues into the circulation, probably by penetrat-
ing the walls of the blood-vessels.

FIG. 60. — ISLET OF THE BACILLUS OF MALIG- PIG-. 61. — TETANUS BACILLI WITH TER-
XAXT (EDEMA (Bacillus septicus) ox A MINAL SPORES.

GELATINE PLATE. (After Mace.)

According to Penzo the bacilli of malignant oedema,
notwithstanding their strict anaerobiosis, develop in ordi-
nary cultures from which oxygen is not excluded, provided
these are simultaneously inoculated with Bacillus prodigio-
sus or Proteus vidc/aris.

The oedema bacilli break up albumen, according to
Kerry, producing the ordinary putrefactive processes, and,
in addition, an exceedingly poisonous oily body, which is
formed by the oxidation of valerianic acid.

Bacillus of tetanus. — The tetanus bacillus was discovered
by Nicolaier in garden mould, and in pus from the wounds
of patients who had died of the disease; but Carle and
Eattone had already established the fact that tetanus is

M 2

164

BACTEEIOLOGY

communicable. The experiments proved that these bacilli
are slender bristle -like rods, having circular spores at one
end and possessing but little power of automatic movement,
which very often range themselves in
chains or clumps (fig. 61). The tetanus
bacillus is rigidly anaerobic and occurs
very often in conjunction with other
anaerobes, so that the disease has been
supposed to be due to the united
action of several of these anaerobic
micro-organisms. This is described as
symbiosis.

The bacilli stand a tolerably high tem-
perature — about 80° C. — without losing
their pathogenic power, but growth takes
place best at incubating heat. This
peculiarity, viz. that the spores can be
subjected to a high temperature without
losing their vitality, enabled Kitasato to
obtain pure cultures of the tetanus ba-
cillus, since the other bacilli cultivated
along with them are destroyed at a tem-
perature of 80° C., so that it is then not
difficult to procure a pure culture of the
tetanus bacilli, which remain alive. A
trace of pus from a patient suffering
from tetanus is smeared upon serum
solidified in the slanting position, or upon
agar, and the cultures so made are then
deposited in the incubator for some days.
They are next transferred for from half
an hour to an hour to a water- bath
heated to 80° C., in order to kill the rnicro-
pfeSer.)Fraenkel and organisms which have grown along with

FIG. 62.— ANAEROBIC GELA-

STREPTOCOCCUS SEPTICUS 165

the bacilli, after which the process of cultivation on plates
is proceeded with, by preference in an atmosphere of hydro-
gen. One or two per cent, of grape-sugar may be added to
the gelatine with advantage. The plates show colonies which
have a halo radiating in all directions, and liquefaction sets
in slowly, being combined with the formation of gas (fig.
62). In high cultures a cloud radiating in all directions
develops, which liquefies later on.

If an infection is made with a pure culture, the rods are
found only on the site of inoculation and in its immediate
neighbourhood.

To destroy the spores, they must be exposed for five
minutes to the action of steam at 100° C.

Streptococcus septicus. — Nicolaier and Guarneri found
the Streptococcus septicus in impure earth. It does not ex-
hibit a chain form in all cases, and occurs in the organs
of infected animals in the shape of diplococci. Gelatine is
not liquefied, and little dots develop on the plate at room
temperature. If mice are inoculated with impure earth
they invariably die within three days ; paralytic symptoms
occur in the posterior extremities before death, and diplo-
cocci are found everywhere in the organs and the blood,
and may block the vessels.

Bacillus anthracis. — This bacillus is also found on the
surface of the ground, and its rods were seen by Pollender
as long ago as 1849 in the blood of animals suffering from
anthrax, although they were first thoroughly investigated
by Koch. According to Pasteur, they are spread by earth-
worms.

They are large even rods with abruptly cut ends,
arranged in chains which consist of segments of varying
length (fig. 68), and are immotile, such movements of
single bacteria as are now and then seen being apparently
only caused by currents in the fluid. They grow at room

166 BACTERIOLOGY

temperature as well as in the incubator, but not below 12°
or 14° C., and their highest limit of vitality is at 45° C. If
the cells are frozen they again recover, according to Frisch,
the capability of further development wrhen the temperature
is raised. The formation of spores can be observed with
distinctness.

The cells stain readily with aniline dyes, and do not
discharge their colour if Gram's method be employed, a
behaviour which is of particular importance for the detec-
tion of the bacilli in the blood or organs. A minute portion
of the spleen is usually taken, rubbed between two cover-
glasses, and submitted to Gram's process of staining ; but

Chains of anthrax bacilli

Solitary bacilli

Spaces between the
^ rods of a chain

Bacilli containing spores
FIG. 63.— BACILLI OF ANTHRAX.

the heating must not be carried too far, otherwise the
protoplasm inside the. limiting membrane of the cell will
undergo fine granulation. Carbolic fuchsine or carbolic
methyl blue are also used for rapid staining. The cells
sometimes have their ends thickened into knobs and with
shallow excavations, so that an oval light spot appears
between the individual cells, or, owing to the thickened
nodes, they assume the figure of a bamboo rod. This latter
appearance is wrell brought out by double staining with
Bismarck brown and methyl blue.

When the anthrax bacillus is grown in bouillon, long
fibres are obtained which are felted together in the form of
a tress of hair, an appearance wrhich becomes visible in

15ACILLUS OF ANTHRAX 167

twenty- four hours. On the gelatine plate little white dots
appear in a day or two, and rapidly liquefy the medium,
floating about on the fluid mass. The colonies are seen
under the microscope to consist of irregularly arranged
filaments, an appearance which is particularly marked at
the border of the colony, and has been compared to the
Medusa's head (fig. 64). Impression preparations of super-
ficial colonies show the shoots and processes very distinctly.
Agar plates after twenty-four hours at incubating tempera-
ture show similar figures to those on gelatine. A liquefaction
beginning at the surface is seen in thrust -cultures, while

" -,'.\ , '',!? Fibrous processes at

fitftf ijto^ir^ the margin

XKsf/^M

Hair-like arrange-
ment of fibres

PlG. 64. — COLOXY OF THE ANTHRAX BACILLUS OX A GELATINE PLATE, UESEMBLIXtt
TIJESSES OF HAIR (THIRD DAY).

from the inoculated track delicate filaments push out into
the gelatine (fig. 65). When liquefaction is further ad-
vanced the bacilli sink to the bottom of the funnel-shaped
excavation, but no skin forms on the surface, and from the
deepest parts of the liquefied area processes push into
the still solid gelatine. Superficial cultures on agar
form a layer which can be easily raised with the platinum
needle. Serum is slowly liquefied, and a dry white coating
develops on potato, with considerable formation of spores.
Disinfected silk threads are often saturated with such
spores, dried, and kept for experimental purposes.

168

BACTERIOLOGY

Infection experiments performed on different animals
by inoculation and inhalation, as well as by admission
through the digestive track after previously neutralising

Liquefaction
Air-bubble

Canal of inoculation with fibrous
processes

FIG. 65.— THRUST-CULTURE IN GELATINE OF THE ANTHRAX BACILLUS (FOURTH DAY).

the gastric juice, result in the death of the animal before
forty-eight hours have elapsed ; but in the case of frogs
infection is successful only when the animals are kept at a

PLASMODIUM MALARI/E 169

higher than their normal temperature. If the bacilli alight
upon a spot invested with epithelium, fhey develop there
locally for some time until the epithelium is broken
through and infection can take place, the result being the
malignant pustule. According to Paltauf and Eiselsberg,
the 'rag-picker's disease/ which affects persons engaged in
sorting rags, especially in paper factories, is identical with
anthrax of the lungs.

Owing to lack of oxygen no spores are formed by it in
the body, but it is possible that they may develop on the
surface of the ground where there is free access of the gas,
and this renders great care necessary in disposing of the
bodies of persons or animals dead of anthrax.

When it is wished to make an experiment on animals the
most convenient for the purpose are white mice, which are
inoculated by a pocket made under the skin near the tail.
The animal dies within forty-eight hours, when the spleen
is found greatly enlarged, and abundant bacilli can be
detected in it as well as in the blood. A trace of the blood,
or of the spleen pulp, is now used to make a gelatine plate,
on which the characteristic islets are found in a few days,
and a pure culture can be prepared from them.

Hankin obtained an excessively poisonous albuminoid
body from cultures of anthrax, while Martin ascribes its
virulence to an alkaloid.

Plasmodium malariae. — The protozoa bearing this name,
whose connexion with malaria has been established, stand
in close relationship to the soil. The names associated
with their discovery are those of Laveran, Marchiafava,
Celli, Golgi, and Guarnieri. If a drop of blood is taken
from a patient suffering from intermittent fever, at the
beginning of the fit, there are found within the red corpuscles
small, roundish amoeboid bodies, difficult to distinguish
from the corpuscular protoplasm (fig. 66). They are

170 BACTERIOLOGY

rendered more easily visible if the blood be smeared on
a cover-glass with the edge of another, allowed to dry,
and stained with watery solution of methyl blue ; or,
according to Celli and Guarnieri, a good method is to dis-
solve the methyl blue in serum or ascitic fluid, and let it
run in from one edge upon a preparation of the blood,
which should not be previously dried. During the attack

Amoeboid figure in a red corpuscle

Remnant of the red corpuscle — - (Rt^fc ^^ Other red corpuscles

FIG. 66. — PLASMODIUM OF MALARIA IN HUMAN* BLOOD, AT THE PERIOD OF AFYKEXIA.

of fever the plasrnodia induce alterations in the corpuscle,
causing a conversion of the haemoglobin into melanine, so
that if the blood is examined after the attack the corpuscles
are found to be paler, -and show in their interior clumps
consisting of minute granules of black pigment (melancemia).
This formation of pigment advances so far that the blood
corpuscles are totally destroyed, and, according to Golgi, it

Pigment

Segmented plasmodium —

f*\ ^sy

: Red corpuscles

FIG. 67.— PLASMODIUM OF MALARIA AT THE STAGE CORRESPONDING TO THK
TIME OF ONSET OF THE FEVER.

goes on between the attacks. Multiplication of plasmodia
takes place by segmentation, and the new plasmodia at first
adhere to the edge of the blood-corpuscle, then become free
and subsequently penetrate again into other corpuscles
(fig. 67). According to Golgi, the process of segmentation
takes two days with some plasmodia, and with others three,
before the stage is reached at which the segmented portions

LAVEEAN'S SICKLES 171

become free, and in this way he explains the occurrence of
tertian and quartan fever respectively, while quotidian fever
is dependent on the presence of both varieties, the new
spores of one kind being set free on the first day, and those
of the other on the second.

In malarial cachexia there are found, besides the plas-
modia, sickle-shaped bodies inside the blood-corpuscles
(' Laveran's sickles ') , and forms provided with flagella are
also occasionally observed (fig. 68).

In the individual plasmodia an outer highly-refractive
part which easily takes the stain (ectoplasm) and an inner
part hard to colour (entoplasm) are distinguished, the latter

FIG. 68. — SEMILUXAR OR SICKLE-SHAPED BODIES, AND FREE BODIES PROVIDED
WITH FLAGELLA. (After Jaksch.)

being enclosed by the former as by a ring. An excentrically-
placed nucleus containing nucleoli is found in the entoplasm
according to Mannaberg and other investigators.

Artificial cultivation has not as yet been successful. It
has only been possible to preserve the plasmodia in the living
leech, in the digestive canal of which, as Eoseiibach found,
they retain their vitality for at least forty-eight hours.

The simplest mode of staining is that of Grassi and
Feletti, and is as follows : — A small drop of malarial blood
is placed upon a cover-glass, which is inverted and laid
upon a slide carrying a drop of aqueous solution of methyl
blue or fuchsine; raising one side of the cover-glass a little
and letting it fall again suffices to effect mixture of the blood

172 BACTERIOLOGY

and the staining fluid. By this method the nucleus appears
stained darker than the rest of the body of the parasite.

Mannaberg has introduced the following method in order
to study the order of events in the life-history of the
malaria parasite : — The preparation after being dried in air
is left for twelve to twenty-four hours in a mixture of equal
parts concentrated solution of picric acid and water, to
which from 3 to 5 per cent, of glacial acetic acid has been
added. It is then deposited in absolute alcohol until com-
pletely decolorised, double- stained with alum hEematoxyline
solution, and differentiated in 25 per cent, hydrochloric acid
in alcohol, and weak ammonia alcohol. The preparations
show the red corpuscles and the protoplasm of the leucocytes
unstained, but the nuclei of the leucocytes and the plasmodia
strongly coloured.

Malachowski lays the cover-glass preparations in alcoho
and stains them in a mixture of equal parts of a 1 per cent,
solution of eosine and* dilute aqueous solution of borax and
methyl blue (-J grm. each of borax and methyl blue in
100 c.cm. water), by which method the red corpuscles show
yellowish-red, the nuclei of the leucocytes violet, and the
plasmodia blue.

Aldehoff recommends that the blood should be spread
out on a cover-glass in the thinnest possible layer, dried in
an exsiccator, and subjected for ten or twelve hours to a
temperature of 120° C. in the hot-air sterilising chamber.
The cover-glass is now left in a concentrated alcoholic solu-
tion of eosine for half an hour, or for two or three minutes if
warmed at the same time, rinsed in distilled water, double-
stained by dipping it several times in a concentrated alco-
holic solution of methyl blue, and rinsed repeatedly in
water. The blood must be examined immediately after
being taken, to avoid mistaking blood-plates for plasmodia.
Other micro-organisms of the soil. — Besides the micro-

ANALYSIS OF PUTREFYING SUBSTANCES 173

organisms here described, a large number of others have
been found which also occur in water or air and have been
described under those heads, e.g. Bacillus ramosus, Bacillus
subtilis, Staphylococcus pyogenes aureus, and broivn mould.
Karlinski also found the typhoid bacillus in the ground,
but these bacilli soon perish on the surface of the soil,
although they can maintain themselves in its deeper str

ANALYSIS OF PUTREFYING SUBSTANCES \P^

Differences in putrefactive processes. — When the condi-
tions determining the decomposition of organic substances
are investigated, it is possible to recognise the presence of
micro-organisms in the products which result. Decompo-
sition must be looked on as including two distinct pro-
cesses, for if the conditions necessary for the formation of
its products be sought for in the mode of life of micro-
organisms, it can very readily be ascertained that one
variety of the process is caused by those microbes which
thrive in the absence of oxygen, that is, is dependent on
the vital activity of anaerobes ; whereas the remaining
variety is essentially a process of oxidation set up by
aerobic micro-organisms.1 In the latter, highly complex
combinations are reduced to the very simplest, as happens
with the manure used in agriculture ; and this can take place
only when oxygen has free access, and in the presence of
the proper amount of water. When there is too much
water the necessary quantity of oxygen cannot gain ad-
mission, and the process reverts to the first -mentioned
(anaerobic) form.

During decomposition the chemical reaction very fre-
quently changes, and there is also a formation of ptomaines

1 [These processes have distinct names in German, for which there are
110 English equivalents, the anaerobic variety being known as Faulnis,
the aerobic as VcrwcsungJ] — TK.

174 BACTERIOLOGY

or putrefactive alkaloids possessing toxic qualities. Phospho-
rescence and a formation of pigment are very often observed
in putrefying substances.

Bacillus fuscus limbatus. — This bacillus was discovered
by Scheibenzuber at the author's Institute, in putrid eggs
smelling distinctly of sulphuretted hydrogen. It consists
of short rods seldom united in threads, which display an
active motility and do not liquefy gelatine. Small brownish
distinctly rounded clumps are at first found on the gelatine
plate, and the bacilli are arranged round these in a trans-
parent circle, forming a clear area like a collar, which is
twice or three times as broad as the diameter of the original
brownish islet in the centre. In thrust-cultures the growth
spreads out over the surface, while along the needle-
track there appear prominences like the teeth of a saw, or
sometimes bowed outwards. The medium shows a dis-
coloration round the puncture in the form of a bag with the
convexity downwards and the constricted opening above.
This appearance, which is characteristic, shows it to be an
organism which, like Bacillus fluorescens non-liquefaciens
and Bacillus melochloros, imparts its colouring matter to
the nutrient medium. On agar and potato a brown growth
appears. It thrives at the temperature of an ordinary room,
as well as at that of the incubator.

Proteus, — The varieties of Proteus described by Hauser
belong to the pathogenic micro-organisms living in putrid
substances. The kinds distinguished are Proteus vulgaris,
Proteus mirabilis, and Proteus Zenkeri, organisms which
set up putrid decomposition. If animals are inoculated
subcutaneously with small quantities of cultures of Proteus
toxic phenomena are evoked, and the animals, whether
rabbits or guinea-pigs, die in a comparatively short time
with the symptoms of a severe peritonitis or enteritis.
The toxic action originates in the decomposition of albu-

PROTEUS 175

minoid bodies, from which poisonous substances are split
off.

The Proteus vulgaris or Bacillus figurans consists of
small, curved, very actively motile rods, which also occur
united in groups ; but sometimes the original form alters,
so that cocci result. Brown colonies first appear on the
gelatine plate, the margins of which carry tufts of processes,
and when these coil after the manner of tendrils, the result
is a figure (wandering islet) which is easily secured by means
of an impression preparation and rendered more distinct
by staining. When examined under a moderately high
power such a preparation shows the elements and the
arrangements formed by their union. A deposit of bacilli
arranged in combinations is found in the liquefied areas
in the interior of the gelatine. Thrust-cultures show a
very rapid liquefaction, the micro-organisms sinking to the
bottom. A coating forms upon agar, and a dirty film upon
potato. Serum is liquefied and undergoes very speedy
putrefaction, as is also the case with masses of meat.

Proteus vulgaris has the power of causing milk to turn
sour, according to Kiihn, or bitter, according to Kriiger. It
has also the power, as shown by J. Schnitzler, of convert-
ing urea into ammonium carbonate.

The Proteus Zenkeri does not liquefy gelatine and grows
also when air is excluded. Its colonies can be readily raised
from the gelatine plate.

Proteus mirabilis liquefies gelatine very slowly. Of the
remaining varieties of Proteus, the Proteus hominis and
Proteus capsulatus must be mentioned.

Bacillus saprogenes, — Kosenbach found bacilli in foul-
smelling secretions, which, when cultivated, exhale a strong
putrid odour, and cause a stinking putrefaction of flesh.
He distinguishes three varieties of Bacillus saprogenes. The
first kind, which he isolated from the white plugs met with

176 BACTERIOLOGY

on the wall of the pharynx, shows on agar a yellowish-grey,
opaque, pap-like streak (Bacillus saprogenes I.). The second
kind, which he found in the foul-snielling perspiration on
the feet, exhibits numerous fine droplets upon agar, which
spread out and form a film as clear as water (Bacillus
saprogenes II.). The third kind was found by him in
septic gangrenous pus. It develops a broad and nearly fluid
film on agar (Bacillus saprogenes III.).

Spirillum concentricum was found by Kitasato in putrid
ox-blood, and shows elements twisted into a screw shape
and furnished with flagella, which impart to them a lively
motility. Koundish, sharply-defined discs occur on the
gelatine plate, and display a concentric stratification con-

Flagella

FIG. 69.— SPIRILLUM RUBRUII WITH FLAGELLA. (After Lbffler.)

sisting of alternate transparent and opaque rings. The
gelatine is not liquefied. In thrust-cultures the microbe
grows mostly on the surface. The layer formed on agar is
firmly adherent ; on glycerine- agar the concentric arrange-
ment is distinctly apparent, while on potato no growth takes
place.

Spirillum rubrum. — This was discovered by Von Esmarch
in a mouse which had died of mouse septicaemia. The
spirilla possess an active motility, and are provided with
flagella, according to Lofner (fig. 69). Von Esmarch grew
them first in roll-cultures on gelatine. The colonies are
grey at the commencement, afterwards red; they grow
with extraordinary slowness, and do not liquefy the gelatine.
A special point is that thrust-cultures show after some time

SPIRILLUM RUBRUM 177

a wine-red colour, which, however, does not extend to the
surface, and hence the pigmentation does not appear to de-
pend on the action of oxygen. Light exercises an inhibitory
influence on its production. On agar a white layer first
forms, which later becomes rose-red. Only small red islets
appear on potato; in bouillon growth occurs, as also in the
water of condensation from serum. The microbes grow
both at room temperature and that of the incubator. .

N

178 BACTERIOLOGY

CHAPTER VIII

MICRO-ORGANISMS IN AETICLES OF DIET

Methods of examining different foods. — As in examining
the micro-organisms in water and in the soil, so also articles
of diet are tested for the bacteria which they contain by
means of the process of plate-culture. Many such articles
are impregnated with bacteria, owing to contamination with
adherent particles of earth or germs from the air ; whilst
probably insects, particularly flies, play an important part
in the transmission of the microbes to them. Many food-
stuffs, again, form a"" very favourable nidus for some of the
bacteria — for example, milk, meat, and soup for the bacilli of
typhoid fever and cholera — while others, such as the milk and
flesh of animals suffering from tubercular disease (Perlsucht),
are directly infectious, owing to the bacteria in them.

In the case of vegetables aqueous infusions are made,
from which samples are taken for microscopic examination.
Cheese is examined by rubbing up a little of it with water,
and using minute particles of the mass to make plate-cultures ;
milk is treated like other liquids, and butter and similar
substances are submitted to examination either with or
without the addition of fluid, according to their consistence.

When it is not possible to prepare plate-cultures imme-
diately, a sample of the substance to be examined is taken
with the sterilised platinum wire, and a thrust-culture in
gelatine made therewith, from which further investigations
can be carried on. Still, this procedure comes far behind

BACTERIA IN MILK 179

the plate method in certainty, and the latter should always
be employed where it is at all possible.

EXAMINATION OF MILK AND ITS PEODUCTS

Methods. — Milk may be contaminated either during
milking or in the subsequent manipulation, and may exhale
an odour owing to the substances so acquired, or become
slimy or stringy, or take on a bitter or an acid taste, and
its colour may turn blue or red. Moreover, various patho-
genic micro-organisms may be imparted to milk from the
animal furnishing it. To examine, Arens recommends that
a loopful of milk be diluted with another of distilled water
upon a cover-glass, dried, and fixed by heat, which should
not be too strong. The cover-glass so treated is now
brought into chloroform methyl blue, prepared by mixing
twelve to fifteen drops of saturated alcoholic solution of
methyl blue with three or four cubic centimeters of chloro-
form. It is then waved to and fro for from four to six
minutes, so as to allow the chloroform to evaporate, and
the adherent methyl blue is rinsed away. In fresh milk and
in cream the bacteria alone are dark blue ; in curdled milk
the flakes of caseine are also stained, but only a pale blue.

Another method of examining milk bacteriologically
consists in treating a drop placed upon a cover-glass with
two or three drops of a one per cent, solution of sodium
carbonate, the fluids being mixed as thoroughly as possible
with the platinum needle, after which the cover-glass is
carefully heated over a small flame until evaporation is
complete. The result of this process is saponification of the
fat, so that the cover-glass appears overlaid with a thin film
of soap, and the preparation so treated can be submitted to
the ordinary staining processes.

Bacillus lacticus (Bacillus acidi lactici). — Pasteur pointed

180 BACTERIOLOGY

out that the conversion of sugar into lactic acid (lactic acid
fermentation) is due to the action of micro-organisms.
Hueppe has investigated this fermentation more thoroughly,
and has described a microbe possessing in an eminent degree
the property of curdling milk. It consists of short, plump,
immotile rods, which usually occur in pairs but are occa-
sionally united in longer chains. Growth can take place in
the absence of oxygen, as well as when it is present. On
the gelatine plate little white points appear, having a surface
like porcelain, which do not liquefy the medium ; they show
under the microscope a yellowish coloration in the centre,
which becomes more transparent and paler towards the
margin. Growth appears along the needle-track in thrust-
cultures ; a coating of considerable extent soon forms on
the surface, and crystals of salt separate out from the
nutrient medium. A whitish layer develops on agar, and a
thick brown dirty deposit on potatoes.

If a small quantity of a pure cultivation of Bacillus
lacticus be added to milk, formation of acid occurs, with
curdling of the caseine, the milk-sugar being split up into
carbonic and lactic acids. This action on milk seems
to be capable of taking place only when air has access,
although the growth of the micro-organism is independent
of it.

Micrococcus acidi lactici.— The micrococcus of this name
described by Marpmann closely approximates in its action to
the Bacillus lacticus. It consists of non-motile cocci which
occur singly or in pairs. In twenty-four hours yellowish-
white punctiform colonies appear on the gelatine plate,
while in thrust-cultures a superficial layer develops which
is thicker in the centre than at the edge, and is rather slow
in growing. Keddening of the milk takes place in twelve
hours, and in twenty-four coagulation and lactic acid fer-
mentation.

BACILLUS AMYLOBACTER 181

Marpmann has also found the Sphserococcus acidi lactici
in milk. It possesses properties similar to those of the
Micrococcus acidi lactici.

Clostridium butyricum, or Bacillus amylobacter. — This is
found in old milk, and is also very widely distributed
elsewhere in nature. It was thoroughly studied by
Prazmovsky. It is very often met with in cheese as well as
in putrid vegetable infusions, and is, as discovered by
Nothnagel, a very frequent inhabitant of faeces. The rods are
actively motile and strictly anaerobic, and develope in
gelatine and agar gases which smell of butyric acid. They
stain blue or dark violet in aqueous solution of iodine, from
which it has been concluded that the protoplasm must con-
tain granulose — hence the name Bacillus amylobacter. Bu-
tyric acid is formed from starch, dextrine, and salts of lactic
acid with simultaneous development of hydrogen and carbon
dioxide gas ; while masses of caseine and other coagulated
albuminoid bodies are liquefied.

Micrococcus acidi lactici liquefaciens. — Kriiger has dis-
covered this micro-organism in caseous butter and in
cheese. It consists of small oval immotile cocci, often
arranged in groups of four, and forms small colonies on the
gelatine plate, which speedily liquefy that medium. In
thrust-cultures there develops on the surface of the funnel-
shaped area of liquefaction a white pellicle, which later
sinks to the bottom. Milk curdles at 25° to 35° C., with
formation of lactic acid and separation of a clear serous
layer on the surface, while after a longer time a smell like
that of paste is given off. The micro-organism grows
whether air is excluded or not.

Acidity and bitterness in milk may be also occasioned,
according to Kiihn, by the Proteus vulgaris, which causes
a thickening of the milk.

Oidium lactis. — In sour milk, and constantly in butter,

182 BACTERIOLOGY

there is found the Oidium lactis, described with considerable
completeness by Grawitz, which forms a white fur like a
mould. The fibres of the mycelium mount upwards, be-
come segmented, and support rows of cylindrical gonidia.
The oidium grows on gelatine without liquefying it, and
forms a white, long-haired fur on the suface of the plate at
20° C., diffusing at the same time an odour as of sour milk.
The thrust-culture appears permeated with fibres. Gela-
tine which has an acid reaction is liquefied. Whitish stars
develop on agar at 30° C., which coalesce and cover the
surface. Growth takes place also on serum, and on milk a
thick coating of mould forms. (See fig. 5.)

Bacillus butyricus. — According to Hueppe the butyric
acid fermentation is caused by this bacillus, which consists
of short curved rods endowed with a lively motility. They
have the power of curdling milk, and originate the above-
named variety of fermentation. The curdled caseine is
dissolved and converted into peptone with simultaneous
splitting off of ammonia and other products of division, the
milk acquiring at the same time a bitter taste. Gelatine is
very rapidly liquefied. Yellowish colonies appear on plates ;
in thrust-cultures a greyish-white skin develops, and also a
yellow coloration of the liquefied gelatine, while on agar a
yellow layer is formed, and a wrinkled, dirty coating upon
potato. Centrally-situated spores develop at incubation
temperature.

A genuine Bacillus butyricus is, according to Botkin, to
be met with in all milk, and by the liberation of free butyric
acid causes rapid curdling with abundant evolution of gas.
To isolate it, the milk is sterilised for half an hour in the
steam steriliser and, after being sealed up air-tight, is de-
posited in the incubator. It is completely decomposed in
two weeks, and is then used to make an anaerobic plate -
culture, best done, according to Botkin, by employment of a

BACILLUS BUTYRI VISCOSUS 183

bell into which hydrogen is conducted in the presence of
pyrogallol, the apparatus being luted with paraffin. The
same observer recommends sugar-agar as a nutrient medium.
After continuing in the incubator for two days, the colonies
have made such progress that they can easily be trans-
planted as pure growths to high cultures in agar. Eound
colonies form, which give the impression of a felt consisting
of closely-intertwined filaments, and are provided with a
considerable number of processes radiating out from all
sides. Gelatine is very rapidly liquefied. On potato
there develops at the depth of a millimeter an abundant
growth of bacilli, which loosen the substance of the medium
to a marked degree, and exhale a peculiar odour resem-
bling that of alcohol.

Bacillus butyri viscosus. — Lafar constantly found short
rods in butter, having rounded ends and not seldom
forming chains. Thrust-cultures spread but little over the
surface of the gelatine, but along the needle-track there
occur figures resembling the scales of fish, in lumps, or often
in clusters, and which do not liquefy the medium. Small
white dots appear on the surface of the gelatine plate, which
grow decidedly more in height than in width, gradually melt
into the form of small lenticular discs, and cover the whole
of the gelatine, which remains solid, with a slimy film about
half a millimeter in thickness. On potato a fine moist
shining layer develops, resembling that formed by typhoid
bacilli. Growth takes place whether air is excluded or
present.

The Bacillus butyri fluorescens, isolated from butter by
Lafar, seems to be identical with the Bacillus melochloros
obtained from air by F. Winkler and Von Schrotter in the
author's Institute. (See p. 118.)

Spirillum tyrogenum. — Deneke cultivated from old cheese
a variety of comma-bacillus which has strongly curved

184 BACTERIOLOGY

elements endowed with rapid automatic motion. On the
gelatine plate small punctiform colonies occur, which liquefy
the gelatine and acquire a yellow colour. They resemble
at first a culture of the cholera vibrio, but are distinguished
from it by the speedier liquefaction of the gelatine, and by
the yellowish tint of the colonies. Liquefaction is, however,
slower than in the case of the Vibrio proteus. In thrust-
cultures the gelatine is liquefied along the whole length of
the inoculated track, and in a few days the bacteria sink to
the bottom and lie there in a loose coil. The bubble of air
in the upper part is larger than in cholera cultivations (see
fig. 53). A thin yellowish coating appears upon agar, a
yellowish film upon potato, and serum and plovers'-egg
albumen are liquefied. If injections of the cultures be made
into the intestinal canal of guinea-pigs, with the precautions
specified by Koch in the case of cholera bacilli, the animals
are destroyed.

Bacillus lactis viscosus. — This very wide-spread spoiler of
milk was first found by Adametz in the water of brooks in
the neighbourhood of Vienna. It forms short rodlets re-
sembling cocci, which possess a thick, highly-refractive
capsule. On gelatine plates round disc-like colonies occur,
sometimes with concentric rings, and on them there forms
at a favourable temperature (16°to 20° C.) a broad, irregularly
serrated hem of a transparent, horny appearance. Surface-
cultures upon agar show narrow whitish stripes, the border
of which is at first smooth, but later finely serrated.
Sterilised milk becomes in four to six weeks viscous like
honey, so that it can be drawn out into long strings (slimy
fermentation} ; in milk which has not been sterilised the
cream alone becomes stringy or slimy, and such cream
yields a white, greasy butter which becomes quickly spoilt
owing to the presence in copious numbers of the butyric
acid bacillus, from which fact Adametz concludes that this

BACILLUS FCETIDUS LACTIS 185

bacillus prepares the soil for that of butyric acid. The
stringy substance comes from the material of which the
envelopes of the bacteria are formed, and is probably meta-
morphosed cellulose, as in the case of Bacillus mesentericus
vulgatus, which also has the power of setting up a slimy
fermentation.

Such fermentation is also originated, as Loffler found,
by the Bacillus lactis pituitosi, which consists of thick curved
rods very rapidly breaking up into segments like cocci.
The colonies on gelatine plates are radially striped, and the
gelatine is not liquefied. On potato a greyish-white coating
develops.

Bacillus actinobacter. — Duclaux very frequently found in
milk fine non-motile rods, partly isolated, partly arranged
in pairs, but always furnished with jelly-like capsules, and
which rendered the milk gelatinous and viscid. The
capsules are retained in artificial cultures in glycerine
solutions, whereas in growths in bouillon and solutions of
sugar they are lost. Development can take place either in
the presence or absence of oxygen.

Bacillus foetidus lactis. — Jensen isolated a bacillus from
milk which imparts to it and its products a nauseous,
sweetish, putrid odour and taste. The bacillus shows short
thick rods, with rounded ends and active motility. Gelatine
is not liquefied. Superficial colonies with the lustre of
inother-of-pearl occur on plates. In thrust-cultures a slimy
growth appears on the surface, and an abundant develop-
ment takes place along the track of inoculation. On agar
there is a formation of large lenticular bubbles of gas.

Bacillus cyanogenus. — The cause for the development of
a blue colour in milk is to be sought for in the Bacillus
cyanogenus, described by Ehrenberg as Bacillus syncyanus,
and which was studied by Hueppe and Neelsen. The
bacillus of blue milk consists of small, actively-motile rods

186 BACTERIOLOGY

provided with abundant flagella, and which do not liquefy
gelatine. Upon the plate there form superficial round
colonies, which cause a dark coloration of the surrounding
gelatine. Thrust-cultivations grow in the form of the nail-
culture, the head of which consists of a thick white deposit.
The gelatine is stained greyish -blue, and finally assumes
a dark tint. On agar a superficial coat forms, and the
medium becomes discoloured. On potatoes there occurs
at first a thick, yellow, dirty layer round the inoculated
spot, and in older cultures a blue coloration of the medium.
The bacillus multiplies by formation of spores occupying
a terminal position. The blue pigment is changed to a
brilliant red by alkalies, but is restored by the action of
acids. According to Gessard, the substance from which it
is derived is lactic acid.

Bacterium lactis erythrogenes. — Hueppe and Grotenfeldt
found this microbe in red milk, and also in the faeces of
children. It consists" of short, oscillating rods, the growth
of which liquefies gelatine. The colonies are of a yellow
colour when first seen on the plate, but after liquefaction
they become rose-red. On thrust-cultures there appears a
whitish, or later, yellowish layer, which finally acquires the
rose-red tint ; when liquefaction has taken place in ten or
twelve days the fluid is rose-coloured, and the coloration
extends also into the substance of the remaining solid
gelatine. A yellowish deposit occurs on agar, and soon
changes to yellowish-red. On the surface of potato a golden-
yellow colour appears in six to eight days at incubating
temperature, whereas at ordinary room temperature the
colonies are yellowish-red. All cultures exhale an un-
pleasant sweet smell.

If a small quantity of pure culture be added to neutral
or feebly alkaline milk, the caseine is separated out and
peptonised, the layer of cream on the surface and the

SARCINA ROSEA 187

precipitated caseine at the bottom remaining white while
between them lies the rose-coloured whey. The formation
of pigment takes place in the dark as well as by daylight.
According to Hueppe, it appears to result from metabolism,
that is, to be a coloured ptomaine, and is seemingly in-
dependent of other influences and conditions.

Ked coloration of milk may also be due to the Bacillus
prodigiosus, which, however, causes only a very slow sepa-
ration out of the caseine, and does not originate any further
changes.

Sarcina rosea. — Menge has found that the agent pro-
ducing the red tint may be a sarcina to which he has given
the above name. In does not, however, appear to be
identical with the micro-organism of the same name de-
scribed by Schroter (see p. 109). In two days small
translucent, perfectly circular colonies form on the gelatine
plate, and soon assume the figure of a rosette having a red
nodule in the centre surrounded by concentric rings. In
the thrust-culture the surface is covered with a thin rose-
red coat with jagged edges, whilst the needle-track remains
colourless. Liquefaction takes place very late. On agar a
coherent growth develops which is white at first but becomes
coloured on the third day ; in the incubator, however, pig-
mentation fails altogether. On potato which has been
rendered alkaline the sarcina grows excellently. The pig-
ment forms in milk independently of light.

The Micrococcus of bovine mastitis. — In different diseases
originated in the lacteal glands of animals by the action of
micro-organisms the latter can be found in the milk also,
and amongst these is the micrococcus of mastitis in the cow
described by Kitt, which is met with in the partly milky
and partly purulent contents of the udder of cows suffering
from this complaint. On the gelatine plate small puncti-
forrn colonies of the size of a pin's head develop, and a thrust

188 BACTERIOLOGY

in the same medium produces a nail-culture without lique-
faction. A wax-like layer appears on potato.

Other pathogenic bacteria in milk. — In the milk of
animals suffering from tuberculosis bacilli may be found, ac-
cording to Bellinger, even before the udders become diseased.
Typhoid bacilli may also find their way into milk along with
water, and may develop abundantly there. The bacteria of
cholera, too, are similarly capable of copious multiplication in
milk without perceptible alteration, but they are destroyed
when lactic acid fermentation sets in. In milk from a cow
suffering from inflamed udder, the Staphylococciis pyogenes
aureus has been detected by Kriiger.

Saccharomyces ruber. — Demme has found a yeast occur-
ring in milk and cheese which produces in the latter red
punctiform accumulations of pigment, and in the milk a red
sediment. This yeast he has described as Saccharomyces
ruber. Gelatine is not liquefied by it, and it develops on the
plate colonies of the ""size of a grain of millet-seed, which
show no red colour until a week has elapsed. In thrust -
cultures the growth is chiefly on the surface. Discs of
potato are covered with a coating of the colour of raspberries
in from eight to twelve days.

Bacillus Caucasicus (Dispora Caucasica, or Kephir bacillus) .
—The name of kephir grains is given to masses of micro-
organisms in a zoogloea, which are used in the Caucasian
districts for the preparation of the drink known as ' kephir '
from milk. In this process the caseine passes into solu-
tion, while the milk-sugar is affected after the manner of
a lactic acid and alcohol fermentation (Adametz). The
granules contain yeast and bacteria capable of peptonising
caseine, viz. the Bacillus acidi lactici, Bacillus butyricus,
Bacillus subtilis, and in addition to these a micro-organism
to which Kern ascribes the principal role, and which bears
the name Bacillus caucasicus. This consists of short

BACILLUS MEGATERIUM 189

cylindrical rods which are destitute of movement so long
as they are in zoogloea, but when isolated possess an, extra-
ordinarily lively motility. The name Dispora was assigned
to the micro-organism from the fact that the protoplasm is
withdrawn to the ends, giving the rod the appearance of
being divided into two spores. By its vital action milk-
sugar is changed into glucose, upon which the yeast can
then begin to work.

EXAMINATION OF OTHER ARTICLES OF DIET

Bacillus megaterium. — This bacillus was found by De
Bary on boiled cabbage-leaves. It consists of long, thick,
somewhat curved rods with rounded angles, frequently
connected in chains, and possessing slightly granular cell-
contents. The motion of the rods recalls the amoeboid
movements of cellular protoplasm. During involution the
shape of the rods loses its distinctness and malformations
occur, which, however, regain their normal outline on

Chain with links containing spores

FIG. 71.— ISLET OP BACILLUS

FIG. 70. — BACILLUS MEGATERIUM WITH MEGATERIUM ON A GELA-

SPORES. TIXE PLATE.

transference to fresh nutrient substances. Their sporu-
lation is particularly suitable for purposes of study, and the
spores also stain easily (fig. 70). On the gelatine plate
there form minute roundish colonies consisting of undulat-
ing filaments, which liquefy the medium slowly, and possess
a uniform or semilunar outline (fig. 71). A funnel-shaped
liquefaction appears in thrust-cultures, in which the bac-
teria sink to the bottom (fig. 72). On agar grey deposits

190

BACTERIOLOGY

develop, which adhere firmly to the mass, and on potato
there grows a dirty whitish-grey layer containing many
involution forms.

Funnel-shaped area of
liquefaction

Needle-track

FIG. 72.— THRUST-CULTURE IN GELATINE OF THE BACILLUS MEGATERIUM.

Besides the above, the Bacillus prodigiosus, Bacillus sub-
tilis, Bacillus amylobacter, and Bacillus butyricus are met

BACILLUS ACETI 191

with on plants. F. Winkler and Von Schr otter found
the Staphylococcus pyogenes aureus in rotten portions of
apples, and the Bacillus melochloros in the excreta of cater-
pillars in worm-eaten specimens of the same fruit.

Bacillus aceti. — As early as 1864 Pasteur brought for-
ward proof that the oxidation of alcohol and its alteration
into acetic acid are connected with the vinegar ferment,
which could only develop in the presence of oxygen.
This ferment consists of short thick rods often uniting in
curved chains, and forming a mass of zooglcea which
is thick and glutinous, the [so-called] 'mother of vine-
gar.' ,

Mace found another bacterium of vinegar, the zoogloea
mass of which is thick, white or slightly rose-tinted, never
wrinkled, and feels almost like cartilage. Numerous rods,
sometimes isolated, sometimes joined in pairs or in threes,
lie in a colourless interstitial substance ; they are without
movement in the membrane, but when free in fluids possess
a slow motility. Gelatine is not liquefied, and a thick,
undulating, rather hard layer develops on its surface. A
less hard, but smooth and non-undulating, layer forms
upon agar.

The bacteria of vinegar are very widely diffused through
nature, according to Duclaux, who assigns an important
part in transmission of the ferment to a kind of fly, the
Musca cellaris, which is met with everywhere.

Bacillus indigogenus was found by Alvarez in infusion of
indigo leaves. It consists of short motile rods surrounded
by an envelope, and has the property of causing the ap-
pearance of blue indigo in decoctions of the leaves of the
Indigo fera. Surface cultures on agar cause cleavage of the
medium, with development of gas.

Pediocoecus cerevisise. — Balcke found the Pediococcus
cerevisicB in beer, in breweries, and in the washings from

192 BACTEEIOLOGY

them. It forms diplococci and tetracocci, and shows
colonies on plate-cultures which are at first colourless
and later brownish, and which do not liquefy the gelatine.
In thrust-cultures a white, leaf-like layer is developed.
An iridescent greyish-white coating occurs upon agar,
while upon potato the growth is scanty. Some formation
of lactic acid accompanies its development.

Sarcina cerevisiae. — A number of sarcinae derived in part
from the air are found in beer. Lintner encountered a
variety in soured beer which, according to Adametz, occurs
also in water, and which sets up lactic acid fermentation.
Gelatine is not liquefied. The islets formed on plate-cultures
are round, colourless, and smooth-edged, and a delicate
fluorescent film develops gradually over the gelatine.
Thrust-cultures show a smooth white coating, and on
potato light granular islets form.

Micrococcus viscosus. — A peculiar disease of wine and
beer, called by the French la graisse, which consists in a
clouding and inspissation of the liquor, so that it becomes
stringy like white of egg, depends, according to Pasteur,
upon the vital activity of the Micrococcus viscosus. The
cells occur singly, or more usually arranged in diplococci or
streptococci. Artificial cultivation succeeds best in solu-
tions containing sugar.

Bacillus viscosus cerevisise. — In viscous beer Van Laer
constantly found slender rods with no tendency to unite in
groups. Gelatine is liquefied. Upon plates there develop
round or oval colonies with uneven edges, which pro-
ject somewhat above the surface. In thrust-cultures a
white, irregularly-edged deposit is observed, while the
track of inoculation also shows an abundant development
of colonies. Growth on agar is very rapid, with the forma-
tion of a broad white slimy layer. These bacteria also occur
in milk and render it slimy. White, watery, and very tena-

MOULDS ON ARTICLES OF FOOD 193

cious colonies develop on potato, and give off an odour of
putrid fish.

Bacillus viscosus sacchari. — Kramer assigns long rods
with rounded corners, which are destitute of motilifcy and
frequently arranged in chains, as the cause of solutions of
sugar becoming slimy. Gelatine is liquefied, a whitish
film develops upon agar, and a firm mass upon potato. No
growth takes place on acid media.

Moulds on articles of food. — The moulds find abundant
opportunity for development and propagation upon vege-
table foods, and the three varieties, Penicillium, Aspergillus,
and Mucor, are all represented. Penicillium glaucum has
already been described under the ' Bacteriological Analysis
of Air' (seep. 103).

The various kinds of Aspergillus flourish on bread and
candied fruits. To obtain the Aspergillus niger, bread
pap is prepared in an Erlenmeyer's flask. After some
time stout fructifying hyphae are found, not ramified at the
end like Penicillium, but swollen so as to resemble clubs.
Upon these are arranged the sterigmata, at the upper ends
of which the spores become segmented off and form aggre-
gations which take the form of rounded, bulging black sweK
lings. The entire flask soon becomes filled with fibres and
grey points. Aspergillus albus and Aspergillus glaucus are
similar, but grow better at incubating temperature (see
fig. 3).

The Aspergillus flavescens is distinguished by its well-
marked fructifications and the greenish colour of the cul-
tures. Aspergillus fumigatus bears very fine fructifications,
and forms an ash-grey fur. Both grow luxuriantly on
bread at incubation temperature. On gelatine plates fila-
ments appear which spread rapidly into the surrounding
parts with liquefaction of the medium.

Of the Mucorinese, the Mucor mucedo, Mucor corymbifer,

o

194 BACTERIOLOGY

and Mncor rhizopodiformis are found upon food-stuffs, par-
ticularly bread. They possess a branching mycelium, with
spore-bearers which resemble flexible tubes, are unseg-
mented, and stand up vertically from the mycelium. At
the upper ends of these are the swollen sporangia, by
bursting of which the spores are set free. In addition
to this process, a kind of conjugation also takes place, in
which two cells (zygospores), developed from the mycelium,
coalesce with one another and form spores.

The Mucor mucedo is one of the commonest moulds, and
is frequently found in animal excreta, especially in horse-
droppings. It grows on acid media, and is not pathogenic.
A dense fur with black fructifications as large as poppy-
seeds appears on the gelatine plate.

The Mucor rhizopodiformis was described by Lichtheim.
It grows very luxuriantly on bread gelatine, which it lique-
fies. The fur is white and bears black fructification heads.
The culture on brea'd is distinguished by the development
of an aromatic odour.

Mucor corymbifer was likewise described by Lichtheim.
It forms a dense snow-white fur upon bread, resembling
teased-out cotton-wool.

The Mucor ramosus, described by Lindt, grows very well
upon agar made with bread infusion, and upon potato.
The fur is at first white, but soon assumes a greyish-brown
tint.

Both Mucor rhizopodiforinis and Mucor corymbifer are
pathogenic. Intravenous injection of fluid containing their
spores causes a fatal disease in rabbits, in which the organs
are always attacked in the following order : viz. kidneys,
intestine, mesenteric glands, spleen. Mucor ramosus acts
quickest, an acute hsemorrhagic disorder being originated
by its injection.

The Aspergillus fumigatus and Aspergillus flavescens in

PATHOGENESIS OF MOULDS 195

like manner exhibit pathogenic properties. Lichtheim
found that a peculiar disturbance of equilibrium occurred
in rabbits and dogs after intravenous injection, and after
death, which took place in twenty-four hours, the germina-
tions were found especially in the myocardium and kidneys.1

1 [The three varieties of Aspergillus may grow in various parts of the body
in man, notably the external ear, in which they produce the disease known
as otomycosis. One or other form has also been found growing in the lungs,
on the nasal mucous membrane, and on the cornea. Such occurrences are,
however, very rare.] — TR.

o 2

196 BACTERIOLOGY

CHAPTEK IX

BACTERIOLOGICAL EXAMINATION OF PUS

Properties and composition of pus. — Pus shows an alkaline
reaction and possesses a high specific gravity, consequently
it furnishes an excellent nutrient medium for the most
widely differing micro-organisms, and that whether it is
derived from exudations or from the surface of wounds, or
is formed in some other inflammation of tissue.

Furthermore, pus may be coloured, the tint being
greenish or brown-red, and it exhales a peculiar odour. In
it are found white and red blood corpuscles, blood pigment,
and crystals of haematoidin, epithelial cells, drops of fat,
fungi, micrococci, and bacilli.

Suppuration may also be excited by bringing certain of
the micro-organisms contained in pus into contact with the
tissues, when their vital activity will start the process.
Amongst these microbes is included more especially the
Staphylococcus pyogenes, which, as wre have already remarked
in the chapter on the ' Bacteriological Analysis of Air ' (see
p. 110), is very widely distributed in nature. Eosenbach
also found the Bacillus saprogenes III (see p. 176) in pus.

Actinomyces. — A fungus has been detected compara-
tively recently in almost all organs, in certain cases of
chronic inflammation combined with suppuration, which
has been named Actinomyces or ray -fungus. It had been
discovered by Langeiibeck as long ago as 1845, but it was

ACTINOMYCES 197

not until 1878 that it was more thoroughly described by
Israel. The disease caused by it is known as actinomycosis.
E. Ullmann states, however, that the actual suppuration in
this disorder is due to the specific organisms of pus, viz.
the staphylococci and streptococci, the actinomyces fungus
being, according to him, merely an accidental occurrence
in the pus.

The fungus forms minute sulphur-yellow nodules of the
size of poppy-seeds, which, if a cover-glass is lightly pressed
upon them and they are examined even with a very low power,
show compact globules having a clustered arrangement.
They were first discovered
by Bollinger in cattle, and
admit of being very easily
transmitted to human
beings. Under a higher
power they are seen to be
made up of filaments re-
sembling hyphae, which for

the most part radiate OUt FIG. 73.— AN ACTIXOMYCES GRANULE.

UNSTAINED PHEPAKATION. (After Jaksch.)

from a central point, each

ray being thickened into an elongated club-shaped enlarge-
ment at the periphery. E. Paltauf describes the actino-
myces fungus as a bacterium, regarding the club-shaped
figures as degenerative forms ; Israel and Wolff count it
amongst the polymorphic fission fungi, on the grounds that
thread-like figures and groups of globular bodies are
visible in addition to the radiating forms ; Petroff and
Flora aim found that the fibres are made up of granules
alternating with small roundish gaps ; and Kabe is of
opinion that a certain alga, Cladothrix canis, presents a very
great similarity to actinomyces. The fungus takes aniline
stains when they are allowed to act on it for some time,
and does not discharge them during the application of

198 BACTERIOLOGY

Gram's method ; but the simplest mode of recognising them
in pus consists in examining the little yellow globular bodies
without staining (figs. 73 and 74).

Cultivations on glycerine agar show, according to
Protopopoff and Hammer, a mass of closely-packed miliary
nodules of the size of hemp-seed at largest, which have a
yellow colour and adhere very firmly to the medium. The
growth on potato is similar, except that the culture looks
quite dry. In broth miliary nodules develop after a short
time, and may attain the size of a hazel-nut. , Gelatine is
slowly liquefied. Growth takes place also in the absence of
oxygen, in eggs, by Hueppe's method.

FIG. 74.— ACTIXOMYCES, STAINED BY CHAM'S METHOD. (After Jaksch.)

F. Winkler has cultivated actinomyces on plover's egg
albumen in the author's Institute. Formation of sulphur -
yellow colonies as large as poppy-seeds takes place all over
the surface of the medium, which is, however, not liquefied.

Bacillus pyocyaneus. — This bacillus is the cause of the
grey or blue colour sometimes seen in pus and in pieces of
dressing saturated with it.

The Bacillus pyocyaneus a, described by Gessard, consists
of small slender rods provided with a flagellum, by means of
which they- move swiftly. Upon gelatine plates roundish
islets of a yellow colour appear in the substance of the
medium, to the whole of which they impart a greenish tint
in about two days, causing at the same time slow lique-

BACILLUS PYOCrANEUS 199

faction. In thrust-cultures the gelatine liquefies along the
needle-track, the fluid mass assuming a greenish colour,
while the part still solid displays a green fluorescence. A
layer forms upon agar which shows a fluorescence, at first
greenish, but subsequently dark green in tint ; while on
potato the deposit appears coloured brown, turning red
when treated with acids, blue with ammonia. According to
Gessard, the generation of pigment depends on the nutrient
medium. The green colouring matter formed, which is
soluble in chloroform, has received the name oipyocyanin.

Babbits die when injected into the peritoneal cavity
with pure cultures of this micro-organism or with
pyocyanin.

The Bacillus pyocyaneus /3, described by Ernst, is not
pathogenic, but is commonly met with in the company of
the foregoing, and when isolated causes the
blue coloration of pus. It liquefies gelatine »°
much more speedily than the last, and shows

£j * »*

a brown tint on potato which changes to *

grev on touching with the platinum wire FIG. 75.— ISOLATED

ELEMENTS OF

.•
***

(the chameleon phenomenon). The reaction

of the potato culture, the more rapid lique-

faction, and the fact that injections produce no result, all

serve to distinguish it from Bacillus pyocyaneus a.

With reference to its physiological relations, Bacillus
pyocyaneus produces coagulation of milk, rapidly peptonises
albumen, and causes vigorous inversion of sugar with fer-
mentation.

Staphylococcus cereus. — Passet found in pus the Staphy-
lococcus cereus albus and Staphylococcus cereus flavus, the
cocci of which are arranged in minute clumps (fig. 75).
The former produces upon the gelatine plate white dots
which spread out superficially and do not liquefy the
medium. In thrust-cultures a white deposit is likewise

200

BACTERIOLOGY

found which gives the surface the appearance of a drop of
stearine (fig. 76). On agar a layer spreads out from the

. Superficial Deposits at the site
deposit of inoculation

Needle-track

Needle-track

FIG. 76.— THRUST-CULTURE ix GELATINE
OP STAPHYLOCOCCUS CEREUS FLAVUS.

FIG. 77. — THRUST-CULTURE ix GELATINE
OF STREPTOCOCCUS PYOGEXES.

streak, in the neighbourhood of which further colonies are
seen. The deposit on potato is similar, but soon becomes
dirty grey.

STKEPTOCOCCUS PYOGENES 201

Staphylococcus cereus flavus is differentiated by the yellow
colour exhibited in its cultures. In addition to these, Von
Schr otter and F. Winkler discovered also the Staphylococcus
cereus aureus, distinguished from both the others by the
orange-red colour of the colonies, and by its slower growth.
These microbes are also constantly found in the nasal
secretion in cases of coryza. The Staphylococcus cereus may
also occur in the interior of pus corpuscles ; but the cells
may then be arranged in diplococci, and in the pus of
urethritis it may be confounded with the gonococcus. The
latter, however, is bleached by Gram's method, whereas the
Staphylococcus cereus retains its colour.

Streptococcus pyogenes. — In phlegmonous suppurative
processes the Streptococcus pyogenes described by Kosenbach
is constantly present, and is probably identical with the
Streptococcus erysipclatis of Fehleisen (p. 113). It is
found, however, not only in erysipelas but also in puerperal
processes occurring in lying-in women. It does not liquefy
gelatine, and forms fine punctiform colonies, while in
thrust-cultures a thin delicate film appears around the
puncture (fig. 77). On agar minute dots resembling dew-
drops occur along the streak, and range themselves into a
ribbon-like stripe. The elements so far alter upon potato
that some individual cells appear larger, and others smaller,
than usual. It grows better at incubation temperature
than at that of ordinary rooms, but growth is usually slow
at best. Transmission of cultures into the lymph-channels
sets up a typical erysipelas, according to E. Fraenkel.

The Micrococcus of gonorrhoea. — The infectious nature of
gonorrhoeal secretion is due, as shown by Neisser, to cocci
which have received the name of Gonococci, and which can
easily be recognised in the secretion owing to their arrange-
ment in pairs. The individual cells are reniform, and may be
found in the pus arranged round the nuclei in the interior
of the cells, but not in groups (fig. 78), and are distinguished

202

BACTERIOLOGY

from similar micrococci which occur in the methra by their
being invariably decolorised by Gram's method, although
otherwise they take the aniline stains very readily.

If it is wished to demonstrate the gonococci in fresh
gonorrhoeal secretion, the following is the best mode of
procedure : — A drop of the pus is placed upon a cover-glass
and spread out lightly, so that the glass is covered with a
thin cloudy film ; but the common method of laying two
cover-glasses one over the other and then drawing them
asunder must not be employed here. The cover-glass is

now stained with a con-
centrated alcoholic solu-
tion of methyl blue,
rinsed in water, dried,
and mounted in Canada
balsam.

Neisser has recom-
mended the following
method of double stain-
ing : — The cover - glass
preparations are brought
for some minutes into
a warm concentrated
alcoholic solution of eosine, the superfluous dye is then
soaked up with blotting-paper, the preparations laid for a
quarter of a minute in concentrated alcoholic solution of
methyl blue, and rinsed in water.

Schiitz's method consists in preparing a filtered satu-
rated solution of methyl blue in 5 per cent, aqueous carbolic
acid, in which the preparations are stained for five to ten
minutes in the cold. After rinsing in water they are laid for
an instant in very dilute acetic acid, again rinsed in water,
and double-stained in very dilute solution of safranine.
The gonococci are blue and the pus-cells salmon-coloured.

FIG. 78.— GOXOCOCCI FROM THE UttETHRAL

SECRETION. (After Jaksch.)

GONOCOCCUS 203

To differentiate them from other diplococci, Stein -
schneider and Galewski recommend that the preparations
should be left for half an hour in a solution of gentian violet
in aniline water, rinsed, laid for five minutes in solution of
potassium iodide, and left in alcohol 'until decolorised, after
which they are again rinsed, dried, and double- stained
in dilute alkaline solution of methyl blue. The gonococci
are pale, the other diplococci stained blackish.

In making a rapid examination of a series of secre-
tions for the presence of gonococci, F. Winkler uses the
following dry ing- on process : — A clean slide is prepared with
a small drop of gonorrhceal secretion, and immediately
passed several times through the flame without being
previously dried in air. The pus is thus desiccated in a thin
film, over which a concentrated alkaline solution of methyl
blue is next poured ; after a half a minute the preparation
is washed in water, dried over the flame, and mounted in
Canada balsam. The pus-cells appear pale blue, the
gonococci dark blue.

The ordinary nutrient materials which prove favourable
to the growth of other micro-organisms fail us completely
in the case of gonococcus, which grows neither on gelatine,
agar, nor potato, though on the other hand, as Bumm has
proved, it thrives on human blood-serum. According to him
the cultures exhibit a greyish-yellow layer, resembling from
its pointed prominences a group of mountains, and whose
margins extend diffusely into the surrounding parts. The
serum is not liquefied. The most suitable temperature lies
between 33° and 27° C. Growth is very slow, and comes to
an end in three days. Cultivation experiments on plover's
egg albumen gave good results in the hands of Von Schrotter
andF. Winkler, a thin, tolerably transparent, whitish deposit
showing itself on the surface of the solidified mass of white
of egg in as short a time as six hours at incubation tern-

204 BACTEKIOLOGY

perature. The growth quickly extends, but ceases after the
lapse of only a few days.

Experiments have been carried on by Wertheim at the
clinic of Professor F. Schauta in Vienna, on the cultivation
of the gonococcus upon a medium consisting of a mixture of
two or three parts peptone glycerine agar and one part
human blood-serum. He obtains the latter from the blood
of the maternal portion of the umbilical cord, which is
severed but not ligatured, and the blood caught in sterilised
flasks. In this way Wertheim was enabled to grow
gonococci in plate-cultures, on which he was able to observe
colonies visible to the naked eye even after
twenty- four hours in the incubator. The
microbes grow rapidly, displaying their
characteristic forms and colour, and give
the best evidence of their specific action
by transmission to human beings. Wert-

FIG. 79. — DEEP - LYING ^ .

COLONY OP GOXOCOC- heirn s whole process vields astonishing

cus ON A SERUM AGAR

hdm' (After Wert" results, and according to his researches

growth takes place decidedly quicker
when deprived of oxygen than when the gas is admitted.

The plate appears diffusely clouded in twenty-four hours,
and assumes the appearance of a delicate flocculent layer
of moss. The majority of the colonies develop in the
substance of the medium, the deep ones appearing whitish -
grey by direct, yellowish-brown by transmitted light, and
in three days show a peculiar nodulation, which is so
regular as to recall the appearance of a blackberry (fig. 79).
The superficial colonies have a compact dot situated exactly
in the centre, which, when examined with a low power,
is found to correspond in structure with the deeper colonies,
but is surrounded on all sides by a superficial film which,
though at first very delicate, transparent, finely granulated,
and colourless, develops in three days round the central

BACILLUS OF SYPHILIS 205

mass numerous minute accumulations of condensed matter
having a brown colour (fig. 80).

Bacillus of syphilis. — In tissues affected with syphilitic
disease and in the layer of pus covering syphilitic ulcers
Lustgarten found small comma-bacilli showing a slightly
S-shaped curvature, but they have not as yet been cultivated
outside the body. In the interior of the tissues they are not
found in the interstices of fibrous bundles or other tissue
elements, but are included in cells which are much larger
than the white blood-corpuscles. The methods of staining
are of particular interest. They are carried out in the case
of tissues only with fine sections prepared from them ; but

Finely

granular

superficial

coating

Central mass

FIG. 80.— SUPERFICIAL COLONY OF G-ONOCOCCUS ON A SERUM AGAR PLATE.
(After Wertheim.)

in the case of secretions a portion is placed upon a cover-glass
and spread out by rubbing. Staining is then done with
Ehrlich's solution of gentian violet in aniline water, in which
the preparations are allowed to remain for twenty-four hours
at ordinary temperature, or for two hours at that of the incu-
bator. They are then placed in absolute alcohol, and after
that for ten seconds in a 1^ per cent, solution of potassium
permanganate, which deprives the tissues of their colour
while the bacilli retain their violet tint. To remove the
precipitate of manganese dioxide which forms upon the
specimens, they are immersed in an aqueous solution of sul-
phurous acid. The preparation can be mounted in Canada

206 BACTERIOLOGY

balsam in the ordinary way, and examined under the micro-
scope. The stained bacilli are decolorised at once by
treatment with glacial acetic acid ; other acids take longer.

A better method than Lustgarten's is that described by
De Giacomi, in which the preparations are treated with
solution of iron perchloride after being stained in Ehrlich's
solution of fuchsine in aniline water. Lewy recommends
staining in carbolic fuchsine and decolorising with distilled
water as the surest and most convenient method. That of
Doutrelepont and Schiitz consists in staining the pre-
parations for twenty- four hours in a one per cent, solution
of methyl violet and decolorising for some seconds in dilute
nitric acid, after which they are transferred for ten minutes
to alcohol of about sixty per cent, strength. The sections
are then double-stained by leaving them for a few minutes
in a watery solution of safranine, and are thoroughly washed
in sixty per cent, alcohol. By this process the bacilli appear
blue and the nuclei and tissue light red.

Marschalko recommends staining the sections of
syphilitic tissue and cover-glass preparations of syphilitic
secretion in Loffler'^s methyl blue for three to four hours at
incubation temperature, or twelve to twenty-four at that of
an ordinary room, washing in water, and double-staining
for one to five minutes in concentrated aqueous solution of
vesuvine.1

Bacillus tuberculosis, — Tuberculosis was regarded as an
infectious disease even by the older physicians ; indeed,
this opinion may have preceded the teaching introduced
in the course of time by the pathological anatomists (such
as Virchow and Eokitansky), which was restricted to the
description of the microscopic and naked-eye appearances

1 [Some authorities believe this to be identical with a bacillus found in
normal smegma prseputiale, &c., which has a similar appearance and similar
peculiarities of staining. The latter is said, however, not to stain by
Doutrelepont's method.] — TB.

TUBERCLE BACILLUS 207

produced by it. But it was Koch who first discovered the
cause of the disease in the tubercle bacilli, recognisable in
all such products of the bodies of men and animals as have
undergone tubercular changes. These furnish a foundation
for the doctrine that tuberculosis is transmissible, inasmuch
as Koch was able to set up a typical tuberculosis experi-
mentally in animals by means of pure cultures. Lortet
and Despeignes are of opinion that the tubercle bacilli
are frequently disseminated by earth-worms.

Tubercle bacilli are fine rods having a length nearly equal
to the diameter of a human blood-corpuscle, somewhat
curved, and often united in groups of two or more, but
seldom in very extensive combinations. They are destitute
of motility. They form spores of an oval outline, both in
artificial cultures and in the bodies of animals, and offer
a high degree of resistance to drying, boiling, and the action
of the gastric juice and of putrefaction. The presence of
carbohydrates or of glycerine is, according to Hammer -
schlag, essential for their growth.

The following is the procedure to be followed when it is
wished to obtain a pure culture of tubercle bacillus : — The
sputum of phthisical patients is mixed with water, and in-
jected into a subcutaneous sac made over the abdomen in
several guinea-pigs. In three or four weeks the tuber-
culosis will be so far advanced as to cause the death of one
or other of the animals, and in the post-mortem examina-
tion tubercular changes will be found in the various organs,
particularly the omentum, spleen, and liver. Another
of the animals is now killed by strangling, a window is
cut in the thoracic wall with all due precautions regarding
disinfection, and a corner of the lung drawn out with the
platinum wire. From this piece of lung some distinct
tubercular nodules are now taken and rubbed between two
slides. (Of course all the instruments used, such as knives,
scissors, and slides, must be carefully sterilised.) The

208 BACTERIOLOGY

glasses infected with the crushed tubercular masses are
now laid upon serum which has been poured out into glass
capsules and inspissated, the tubercular mass is smeared
over the surface of the serum with a strong platinum wire,
and the capsules are covered with glass plates and placed in
the incubator. The colonies are perfectly formed in about
three weeks, when further cultures can be made from them
in test-tubes. When examined under a low power, the
cultures show figures coiled in the shape of an S and
thickened in the centre, which consist of bacilli massed
together (fig. 81). The colonies form dry, white scales,
having a dull surface and not larger than poppy- seeds, which
adhere but loosely to the surface of the medium, never
penetrate into its substance, and do
not liquefy it (fig. 82).

Pastor gives the following pro-
cess for obtaining pure cultures of
tubercle bacilli from sputum : — A
patient is chosen whose sputum is

FIG. 81. — IMPRESSION PREPARA-
TION FROM A CULTURE OF TU- very rich in bacilli and shows coin-

EERCLE BACILLUS ON SERUM.

paratively little contamination with

other micro-organisms, and he is made to rinse out his
mouth and pharyngeal cavity repeatedly with distilled water,
and then to expectorate into a sterilised test-glass. The
sputum, or more properly the liquid contents of the pul-
monary cavities, is shaken up with sterilised water and
filtered through fine gauze to remove the coarser particles.
A few drops of the filtrate are mixed with melted nutrient
gelatine in such a manner as not to render it very
turbid, and the mixture is poured out on plates which are
left under bell-glasses at room temperature. In from three
to four days the various colonies of bacteria contaminating
the sputum form. The portions of gelatine between the
colonies, and which still remain clear, are now sought out

PREPARATION OF PURE CULTURES

209

with a lens, carefully cut away with a sterilised knife, and
transferred to the surface of serum which has been made
to solidify in a slanting position.

The following is Kitasato's method of
preparing pure cultures from sputum :—
The patient is requested to evacuate his
morning sputum into sterilised double
capsules. A flake derived from the
deeper parts of the respiratory appa-
ratus is isolated with sterilised instru-
ments, and carefully washed in at least
ten watch-glasses full of distilled water,
one after the other, fco remove the
bacteria taken up in passing through
the cavity of the mouth. The flake is
now transferred to glycerine agar or
serum. After it has been kept for some
fourteen days in the incubator the first
colonies form, appearing as circular pure
white transparent specks, which project
above the surface of the medium. Ob-
tained by this method the colonies are
flat, shining, and smooth, whereas those
obtained by the earlier method from
tubercular organs are from the first dry,
dull, and wrinkled.1 The difference,
however, disappears as growth advances,
and the whole of the medium becomes
covered with a coat consisting of trains
of bacilli many times coiled.

Hammerschlag obtains a luxuriant
growth by the addition of mannite and

:G. 82. — C ULTURK OF
TUBERCLE BACILLUS
ON SERUM. (After
Baumgarten.)

1 [The bacilli of tuberculosis in birds form a permanent wrinkled mem-
brane on the medium ; but they differ so much in other respects also that,

P

210 BACTERIOLOGY

grape-sugar to the agar. Pawlowsky found that white
detachable colonies appear on the surface of potatoes in
twelve to twenty days, but the potato discs must be pro-
tected from evaporation in air-tight glass tubes. The bacilli
grow in the most luxuriant manner, according to Koch, in
an infusion of veal rendered feebly alkaline, and to which
an addition of 4 or 5 per cent, glycerine and 1 per cent,
peptone has been made. Inoculation is done by floating
a fairly large piece of the seed-culture upon the surface of
the fluid, and when the cultivation has been kept for
several weeks in the incubator, the surface becomes covered
with a tolerably thick skin, dry above, and often wrinkled,
which a few weeks later becomes moistened by the fluid,
breaks up into single ragged pieces, and sinks to the
bottom. Growth requires from six to eight weeks for its
completion.

With regard to the staining of tubercle bacilli, it is of
considerable importance, according to Koch, that the colour-
ing fluid should have an alkaline reaction, as these micro-
organisms will only take the aniline dyes when acted on
simultaneously by alkalies. In order to protect the ob-
server, Pampukes recommends that the tubercular sputa
should be sterilised at 120° C. before being examined, as
this does not impair their power of absorbing stain.

For practical investigation, the observation of Dahme
is of importance, that the flakes which lie at the bottom of
the sputum contain the greatest number of bacilli (fig. 83).

According to the method of Koch and Ehiiich, the
sputum to be examined is poured upon a dark surface, and
the tenacious yellowish particles are picked out. By
means of a penholder containing a nib one half of which
has been broken away the smallest possible lump of

although identical in appearance and staining, they are now generally re-
garded as a distinct variety.]— TE.

STAINING OF TUBERCLE BACILLI 211

sputum is taken up, and is then rubbed between two cover-
glasses. When the sputum is very viscid, the cover-glasses
between which the lump is spread out are, before being
drawn apart, laid on a hot plate at a temperature below
100° C. until a slight clouding takes place, betokening
coagulation. The cover-glass preparation, after drying, is
passed three times through the flame, and stained in a warm
solution of fuchsine or methyl violet in aniline water for a
quarter to half an hour. To decolorise, the cover-glass
is brought into an acid mixture containing one part nitric
acid, two parts water, and two parts sulphanilic acid, or into

Fio. 83.— TUBERCLE BACILLI IN SPUTUM. (After Jaksch.)

a solution of 3 per cent, of hydrochloric acid in 9 per
cent, alcohol, after which it is rinsed in 60 per cent,
alcohol and double- stained in methyl blue or malachite
green if the tubercle bacilli have been stained red, or in
vesuvine or Bismarck brown if they have been stained
blue. It is then rinsed again, dried, and mounted in
Canada balsam.

Kaatzer has modified the process in the following
manner : — The sputum — best that first expectorated in the
morning — having been received into. a completely empty
spitting-cup, is spread out upon a black plate or a piece of
black glazed paper. A very minute particle is now picked

212 BACTERIOLOGY

up from a yellow purulent spot by means of a platinum
needle with the point flattened like that of a scalpel, and
which has previously been sterilised at a red heat ; this
particle is rubbed between two cover-glasses, which are
then passed three times through the flame. Staining is
done with an aniline water solution of gentian violet which
has first been warmed, and the preparations are decolorised
by transferring them from the gentian violet, as soon as it
has grown cold, to a hydrochloric acid alcohol containing
20 parts water and 2 parts hydrochloric acid to 100 parts
alcohol. The preparations remain in this mixture for half
a minute to a minute, are then transferred to concentrated
alcohol for the same length of time, and afterwards rinsed
in water. They are now dried and placed for a half to one
minute in a filtered concentrated solution of vesuvine in
water by way of double-staining, rinsed in 96 per cent,
alcohol, and brought under the microscope in a drop of
water. The tubercle bacilli appear of a dark violet colour,
or even nearly black, as the aniline brown absorbs the
blue part of the spectrum; so that a blue object must seem
black in a brown solution. The other portions of the
preparation are brown.

The following is the method employed by Giinther : —
The cover-glass, having been prepared with sputum and
fixed in the flame, is deposited face downwards in a watch-
glass filled with solution of fuchsine in aniline water, the
centre of which is now heated over a very small flame while
being kept moving vertically up and down. When the
fluid begins to give off bubbles, heating is stopped, the
watch-glass placed on the table, and let stand for a minute.
The process of heating is repeated about five times with
intervals of one minute's standing, after which the cover-
glass is taken from the stain and laid with the film of
sputum uppermost in a watch-glass containing 3 per cent.

ZIEIIL-NEELSEN METHOD 213

hydrochloric acid in alcohol, in which it is moved to and
fro for one minute, and is then rinsed in water. Some
drops of a dilute aqueous solution of methyl blue are now
poured upon it, when, after again washing and drying, it is
passed three times through the flame and mounted in
Canada balsam.

In the Ziehl-Neelsen process, the cover-glass with the
sputum is seized in a forceps, covered with an alcoholic
carbolic fuchsine solution, warmed over a feeble spirit flame
until bubbles appear, washed in water, and flowed with a 5
per cent, solution of sulphuric acid. It is then rinsed in 70
per cent, alcohol, dried, and double-stained with aqueous
solution of methyl blue or malachite green.

According to Friedlander's method, some sputum is
smeared on two slides, which are drawn three times through
the flame. The sputum is then covered with two or three
drops of carbolic fuchsine, passed afresh through the flame,
moistened with water and a few drops of nitric acid alcohol
(3 per cent, of nitric acid in 90 per cent, alcohol), rinsed in
water and watery solution of methyl blue, and examined
without a cover-glass under the oil-immersion objective.

Kiihne recommends shaking up the sputum thoroughly
in a glass with a concentrated solution of borax, so as to
render it fluid. The cover-glasses prepared with this are
stained in carbolic fuchsine for five minutes, decolorised in
30 per cent, nitric acid, rinsed in water, dried, and examined
in a drop of aniline oil coloured light yellow with picric acid,
which is best done by adding two or three drops of a con-
centrated solution of picric acid in aniline oil to a watch-
glass full of pure aniline oil. To obtain permanent pre-
parations the double-staining should be done by transference
for a few minutes to an aqueous solution of picric acid after
decolorising with nitric, after which the specimen is dried
and put up in balsam. An addition of 4 per cent, citric

214 BACTERIOLOGY

acid is advisable, in order to increase the solubility of picric
acid in water. '

In the process of B. Frankel, and in that of Gabbet,
the staining and decolorisation are carried on simul-
taneously. The former is done with fuchsine in aniline
water, from which the preparation is directly transferred
into a filtered mixture of 50 parts alcohol, 30 parts water,
20 parts nitric acid, and as much methyl blue as will dis-
solve with repeated shaking. If aniline water gentian
violet has been used for staining, the preparation is
transferred to a vesuvine solution, consisting of a filtered
mixture of 70 parts alcohol, 30 parts nitric acid, and as
much vesuvine as will dissolve. Staining is finished in a
short time — one or two minutes — and the preparation is
rinsed in water or alcohol rendered feebly acid with acetic
acid, and thoroughly dried.

In Gabbet's modification, the Ziehl-Neelsen carbolic
fuchsine is used instead of the aniline water solution, and as
the second stain methyl blue in sulphuric acid, consisting
of 1 or 2 parts methyl blue to 100 parts of 25 per cent,
sulphuric acid.

Arens pours three drops of absolute alcohol over a
crystal of fuchsine about the size of a millet- seed in a watch-
glass, so as to obtain a saturated alcoholic solution, which
is mixed with 2 or 3 c.cm. chloroform. This causes a tur-
bidity of the solution, which begins to clear with separation of
the fuchsine in the form of flakes. Cover-glass preparations
treated in the usual way are laid in the solution, when clear,
for four to six minutes, and, after allowing the chloroform
to evaporate, are decolorised in a watch-glass full of 96
per cent, alcohol, to which three drops of hydrochloric acid
have been added. They are now rinsed in water and
examined in the same medium, or finally double-stained
with methyl blue.

BAUMGARTEN'S METHOD 215

Gibbes gave the following recipe : 2 grms. fuchsine and
1 grm. methyl blue are slowly introduced into a solution
of 3 c.cm. aniline oil in 15 c.cm. absolute alcohol until they
are completely dissolved, and 15 c.cm. water are then
added. A few drops of this liquid are heated in a test-tube
and poured out into a watch-glass, in which the cover-glass
is laid for five minutes, being then washed in alcohol until
no more colour is discharged. The bacilli are red on a
blue ground. Further staining may be well done with a
concentrated aqueous solution of eosine.

Occasionally it is of advantage to subject the cover-glass
preparations to different methods of examination. Kaatzer
recommends staining the same preparations first with
carbolic fuchsine after the Ziehl-Neelsen or B. Frankel's
method, and examining them with the oil immersion lens,
•after which the cedar oil should be removed with blotting-
paper and alcohol, the preparation dried in the flame,
stained in hot aniline water gentian violet, decolorised in
hydrochloric acid and alcohol, and double-stained in vesu-
vine. The tubercle bacilli, which were red when first
examined, are turned dark violet on a light-brown ground
by the second staining.

In cases where staining yields doubtful results, the pro-
cess of Baumgarten may be employed as a control observa-
tion. The preparation of sputum, having been dried in
air and passed three times through the flame, is first laid
for a few minutes in a watch-glass containing chloroform
to free it from fat, rinsed in absolute alcohol, and, after this
has evaporated, is placed on a slide in a drop of potash
solution, prepared by adding two or three drops of a 33
per cent, solution of caustic potash to a watch-glass of
water. If the microscopic examination (without oil im-
mersion) reveals the presence of bacilli, the cover-glass
is removed from the slide, dried in the air, passed three

216 BACTERIOLOGY

times through the flame, moistened with a few drops of
dilute aqueous solution of methyl violet, and examined at
once under the microscope. The tubercle bacilli, if present
in the preparation, remain completely colourless if the
examination is not protracted beyond five or ten minutes,
whereas all the other bacteria almost instantly assume a
blue colour.

In very doubtful cases in which the ordinary methods
of staining are at fault, preparations must be made from
the sediment. According to Stroschein's method, a table-
spoonful of sputum is vigorously shaken in a test-glass with
three tablespoonfuls of a mixture of 1 part concentrated
solution of boric acid and 3 parts water until the sputum
is liquefied, when it is poured into a glass ending in a point
below. After standing for twenty-four hours, the clear
supernatant liquid is poured off and a cover-glass prepara-
tion made from the sediment at the bottom.

Biedert recommends that a tablespocnful of sputum
should be boiled with two tablespoonfuls of water and seven
or eight of solution of caustic potash ; four more table-
spoonfuls of water are then added, and boiling is repeated
until the mass has become evenly fluid. This remains
standing for three or four days in a pointed glass covered
over, and is then poured off until a depth of only five to
eight millimeters is left. The sediment is energetically
stirred up with some fresh white of egg, and some of the
mixture is rubbed on a cover-glass, dried at a moderate
heat, and stained by the Ziehl-Neelsen method. A small
quantity of untreated sputum from the same source may
be used instead of the white of egg for fixing the sediment
to the cover -glass.

Sedimentation is particularly important in the examina-
tion of fluids which are poor in corpuscular elements, and is
most conveniently done by centrifuging. In the absence,

TUBERCLE BACILLI IN SECTIONS OF TISSUE 217

however, of an apparatus for this purpose, Des Vos mixes
white of egg with four times its bulk of distilled water, which
causes the globulines to sink to the bottom as a coarsely
flocculent mass. Up to 10 c.cm. of the supernatant opales-
cent liquor, which consists of dilute albumen, are now added
to the fluid to be examined, and the whole is well shaken
up and heated in the water-bath until the white of egg
coagulates. In a short time, according to the quantity of
white of egg added, a greater or less amount of a finely
flocculent sediment forms, which is to be examined for
bacilli.

To examine milk for tubercle bacilli, the most con-
venient mode is to place a drop of the suspected milk upon
a cover-glass and add to it two or three drops of a 1 per
cent, solution of sodium carbonate. The whole is well
mixed with a platinum needle, and the cover-glass is then
warmed carefully over a small flame until complete evapo-
ration has taken place. The fat is in this way saponified,
so that finally a thin film of soap remains behind on the
glass, which is stained, &c., like an ordinary cover-glass
preparation.

Kegarding the staining of tubercle bacilli in sections of
tissue, the same methods hold good with suitable modifica-
tion, for a more detailed description of which the reader
may refer to the general section (see p. 84 et seq.) . Here it
is only necessary to remark that the usual method of stain-
ing employed is that with Ziehl's solution, but the sections
must be left lying in it for an hour or even longer. Bleach-
ing is carried out in 10 per cent, nitric acid for a half to
one minute until the red colour has vanished, after which
the sections are transferred to 70 per cent, alcohol until
they acquire a rose-red tint or become completely colourless.
They are then double-stained in methyl blue, thoroughly
washed in alcohol, and put up in Canada balsam. The

218 BACTERIOLOGY

bacilli are distributed through the tissue, or lie in masses
between the lymph cells, or are taken into their interior,
and they are also found enclosed in the so-called giant-
cells.

In order to obtain the active principle tuberculine, Koch
evaporated pure cultures of tubercle bacilli, made in
glycerine and veal infusion, to a tenth part of their volume
in a water-bath, and filtered the fluid through an earthen-
ware filter. An albumose can be procured from tuberculine
after the process of Klebs, which this observer has named
tuber culocidine. The tuberculine is treated with platinum
chloride, or with the so-called alkaloid reagents, and the
albumose remaining in the solution thus formed is precipi-
tated out with alcohol.

Bacillus of glanders. — This bacillus was discovered by
Loffler and Schiitz to- be the cause of the disease called
glanders or malleus, which may be transmitted from horses
and donkeys to human beings. The micro-organisms are also
found in what are called glanders nodules, from which they
penetrate into the surrounding parts and originate morbid
phenomena, and which occur as a rule in the cavity of the
nose, and form deep ulcers on the mucous membrane cover-
ing the turbinated bones, on that of the larynx, and in the
lungs. The neighbouring lymphatic glands are swollen
and rendered hard, as well as the cutaneous lymphatic
vessels, which often burst outwards, forming ulcers.

The bacilli are slender rods with rounded ends. They
are about the size of tubercle bacilli, are without power of
automatic movement, and grow upon our nutrient media
only at incubation temperature. Development is suspended
at a temperature above 42° C. In consequence of this
peculiarity gelatine cannot well be used, as growth is very
scanty at a lower temperature, but the micro-organisms
thrive very well on glycerine agar at 37° C., showing pale

BACILLUS OF GLANDERS 219

yellow or whitish round colonies on the plate as early as
the second day, while along a streak of inoculation made
on the same medium a shining coating appears in four to
five days. On the surface of blood-serum there are de-
veloped yellowish deposits which coalesce with one another
and form a slimy covering. The cultivation on potato is
particularly important : a deposit appears at incubation
temperature which is at first yellowish and transparent,
but which gradually increases and assumes a darker tint.
In about a week the colour changes to reddish-brown, and
finally passes into red. The bacilli readily take up the
aniline dyes, but discharge them again when Gram's method
is employed (fig. 84).

©a 0

'

FIG. 84. — BACILLI OP GLANDERS IN HUMAN BLOOD. (After Jakscli.)

The staining process of Loffler consists in colouring the
cover-glass preparations for five minutes in a hot alkaline
solution of gentian violet or fuchsine in aniline water con-
taining an addition of 1 per cent, of caustic soda solution.
Bleaching is done in 1 per cent, acetic acid coloured to a
wine yellow with a watery solution of tropseoline 00, in
which the preparations remain for an hour, and are then
rinsed in water.

Kiihne treats the cover-glasses with hot carbolic fuchsine
or carbolic methyl blue, and decolorises in water contain-
ing two drops of hydrochloric acid per 100 grams.

For staining glanders bacilli in sections, Kiihne has
•devised the following method, which is reliable and easy in

220 BACTERIOLOGY

use : — The sections, having been stained with carbolic
fuchsine and soaked in water, are quickly decolorised in an
aqueous solution of hydrochloric acid (3 per cent.), well
rinsed in water, and then either dipped lightly into alcohol
or freed as far as possible from water by pressing them
with blotting-paper Aniline oil mixed with 20 per cent,
of oil of turpentine is now allowed to act on the sections in
a watch-glass for eight to ten minutes, during which they
remain adherent to the cover-glasses, and thence they are
transferred to oil of turpentine, xylol, and balsam.

Noniewicz recommends that the sections should be
stained for a few minutes in alkaline methyl blue, rinsed
in water, and decolorised in a mixture of 3 parts of i per
cent, acetic acid, with 1 part of a ^ per cent, solution of
tropseoline 00 in water. Thin sections are only dipped in
quickly, but thick ones may remain in for two to five
seconds or longer. They are then washed in water, spread
out upon a slide, freed from moisture with blotting-paper,
dried in air, cleared with xylol, and mounted in Canada
balsam. The glanders bacilli appear black on a more or
less blue ground. By this method the discovery was made
that the characteristic bacilli appear in the acute form of
the disease, whereas in the chronic form they are sparser,
and round bodies, which stain intensely, appear in their
stead in large numbers.

The glanders bacilli are also found in the interior of
dead cells. The vessels are free, and but few appear in the
blood. Transmission experiments made with pure cultures
have yielded successful results, the infection spreading most
from wounds in the skin. The cornmunicability of the
disease is considerable, and the infecting power of the virus
is not altered by drying for three months.

Other microbes of pus. — It need hardly be said that the
pus from wounds in other specific infectious processes con-

OTHER MICROBES OF PUS 221

tains the corresponding micro-organisms, e.g. Bacillus
anthracis and the bacillus of tetanus ; but besides these, a
considerable number of other microbes have been found
which have the power of originating suppurative processes.
Thus Weichselbaum discovered the Micrococcus intracellu-
laris meningitidis ; Neumann and Schaffer the Micrococcus
meningitidis purulentae ; Becker the Micrococcus osteomyeli-
tidis, and Heydenreich the micrococcus of Pende's ulcer
(Micrococcus Biskra) ; while the Streptococcus contagiosae
coryzse equorum was detected by Schiitz in suppuration
occurring in animals, and Pfeiffer found in the abdominal
cavity of a guinea-pig which died spontaneously a tenacious,
puriform exudation consisting of a pure culture of Bacillus
capsulatus.

Some of the methods invented by Unna for staining
micro-organisms in cutaneous abscesses (see p. 90) are
excellently adapted for showing those in pus. The follow-
ing process may be especially recommended : — The cover-
glass preparations are stained in carmine, transferred for
two minutes to borax methyl blue (1 part each borax and
methyl blue to 100 of water), rinsed in water, and placed in
alcohol to which a few drops of Spiritus saponis kalini l
have been added. They are finally brought once more
into alcohol, and may then be submitted to examination.

1 [See p. 91, note.]- TR.

222 BACTEEIOLOGY

CHAPTER X

BACTERIOLOGICAL EXAMINATION OF THE ORGANS AND CAVITIES
OF THE BODY AND THEIR CONTENTS

Micro-organisms of the living body, — In this section a
series of micro-organisms will be dealt with, which find a
congenial pabulum in the organs and cavities of the living
individual. They are derived partly from the air, partly
from water or other surrounding media, and may occur
either as saprophytes or parasites.

I. THE SKIN

Micro-organisms of the skin. — Most of the micro-organ-
isms met with on the skin in the cutaneous secretions are
saprophytes, but many pathogenic varieties also occur in
this situation. The space beneath the nails, in particular,
is rich in forms, containing especially germs, according to
Preindlsberger, which are found also on other regions of
the cutaneous surface as well as often in the air, and hence
may penetrate with it into the respiratory organs. Patho-
genic germs are found amongst them. Penetration of
micro-organisms into the unbroken skin probably takes
place by the openings of the glands, or perhaps by the
agency of stinging insects.

Amongst the cutaneous micro-organisms the bacteria
of the female generative organs are also included. Straus
and Toledo have found that microbes do not occur either in
the parietes or secretion of the normal uterus, and it is
only in the vagina, where there are accumulations of

METHODS OF EXAMINATION 223

epithelial masses, that the bacteria find a favourable medium
upon which to develop. In normal vaginal secretion the
vaginal bacillus of Doderlein is regularly found, and the
thrush fungus and yeast cells are often met with, while Sta-
phylococcus pyogenes albus, ritreus, and aureus are no rarities.

Micro-organisms are also found in the sebaceous and
sweat glands. Brunner discovered, for example, that strep-
tococci may be detected in the sweat during suppurative
processes, and Garre conversely was able to start a phlegmon
by rubbing a culture of staphylococci into his unwounded
arm. The appearance of coloured sweat is due to the
presence of Bacillus pyocyaneus and Micrococcus htematodes,
whilst in the foul-smelling perspiration on the feet Kosen-
bach found the Bacillus saprogenes II. (see p. 176).

Preindlsberger isolated from the sub-ungual space the
Micrococcus cereus albus and flavus, the Diplococcus lique-
faciens albus and citreus, Micrococcus candicans, Sarcina alba
and flava, and white yeast ; and of pathogenic micro-organ-
isms the Staphylococcus pyogenes aureus and Streptococcus
pyogenes.1

Methods of examination. — In order to stain the micro-
organisms in the skin, the objects to be examined must
first be freed from fat in alcohol and ether, after which they
are stained in glycerine methyl blue by spreading them out
with needles upon a slide and letting the stain act for some
minutes ; the micro-organisms appear blue, the epidermis
colourless. In Bock's process the epidermic scales, after
being freed from fat, are deposited for half a minute to
several minutes in borax methyl blue, then for half a
minute to a minute in a weak aqueous solution (J to 1 per
cent.) of resorcine, and then for some minutes to an hour
in alcohol. To make the fungi stand out clearly, the

1 [The Trichophyton tonsurans also occurs in this situation, causing the
disease known as onychomycosis.~] — TE.

224 BACTERIOLOGY

epidermis is decolorised for some seconds in a weak solution
of hydrogen peroxide and then transferred to alcohol, xylol,
and Canada balsam.

Unna devised the following method for the rapid recog-
nition of fungi in epidermic scales : — The horny scale
to be examined (crust or comedo) is laid upon a slide,
moistened with a drop of acetic acid, and rubbed to a pulp
by means of another slide placed crosswise upon the first,
after which the two slides are separated and dried rapidly
over the flame, so that all the acid is evaporated off
again. Some drops of ether and alcohol are now poured
upon the free upper end of the slide, in order to wash out
the fat, and then two drops of solution of borax and methyl
blue are at once placed upon one slide, which is again
covered crosswise with the other, and the pair are held over
the flame for ten to .twenty seconds. The preparations
are now either immediately subjected to a further process
of bleaching, or are dried over the flame. Styrone, glycol,
or equal parts of glycerine and ether are used as bleaching
agents. By the first or styrone method the preparations
stained in methyl blue are rinsed in alcohol, decolorised for
two minutes in styrone, again rinsed in xylol, and put up in
balsam ; by the second method, the stained preparations are
rinsed in water, decolorised in glycol for two to five minutes,
rinsed again in water and then in alcohol, dried over the
flame, and mounted in balsam. One per cent, solution of
acetic, oxalic, citric, or arsenic acid, or of hydroxylamine, or
one per cent, aqueous solution of soap, one per cent, salt
solution, five per cent, aqueous solution of resorcine, and one
per cent, alcoholic solution of hydrochinone, may be also
used for decolorising.

Diplococcus subflavus. — Bumm very frequently found
in the vaginal secretion diplococci closely resembling the
gonococcus in appearance. They thrive fairly well upon

MICROCOCCUS LA GTE US FAVIFORMIS 225

our culture media, but grow slowly ; and gelatine is lique-
fied, also slowly. Not until a week has elapsed do sulphur-
yellow punctiform colonies, whose border appears fibrous
under low powers, show themselves on the gelatine plate.
In thrust- cultures a yellowish raised deposit with a dull
surface appears after three days, and gradually spreads ; no
growth can be seen along the nee die -track. A greyish-
white raised layer develops upon agar even in twenty-four
hours, and assumes a yellow colour after some days. On
potato a smooth, spherical, sharply-defined deposit forms,
which adheres to the needle and is very viscid. Serum is
liquefied.

Irrespective of these biological peculiarities, the Diplo-
coccas subflcivus may be distinguished from the gonococcus
by the fact that it does not give up its stain under Gram's
process.

Micrococcus lacteus faviformis. — Bumm has very often
found in the vaginal secretion diplococci which show a
tremulous motion. When examined in the hanging drop
they exhibit a propensity to unite in a clump which forms the
figure of a honeycomb. Gelatine is not liquefied, and the
colonies on the plate are small and evenly circular. Little
white dots form upon thrust-cultures even in one day, and
later unite into milk-white islets. A milk-white layer
develops also upon agar and potato. The diplococci do not
surrender their colour under the application of Gram's
process.

Diplococcus albicans amplus. — A not uncommon guest in
the vaginal secretion is the coccus of this name discovered by
Bumm, the elements of which are differentiated from the rest
of the diplococci by their considerable size. Gelatine is slowly
liquefied, and the gelatine plate shows greyish -white, some-
what elevated islets. In thrust- cultures a moist coating
appears on the surface and a white stripe along the needle-

Q

226 BACTERIOLOGY

track. On agar, a stripe-like grey deposit develops rapidly
at 35° C.

Diplococcus albicans tardus. — Unna and Tommasoli found
this microbe in cases of eczema. The diplococci are im-
motile, sometimes arranged in clumps and chains, and do
not liquefy gelatine. Eoundish colonies form on plate -
cultures, presenting an uneven lumpy surface when
examined under the microscope. The colonies gradually
become greyish-yellow, lighter in the centre and at the
margin, so that they appear girdled by a light zone.
Thrust-cultures show a superficial yellow coating, surface-
cultures on agar a greyish -yellow stripe with uneven edges.

Diplococcus citreus liquefaciens. — In cases of eczema Unna
and Tommasoli also found immotile cocci which liquefy
gelatine by their growth. Whitish dots develop on the
plate, which rapidly become fluid and assume a lemon -
yellow colour. Thrust-cultures show a yellowish coating
at first, but after a few weeks a portion of the lemon -
yellow culture is seen floating in the liquefied mass, while
the rest, which has also a yellow tint, is found at the
bottom. The lemon-yellow colour on the surface of the
fluid mass is particularly characteristic. A yellowish layer
forms upon agar, and also on potato.

Diplococcus flavus liquefaciens tardus. — Besides the micro-
organisms just described, the same observers found in cases
of eczema diplococci shaped like a roll of bread, which liquefy
the gelatine rather slowly. Punctiform brownish-yellow
colonies form on the plates, and yellowish masses float on
the surface of the gelatine when it has become fluid. In
thrust-cultures also liquefaction progresses, but with such
extreme slowness that two months elapse before half a
cubic centimeter of gelatine is liquefied. A thick slimy
coat of greenish-yellow colour develops on agar, and a
lumpy sulphur -yellow deposit upon potato.

MICBOCOCCITS OF TRACHOMA 227

Micrococcus haematodes. — Babes isolated from the foul-
smelling sweat of the axilla, which leaves a red spot upon
the under-clothing, a micrococcus the elements of which are
circular or oval, and united to one another by means of a
transparent jelly-like red mass. Babes cultivated them at
incubation temperature upon coagulated white of hens'
eggs, on which medium they form blood-red confluent islets.
Micrococcus of trachoma. — Battler and Michel believe
that the cause of the disease of the conjunctiva and cornea
known as trachoma, or Egyptian ophthalmia, is a diplo-
ooccus distinguished from the gonococcus by its small-
ness and the feeble marking of its line of division. It does
not lose its colour under Gram's process, and does not
liquefy gelatine. On the plate little white nebulosities
develop, and in thrust-cultures a row of globules is seen
along the track, resembling a string of pearls, while a
shining white deposit, which later becomes yellow, forms on
the surface. A grey coating develops upon agar, and a
white ribbon-like stripe upon blood-serum. Experimental
transmission to the human conjunctiva produces a typical
trachoma.

Diplococcus of acute pemphigus. — Demme found in the
vesicles of pemphigus cocci arranged in pairs, which lie
together in dense masses. The micro-organism seems to
be pathogenic, since, when pure cultures are transmitted to
guinea-pigs, inflammatory nuclei form in the lungs, with
emaciation ; and injection into the blood produces similar
phenomena. The diplococcus only grows at incubation
temperature, and is best cultivated upon agar. Bound
milk-white colonies form on the plate, sending out lateral
processes towards the surface and in a radial direction,
which cause the islets to show irregular prominences. The
appearance of superficial cultures on agar is similar. On
serum and potatoes growth is very slow.

Q 2

228 B ACTERIOLOG Y

Vaginal bacillus. — Doderlein detected slender rods, not
endowed with motility, in the normal vaginal secretion
of the adult ; that of new-born infants is destitute of
bacilli. Vaginal bacilli are facultative anaerobes, growing
better in the absence of oxygen. Since their growth
does not advance except at the heat of the body they
cannot be cultivated to advantage upon gelatine, but upon
agar an exceedingly delicate film develops, consisting of fine
minute drops which are clear like water. The culture is
very sensitive to dryness and to degrees of temperature
lower than that of the body. In the incubator the layer
develops into somewhat flat elevations, which always
remain clear and transparent. Cultivation succeeds also
in bouillon, in milk, and upon blood- serum, but no growth
takes place on potato. The bacilli are active acid-formers,
and the acid contained in the vagina (0'5 per cent.) is due
to their vital energy, the secretion of new-born infants,
which is free from them, showing a neutral reaction.

Bacillus of symptomatic anthrax (Rauschbrand) . — Sym-
ptomatic anthrax is a disease occurring in calves and lambs,
which are killed by it in two days. The cause has been found
by Bollinger, Thomas, Kitasato, Kitt, &c., to be bacilli met
with in the sero-sanguineous fluid from the subcutaneous
cellular tissue, which resemble cocci or have a drumstick
form, possess the power of automatic movement, and belong
to the anaerobes. The motility is due to the presence of
numerous flagella. The bacilli are decolorised by Gram's
process ; their growth liquefies gelatine. Warty irregular
islets appear on plate-cultures, and in high cultivations
development takes place along the needle-track, with gene-
ration of gas. The islets appear as globular cavities filled
with fluid, from which whitish fibres penetrate into the
gelatine. On agar an active generation of gas takes
place at incubating hsat, and in blood-serum peculiar

LEPRA. BACILLUS 229

spirally twisted tress-like figures form, which show the
greatest difference in size, and consist, according to
Loffler, of flagella which have been torn off and compacted
together (fig. 85). The surface of potatoes acquires a moist,
polished appearance, in consequence of a thick white
coating which can be easily detached. Bouillon cultures
smell of rancid butter, as also those on agar.

Transmission to .sheep, cattle, and goats is attended
with success, but pigeons and mice are nearly immune.
Owing to the anaerobic character of the micro-organism,
inoculation can only be done subcutaneously. Cultures
on solid media retain their virulence longer than those in
fluids.

Flagella Mass of flagella resembling a tress of hair

FIG. 85.— BACILLUS OF SYMPTOMATIC ANTHRAX, FROM A CULTURE ON SERUM.
Magnified 1,100 times. (After Loffler.)

Lepra bacillus,— Hansen found in leprous tissue, and
particularly in the lepra cells, small slender rods which
have club-shaped ends and approximate nearly to tubercle
bacilli in size. They are immotile, and are principally
distinguished from the tubercle bacilli by their property of
staining readily in aqueous solutions of the aniline dyes.
Besides this, however, they also stain by the methods
devised for tubercle bacilli, as well as by Gram's process,
but staining succeeds with much greater ease with bacilli of
lepra than with those of tubercle.

By Baumgarten's process, cover-glass preparations are
stained for six or seven minutes in a dilute alcoholic
solution of fuchsine, decolorised in a mixture of one part of

230 BACTERIOLOGY

nitric acid and ten of alcohol, rinsed in water, and double-
stained in an aqueous solution of methyl blue.

According to Lustgarten, cover-glass preparations, after
staining in aniline water solution of fuchsine or gentian
violet, are decolorised in one per cent, sodium hypochlorite
and rinsed in water.

Unna stains sections for twelve to twenty-four hours in
aniline-water fuchsine, and decolorises ,in a ten to twenty per
cent, aqueous solution of nitric acid until the preparations
have acquired a yellow colour, after which they are
transferred to dilute alcohol, and left there until the tint
has turned red. The acid still present is now removed by
immersion in a weak solution of am-
,. monia in water, and the water is ab-
|fe sorbed up with blotting-paper, after
which the preparations are mounted

in balsam free from oil, dissolved in
l^~

chloroform.

FIG. 86,-isLET OF LEPRA The Lutz-Unna method consists in

BACILLUS ox A GELA-
TINE PLATE. (After staining the preparations in a warm

aniline-water solution of gentian violet
until they have become darkly-coloured, after which they
are decolorised by successive immersion in solution of
iodine and potassium iodide, in alcohol and nitric acid, and
in plain alcohol. This procedure must be repeated until
the preparations show a bluish slate-colour, after which
they are mounted in balsam dissolved in oil of thyme or oil
of cloves.

Cultivation of the bacilli succeeded in the hands of
Bordoni-Uffreduzzi upon peptone glycerine serum, which
was inoculated with marrow from the bones of a person
who had died of leprosy. From this it was possible to
transfer the microbes to gelatine, on which they form
rounded colonies at 20° to 25° C. without liquefying the

BACILLUS SYCOSIFEEUS FGETIDUS 231

medium (fig. 86). A surface-culture on agar likewise shows
roundish colonies, prominent in the centre. Those on serum
are ribbon-like with jagged edges, and do not cause lique-
faction. Cultivation experiments are best carried on at
37° C.

The disease can be transmitted to men and animals.
In the tissues, the bacilli are found in the cutaneous con-
nective tissue, nerve-sheaths, spleen, and lymphatic glands,
but do not occur in the blood. According to Unna they
are found in the lymph-paths of the tissue, and he also
believes the lepra cells to be transverse sections of lymphatic
vessels.

The other methods of staining the micro-organisms of
the epidermis, which can also be used for abscesses in
the skin, have already been dealt with in the general section,
to which the reader is referred (p. 90).

Bacillus sycosiferus fcetidus was found by Tommasoli
on the hairs of the beard of persons suffering from sycosis.
The rods are short, rounded at the ends, and immotile,
and do not liquefy gelatine. Growth makes slow progress
at room temperature. On plate-cultures white punctate
•colonies form, and gradually spread out like a slimy veil.
A white nail-head growth appears in thrust-cultures, and a
.greyish-white veil-like layer in surface-cultures on agar,
this being formed by the coalescence of single isolated
specks, and often showing wavy stripes. The colonies
on potato are raised, and coalesce with the formation of a
yellowish-white colour. Pure cultures may be obtained
from plucked-out hairs or from the pus in a bacillary sycosis ;
and if, on the other hand, some of a pure cultivation be
rubbed on the skin, there result reddening and intense
itch, and vesicles form in the neighbourhood of the hair.

Ascobacillus citreus. — This was found by Unna and
Tommasoli in cases of eczema of the skin, and consists of

232 BACTEEIOLOGY

motile rods whose propagation but slowly liquefies the
gelatine. Dark colonies form on the plate, and in thrust-
cultures there appears a lemon-yellow coating, which after
some weeks floats on the surface of the liquefied gelatine,
while a few greenish flakes show themselves at the bottom.
On agar a yellowish layer develops, formed by the
confluence of several globular colonies. The growth on
potato is more rapid, and in about two weeks the yellow
coating shows in the centre a greenish-yellow colour with
fine light venules, so that the whole resembles a vine
leaf.

Bacillus xerosis. — E. Frankel, Leber, Neisser, Colomiatti,
and several others found in xerosis of the conjunctiva short
bacilli, devoid of motility, which will not grow on gelatine
or potato. A veil-like film develops on agar at incubation
temperature, and on s'erum there forms a focus of rosette-
like figure. The bacilli are found in the white fatty-looking
scales on the conjunctiva, which consist of horny and fatty
degenerated epithelial cells, free fat drops, and the short
bacilli.

Trichophyton tonsurans. — Malmsten and Gruby found
in the scaly accumulations of Herpes (tinea) tonsurans a
fungus designated as Trichophyton tonsurans, [which
occurs in the hair-follicles and hair-substance, and in the
epidermis]. Leslie Eoberts cultivated it upon infusion of
malt containing sugar and on alkaline veal-bouillon. The
portion of skin affected by the disease was cleansed with
corrosive sublimate, several short stumps of hairs were pulled
out with sterilised forceps, and their bulbous extremities
snipped off with a pair of scissors (previously sterilised at a
glowing heat) in such a way that they fell directly into
a corresponding number of flasks prepared with infusion
of malt or bouillon. Even in twenty-four hours colourless
threads radiate on all sides from the yellow hair-bulb,

TRICHOPIIYTON TONSURANS 233

and grow together into a colourless tuft of mycelium.
If they rise to the surface, the part exposed to the
action of the air becomes very speedily covered with
a snow-white powder, which lends the surface, seen from
above, the appearance of a, membrane, and which gradu-
ally becomes yellow. Already in the earliest stages of
development there occur in the mycelial tubes dilatations
resembling ampullae and filled with a granular mass, and
these are taken to be the spores of the mycelium. The
tubes enlarge and become segmented, which causes a dis-
tinct resemblance to a string of pearls ; the mycelium now
gradually becomes finer, the divisions are small, and show
smooth walls with bulgings. Yerujski has shown experi-

FIG. 87.— MYCELIUM FILAMENTS OF TIUCHOPHYTOX ToxsuitAxs, FROM A PURE
CULTURE ov INKUSIOX OP MALT. (After Leslie Roberts.)

mentally that glucose, and not, as Grawitz supposed, a
nitrogenous body, is the medium suitable for the tricho-
phyton. The development, therefore, of the tonsurans
fungus in the epithelium of the skin covering the body does
not progress by means of spores, but merely by the swelling,
constriction, and final separation of fibres (fig. 87) .

This fungus is also, according to H. von Hebra, the
exciting cause of impetigo contagiosa (an exanthem charac-
terised by the formation of pustules), as well as of eczema
marginatum, [and it is now agreed that tinea sycosis, tinea
circinata (common ringworm), and onychomycosis (an
affection of the nails) are due to it. (See also note at end
of chapter.)]

Gelatine is quickly and actively liquefied, according to

234

BACTERIOLOGY

Grawitz, and the fungus fur floats upon the surface as a
thick coating, white above and yellow underneath.

The fungus of favus (Achorion Schcenleinii) . — In the scaly
accumulations from persons suffering from
favus, Schonlein discovered a fungus to
which the name of Achorion Schcenleinii
has been given. The favus crusts or
scutula show the mycelium of the organ-
ism, which is remarkable for the fact that
the fibres grow up perpendicularly from
the horny layer. The right angle formed
by these fibres with the substratum is
characteristic of the scutula of favus, since,
in the horny scales permeated by the fungus
of pityriasis versicolor or by the Tricho-
phytoii the fibres of the organism form
acute angles, or run parallel with the cells
of the horny layer.

Unna differentiates three varieties of
favus fungus — the Achorion euthythrix, with
straight fibres and abundant formation of
spores ; the Achorion dicroon, with forking
hyphae ; and the Achorion atacton, the hyphse
of which run an irregular course and pos-
sess peculiar knotty ramifications and many
sharp angles. The first forms voluminous
greyish-yellow scutula (favus griseus), the
second sulphur-yellow scutula, which are
slow in developing (favus sulphur 'eus tar-
dus), and the third sulphur-yellow scutuia
also, which form more quickly (favus sul-
phureus celereus). Unna used for ctfltiva-

"

tion a medium composed of 4 per cent, of
agar, 1 per cent, of peptone, and 5 per cent.

IG. ss. — SURFACE

ClTLTURE OF THE

THE FUNGUS OF FAVUS 235

of levulose or J per cent, of common salt. In order to
obtain pure cultures dry media only must be employed, and
they must accordingly have been kept in a warm place for
a considerable time before use. All other micro-organisms
which might spoil the pure culture of favus require a tolerably
large amount of water, and soon cease to grow upon very
dry soils. The Achorion euthythrixfoms dense woolly white
furs, half a centimeter in height, upon the nutrient medium,
and grows but a short way into its substance, whereas
the growth of Achorion dicroon lies for the most part in
the medium and only raises a thin coating of mycelium
.above the surface. The Achorion atacton occupies an inter-
mediate position between the other two. Those varieties
of favus fungus which are marked by their deep growth
in artificial cultures grow also into the hair-follicles and
are more difficult to treat.

Gelatine is liquefied very late and only very slightly by
the growth of the favus fungi. On agar plates there
develop in twenty-four hours at incubation heat, according
to Plaut, very small shining fibres resembling cotton wool,
which are recognisable under the microscope as minute
masses of conidia. In forty-eight hours the colonies have
changed to round milk-white opaque discs possessing a
border of very fine and quite short threads. On potato
Plaut obtained, at incubation temperature, an irregular dis-
coloration on the site of inoculation, from which the fun-
gus grows into the substance of the potato. Similar
-coatings to that upon potato appear on boiled white of egg,
.and on blood-serum a greyish-white, thin, coherent film
develops below the surface and sends out long thin pro-
cesses downwards and parallel to it. The serum is neither
liquefied nor discoloured (fig. 88).

[Microsporon furfur is found in the yellowish or brownish
,-scaly patches on the skin in pityriasis versicolor. When
some of the scales are examined under the microscope in a

236 BACTERIOLOG Y

drop of potash solution, abundant mycelial threads are-
seen, together with spores, which are gathered in groups
resembling clusters of grapes. Cultivation on solid media
has not yet succeeded.] — NOTE BY TRANSLATOR.

Certain observers, most recently M. Sabouraud, believe
that Trichophyton tonsurans really includes several distinct
varieties. The latter states as the result of an extended
research that there are two principal kinds, distinguished by
the size and arrangement of their spores and by their bio-
logical characters. The first, called by him Trichophyton
microsporon, has spores 3//, in diameter which lie in no>
particular order, and is apparently destitute of mycelium.
It occurs only in tinea tonsurans, causing about 60 per cent,
of the cases, and is most difficult to treat. On beer-wort
and agar it produces ,a growth which is at first white and
downy, but from the fifteenth to the eighteenth day becomes
dry, floury, and yellowish ; on potato it is dry and yellowish-
brown from the first.

The other variety, Trichophyton macro- or megalosporon^
which causes all the remaining forms of tinea, is distinguished
by spores IJJL to Sfj, in diameter and arranged in lines in the
branches of its mycelium. Cultures show a white downy
growth, which appears a little later than in the case of the
former variety, and does not alter. On potato a reddish-
brown spot, like dried blood, appears ten days before the
white down develops. The macrosporon variety, however,
seems to include many species distinguished by further dif-
ferences in growth and producing distinct clinical phenomena.
One such causes about half the cases of tinea circinata, the-
others are rare.

The above-named varieties always reproduced the same
species, and the former could not be made to grow on any
part of the body except on the head.

(See Ann. de Derm, et de Syph., vol. iii. Nov. 1892 ; also Brit. Joiirn. of
Derm., vol. v. Jan. 1893, and Med, Week, vol. i. Feb. 24, 1893.)— TB.

CHAPTEE XI

THE ORGANS AND CAVITIES OF THE BODY AND THEIR

CONTENTS (contin ned)

II. THE DIGESTIVE TRACT
The Cavity of the Mouth

Micro-organisms of the mouth and their examination.—
A number of micro-organisms are found in the mouth,
which are derived from the air and are taken into
the digestive tract with the food. Their occurrence in the
buccal cavity is so constant that in each examination of
saliva cocci, bacilli, spirilla, and other fungi are encountered
in large numbers. When saliva is freely smeared over the
surface of a cover-glass, allowed to dry, fixed in the flame,
and stained in a dilute alcoholic solution of an aniline dye,
micro-organisms will be found which belong in part to the
mouth fungi, the properties of which we know by cultivation,
but a large number have not as yet been successfully grown
on our nutrient substances. Besides the saliva, the particles
of food which remain behind in the mouth, and especially
between the teeth, play an important part in the retention
of bacteria in this cavity, and to these must also be added
changes in the teeth and gams which promote the develop-
ment of micro-organisms. The importance of the saliva to
the life of the micro-organisms lies in the alkaline reaction
which it commonly possesses, since if an acid reaction sets
in, its capability of serving as a favourable nutrient
medium for microbes becomes diminished ; but in this case,

238

BACTERIOLOGY

on the other hand, the teeth become so altered by the acidity
as to furnish a suitable nidus for their growth (Miller) .

If solution of iodine and potassium iodide be added to
a sample of fresh saliva, many fibres are seen which are
coloured yellow and intertwined with one another. These are
described as Leptothrix buccalis. Others stain bluish-violet
with tincture of iodine, appear in tufts, and are called the
Bacillus buccalis maximus (fig. 89). Micrococci which
appear bluish-violet after the addition of tincture of iodine
bear the name of lodococcus, amongst which Miller distin-
guishes by cultivation an lodococcus magmis and an lodo-
coccus parvus. Some-
times cocci are stained
blue and their sheath
yellowish by tincture
of iodine (lodococcus
vaginatus), and some
stain rose-red. Infec-
tions of the teeth
leading to caries are
supposed by Miller to
be due to the fact that
in acid solutions the enamel loses the lime it contains
and grows soft, this softened substance then forming the
gelatinous ground -substance which is altered just like
gelatine by the action of micro-organisms, so that damage
to the tooth results, leading to caries.

Micrococcus salivarius septicus and Bacillus salivarius
septicus. — Biondi found both micro-organisms in saliva, the
bacillus in healthy as well as diseased individuals, the
micrococcus in puerperal septicaemia.

The Micrococcus salivarius septicus consists of round or
sometimes oval cocci, the growth of which does not liquefy
gelatine. Upon plates round colonies form which are dark-

FIG. 89.— LEPTOTHRIX BUCCALIS. (After Jaksch.)

BACILLUS ULNA 239

coloured ; in thrust-cultures the growth is granular, owing
to the close apposition of the punctiform colonies, and on
agar there is an abundant superficial growth.

The Bacillus salivarius septicus shows short rods with
pointed ends which at times lie in chains, at others in clumps,
and grow best when a little phosphoric or hydrochloric
acid (0*04 per cent.) is added to the medium. Gelatine is
not liquefied. Eound colonies with a transparent periphery
develop on the plate, and the islets show a zigzag network
under a high power. A thrust-culture appears as a clear
band with peripheral dots. If a transmission be made to
agar from a gelatine culture, a transparent coating develops.

Both have a pathogenic action upon mice, guinea-pigs,
and rabbits, and the animals die of septicaemia, sometimes
in as short a time as forty hours.

Bacillus ulna. — Vignal found in the mucus from his
mouth rods with rounded ends, which liquefy gelatine in
growing. The colonies are round, and appear on the plate
like two concentrically- arranged stripes separated by a clear
line. Several zones appear in the liquefied part and towards
the border, the outer one of which looks like a tangle of
fine fibres. In thrust-cultures a funnel-shaped area of
liquefaction appears along the needle-track, on the bottom
of which lie crumbling accumulations of bacteria, and in a
few days a shining membrane forms on the surface. Growth
progresses best at 20° C. On agar there appears at incuba-
tion temperature a deposit in the form of a coherent pellicle,
which can with difficulty be raised from the surface of the
medium. A coating develops on potato at the same tem-
perature. Serum is liquefied. The smell of the cultures
resembles that of decomposed pus.

Bacillus gingivae. — Miller isolated thick rods with
rounded ends from the deposit in a mouth not kept properly
clean, and in the suppurating pulp of a tooth. They form

240 BACTERIOLOGY

at ordinary temperature round colonies on the gelatine
plate, which liquefy in two days. Thrust-cultures show
a funnel-shaped excavation. On agar there forms a
greenish - white layer. Intraperitoneal injections into
white mice prove fatal with the symptoms of peritonitis,
and subcutaneous injections lead to the formation of
abscesses.

Bacillus diphtheria. — Klebs was the first to draw atten-
tion to the presence of bacilli in diphtheritic disease, and
Loffler has succeeded in producing similar morbid conditions
in animals by the injection of pure cultivations. The
bacillus is an imniotile straight or slightly curved rod of
the same length as the tubercle bacillus, but much thicker.
According to Loffler, it grows upon a nutrient medium
composed of 8 parts serum of sheep's blood, 1 part
neutralised veal bouillon, 1 per cent, of peptone, 1 per cent,
of grape sugar, and J per cent, of common salt ; and Kolisko
and Paltauf state that luxuriant growth takes place upon a
nutrient bouillon containing sugar, while Kitasato obtained
an abundant development upon glycerine agar. It should
be observed that the best temperature for all these experi-
ments is 33° to 37° C., and, speaking generally, the diph-
theria bacilli require for their development a heat of over
20° C. They retain their vitality even when completely dried,
and Loffler found them still capable of developing after 101
days. If diphtheritic membranes are protected from the
action of light and kept dry, cultures retaining their viru-
lence can be prepared from them after as long a time as
three months. They form upon gelatine small, round,
white colonies, with a coarsely granular texture and irregular
edges, and do not liquefy the medium. Little white dots
may be observed in the thrust-culture, and the rodlets often
assume altered shapes. The growth upon ordinary agar is
scanty, although luxuriant upon glycerine agar. A greyish-

BACILLUS DIPHTHERIAS 241

white deposit is visible in forty-eight hours upon potatoes
which have been rendered feebly alkaline.

For staining the bacilli in sections, the best solution to
employ is composed of 30 c.cm. alcoholic solution of methyl
blue in 100 c.cm. of O'Ol per cent, caustic potash (1 part
of caustic potash in 10,000 parts of water). A few
minutes are required for staining, after which the sec-
tions are placed for some seconds in Jper cent, acetic acid,
and then in absolute alcohol. Finally, they are treated with
cedar oil and mounted in balsam. The ordinary aniline
dye solutions have no effect. Besides the method of Lofner,
the negative result obtained with Gram's process is to be
considered a criterion of the presence of Lofner's diphtheria
bacilli.

Various animals exhibit a high degree of sensibility
to infection with pure cultures of this micro-organism.
When the conjunctiva of young rabbits is slightly abraded
and smeared with a pure culture, they soon die, according
to Babes, and subcutaneous injections act with fatal effect
upon guinea-pigs in two days. Bacilli can be detected at the
point of inoculation, but all the remaining organs are free.

Loffler obtained a whitish substance from the cultures
by extraction with glycerine and precipitation with alcohol.
It is soluble in water, and one or two decigrams when in-
jected hypodermically set up a hsemorrhagic oadema and
cutaneous necrosis. Brieger and C. Friinkel state that
the poison extracted from the diphtheria bacillus belongs
not to the alkaloids but to the toxalbumins.

Numerous cocci are often found in the false membranes
of diphtheria, and these were formerly looked upon as the
cause of the disease ; some of them are, however, strepto-
cocci, the rest are saprophytes. According to Loftier and
Von Hoffmann, bacilli are frequently detected in the cavity
of the mouth and pharynx which morphologically and

242 BACTERIOLOGY

biologically resemble those of diphtheria, but are devoid of
virulence, and these are consequently described as pseudo-
diphtheritic bacilli. Eoux and Yersin, however, believe
them to be genuine diphtheria bacilli the virulence of which
has become very much attenuated.

Spirillum Milleri. — Miller isolated from a carious tooth a
variety of bacterium which appears to be identical with the
vibrio described by Finkler and Prior. It consists of jointed
rods, sometimes more and sometimes less curved, the figure
of which resembles S or O. The spirilla are destitute of
inotility. Upon the gelatine plate there appear scattered
colonies, which speedily liquefy. Thrust-cultures show no
air-bubble at the surface, owing to the rapidity with which
liquefaction progresses, but there soon forms a tolerably ex-
tensive funnel-shaped fluid area, on the bottom of which the
masses of bacilli lie (compare fig. 52). A superficial layer
forms upon agar. The micro-organism seems to take a
prominent part in the softening and liquefaction of the
enamel organ which occurs in caries.

Spirochaete dentium (Denticola). — There is found in the
cavity of every mouth, according to Miller, and particularly
below the edge of the gum, a variety of spirillum which is
also met with in the secretion of the nose, and is remark-
able for the fact of having pointed ends. This peculiarity
is only seen in two of the forms of spirilla discovered up to
the present, viz. the Spiroch&te dentium and the spirillum
of recurrent fever. Artificial cultivation has not yet been
successful.

Vibrio rugula. — Vignal found also an anaerobic micro-
organism in the mouth, which, however, can only be pre-
served in an indifferent gas, such as hydrogen. Yellowish-
white globular colonies develop on the gelatine plate, and
liquefy outwards from the periphery. In thrust-cultures
little white nodules form on the surface as early as the first

THRUSH-FUNGUS 243

day, and liquefaction gradually sets in, a white mass lying at
the bottom. A white wrinkled coating develops on potatoes
and serum, and the latter is speedily liquefied. All cul-
tures generate a gas with a most offensive feculent odour.
According to Prazmowski, the spirillum develops terminal
spores. Its growth causes curdling in milk, and a very
rapid decomposition of albuminoid bodies and of cellulose.

The fungus of thrush (Soorpilz) . — The patches known as
thrush, which occur in the mouths of infants at the breast
and of persons greatly reduced by disease, are white in the
uncontaminated state, resembling curdled milk, but when
they have become impure display various colours. They
consist of fibres, conidia-spores, bacteria, epithelial cells,

Conidia

Filaments
PIG. 90. — FUNGUS OF THKUSH (Monilia Candida).

and white blood-corpuscles. The thrush-fungus was for-
merly assigned to the group O'idium, and described as
O'ldimn albicans ; but according to Eees, Grawitz and
Kehrer it belongs to the yeast fungi, and must therefore be
spoken of as My coder ma albicans. Plaut believes it to be
identical with a yeast very widely distributed in nature, the
Monilia Candida (fig. 90).

According to Grawitz snow-white colonies develop on
the gelatine plate, but do not liquefy the medium. In thrust-
cultures white islets form, from which processes radiate out
on all sides, so that the culture has the appearance of a fine
brush, while filamentous mycelium develops in the deeper
part. The fungus grows on potato in a thick white coating ,

R 2

244 BACTERIOLOGY

consisting of little masses from the size of a millet seed to
that of a lentil strung together in chains. According to
Klemperer, guinea-pigs into whose veins a pure culture has
been injected perish within from twenty- four to forty -eight
hours.

Other bacteria of the mouth.— Besides these, Yignal found
in the cavity of the mouth a considerable number of other
micro-organisms, such as the Staphylococcus pyogenes albus
and aureus, Bacillus subtilis, Bacillus mesentericus, and a
number of chromogenic bacteria, especially those producing
yellow, green, red, and brown pigment. Vignal and Biondi
have also found the Diplococcus pneumonia almost con-
stantly in the cavity of the mouth and the saliva. Rosen-
bach found Bacillus saprogenes I. in white plugs (see
p. 175).

The Tympanum

Micro-organisms of the tympanum. — We subjoin the
examination of the cavum tympani to that of the mouth,
taking the view of Urbantschitsch that it is a diverticulum
from the latter.

Netter found the Streptococcus pyogenes in diseased con-
ditions of the middle ear, as well as Staphylococcus pyogenes
and the Pneumococcus, but he encountered them not only
in middle-ear inflammations in adults, but also in the
middle ears of new-born infants. Gradenigo and Penzo
very often found the Micrococcus cereus albus and Bacillus
lactis aerogenes, and further the Micrococcus subflavus,
Micrococcus candicans, Micrococcus flavus tardigradus, Micro-
coccus urcte liquefaciens, Diplococcus lacteus faviformis,
Bacillus fluorescens, &c. ; so that these observers could
find no pathogenic micro-organisms normally present in
the middle ear of new-born infants and those at the breast.
In acute otitis media, Kanthack found chiefly the Diplo-

GASTRIC MICRO-ORGANISMS 245

coccus pneumonia, and also the Bacillus pyocyaneus, amongst
others.

The Stomach

Micro-organisms of the stomach. — By examining vomited
matters, or by washing out the stomach, some micro-
organisms are found in its contents, which are in part de-
rived from the air, having made their way into the region of
the pharynx by means of respiration, and passed on thence
into the stomach by the act of swallowing ; and similarly
micro-organisms can be conveyed into the stomach along
with water or articles of food, or also from the mouth by
means of the saliva. Neither the surface of the mucous
membrane lining the stomach, nor its contents, forms, as a
rule, a favourable nidus for the various micro-organisms,
since the acid contained in it has a destructive action on
most of them. Other microbes, on the contrary, resist the
acid of the gastric juice, and are also capable of thriving
when but little oxygen has access, and hence can grow
and maintain themselves for a considerable time in the
stomach.

Abelous found certain microbes which are normally
present in the stomach. To these gastric bacteria be-
long the Sarcina ventriculi, Bacterium lactis aerogenes,
Bacillus pyocyaneus a, Bacillus subtilis, Bacillus amylobacter,
Bacillus megaterium, Bacillus mycoides, and Vibrio rugula.
Besides these, other bacteria have been described by this
author which are not yet thoroughly known, but amongst
which bacilli preponderate. Before Abelous, De Bary had
subjected the contents of the stomach to an investigation,
and, in addition to different bacteria, discovered the O'idium
lactis and the Leptothrix buccalis.

It happens in many cases that the acid constituent of
the contents of the stomach is reduced to a minimum, or
the reaction may even change to alkalinity, so rendering

246 BACTERIOLOGY

the conditions decidedly more favourable for the growth of
micro-organisms, which can pass on into the intestinal canal
along with the substances which the stomach contains.

According to Arnold, no micro-organisms are found in
the stomach of new-born infants, but on the other hand
they can be detected after even twenty-four hours of
life.

Sarcina ventriculi. — This sarcina was found by Goodsir
in the contents of the stomach, and shows cells resembling
cubes with rounded angles and edges, and which are arranged
in fairly large-sized packets (see fig. 1). On the gelatine
plate rounded yellowish colonies appear in from one to two
days, which show diplococci and tetrads under the micro-
scope, but no packets. The gelatine is not liquefied. The
thrust -culture shows a growth along the track of the needle
and a prominsnce upon the surface (nail-culture). A coating
of a yellowish-brown colour, consisting of round islets, ap-
pears upon agar. On potato a yellow colour becomes visible
round the place of inoculation, followed gradually by a warty
deposit, and the inoculated spot itself is chrome-yellow. The
envelope surrounding the sarcina when in packet shape
distinctly exhibits the cellulose reaction, which consists of JL
red colour under the action of iodine and sulphuric acid.
While the form of the micro-organism is altered when it is
grown on the ordinary culture media, the packet figure is
retained in cultivations on neutralised infusion of hay, and
can be obtained for observation partly from the pellicle and
partly from the precipitated masses. If inoculation be
effected from a gelatine plate into the infusion of hay, the
packet form will again be assumed by the cocci, which have
been arranged on the gelatine as diplococci and tetrads. The
sarcina thrives better upon a hay infusion to which two per
cent, of cane sugar or glucose have been added (fig. 91).

The Sarcina ventriculi very readily takes up the aniline

SARCINA VENTR1CULI

247

colours, and does not discharge the stain under Gram's
process. •

Colony on the gelatine
plate

_ _ Tetrad form of the sarcina
ljj? giown on gelatine

Superficial prominence

Needle-track

FIG. 91.— THRUST-CULTURE ix GELATINE OP SAUCIXA VEXTRICULI, FOUR DAYS OLD.

The growth of this microbe does not affect albuminoid
bodies, but it has the property, on the other hand, of pre-
cipitating caseiue and changing milk sugar into lactic acid.

248 BACTERIOLOGY

Micrococcus tetrageaus mobilis ventriculi. — Mendoza
found in the contents of the stomach motile cocci which
tend to arrange themselves in fours, and appear as tablet-
cocci. Bound colonies form on the gelatine plate, which
appear finely granular under a low power and are sharply
defined, while on thrust-cultures a dirty-white layer is
visible. When the cultures stand for some time they take
on a yellowish-brown colour, the sugar tint. The gelatine
is not liquefied.

Bacterium lactis aerogenes. — In the normal small in-
testine of young animals and sucklings Escherich found a
micro-organism which Abelous detected almost as a rule
in the stomachs of adults also. They are short rods with
rounded ends and destitute of motility. On the gelatine
plate roundish colonies form, which appear yellow7 in the
deeper parts. In thrust- cultures growth takes place along
the canal and on the surface a prominence develops (nail-
culture). The gelatine is not liquefied. A shining layer
develops upon agar, having the centre whiter and denser than
the periphery. This layer spreads out very rapidly over
the surface, and soon appears permeated by bubbles of
gas. On serum there forms a greyish moist deposit with
irregular edges, which rapidly extends without liquefjing
the medium, On potato there develops a white coating per-
vaded by gas-bubbles, which becomes yellowish and fluid.
Gas-bubbles appear on other media also, but on gelatine
and agar only when glycerine is added. It grows 011 milk
even when oxygen is excluded. Curdling of milk is caused
by the growth of the bacterium, and the lactose is very
rapidly and energetically converted into grape-sugar. It
has no action on albuminoid bodies.

Bacillus indicus. — Koch found in the gastric contents of
an Indian ape a fine, very short bacillus with rounded ends,
and characterised by a tolerably distinct motility. The

INTESTINAL MICRO-ORGANISMS 249

gelatine is very rapidly liquefied. Round yellow colonies
with bulging edges form on the gelatine plate, being larger
on the surface than in depth. In thrust -cultures the upper
part of the liquefied mass is coloured brick-red, and on
agar there develops a brick-red deposit with white edges.
Serum is liquefied, sometimes with, sometimes without,
pigmentation. On potato a brick-red deposit forms on the
place of inoculation. Pigmentation depends upon the access
of air. This bacillus is distinguished from the Bacillus
prodigiosus, to which it has otherwise considerable resem-
blance, by its pathogenic action on animals, which it kills
with the symptoms of a severe gastro-enteritis.

The Intestine

Intestinal micro-organisms, — Passing from the stomach
along with the chyme, the micro-organisms reach the
internal surface of the intestine, an occurrence which, how-
ever, only becomes possible from the fact that certain of
them are capable of preserving their vitality notwithstanding
the gastric juice, or that the reaction and other qualities of
the juice itself are not such as to exert a prejudicial influence
on the microbes. The internal surface of the intestinal
mucous membrane contains very many micro-organisms,
and seems as a rule to afford a very favourable environment
for them, while the contents of the gut, whether liquid or
firm, show microbes in such abundance even under normal
conditions that of all parts of the body they are richest in
.such organisms.

The alkaline reaction of the intestinal secretion and the
length of time which food takes to pass through the intes-
tinal canal are important factors in favour of the growth of
micro-organisms. Duclaux ascribes to them a certain
amount of digestive significance, on the ground that their
activity is capable of assisting the physiological digestive

250 BACTERIOLOGY

function, and he believes that the duty has fallen to
them of carrying on a ' digestion bacterienne ' by their vital
action.

Under normal conditions the microbes lie on the inner
surface of the intestine, but in pathological processes the
parasites penetrate between the epithelia of the villi, thence
into the interior of the villi, and eventually through the
intestinal parietes to the peritoneum.

Articles of food which contain bacteria increase the
number of colonies obtained from the contents of the gut
to a very considerable extent, while the ingestion of sterile
food-stuffs or of red wine causes, according to Arnold, the
appearance of a striking diminution in the number of
germs.

According to Duclaux, only those bacteria can develop
luxuriantly in the intestine which have no stringent need of
oxygen ; but Abelous sets up the hypothesis that although
the oxygen necessary for the existence of aerobes occurring
in this situation can, in point of fact, reach the intes-
tine with the saliva, the intestinal micro-organisms have
the power of accommodating themselves to a deficiency
of it.

Most of the bacteria living upon the mucous membrane
are saprophytes ; the pathogenic varieties are present only
in small numbers, and often perish by being overgrown by
the former, while many pass away in the faeces. According
to Nothnagel, a very large number of yeast- cells are found
in the contents of the gut, so much so that Brefeld seeks the-
normal habitat of yeast in the large intestine.

Micrococcus aerogenes. — Miller found in the digestive
tract fairly large non-motile cocci of oval outline, which are
distinguished for their marked power of resisting acids,
and hence do not lose their vitality in acid digestive juices.
Gelatine is slightly liquefied. The colonies on plate-cultures.

BACILLUS COPROGENES FCETIDUS 251

are round, and have on their surface several specks, which
under a low power appear sometimes light and sometimes
dark. In thrust-cultures the growth takes the form of
a nail, and is of a greyish-yellow colour. A yellowish-white
deposit develops upon a gar ; that on potato shows irregular
prominences. Generation of gas takes place in the presence
of carbohydrates.

Bacillus putrificus coli. — Bienstock found a micro-organ-
ism in the contents of the intestine which has the power of
decomposing albuminous substances with formation of am-
monia, a process which takes place whether air is excluded
or is present. It consists of short motile rods, the growth
of which does not liquefy gelatine. A yellowish coating
develops upon agar after some time.

Bacillus coprogenes foetidus. — In the intestine of pigs
attacked by erysipelas, Schottelius very often found short
immotile rods with rounded ends, which play no part in
the erysipelas, but can very readily make their way into
the blood and neighbouring organs in consequence of the
intestinal ulcers. Gelatine is not liquefied, and small pale
yellow islets develop in it, which coalesce and form a grey
transparent coating. The culture gives off a very unpleasant
smell.

Bacterium Zopfi. — Kurt and Fliigge discovered in the
intestine of chickens actively motile bacilli, the growth of
which does not liquefy gelatine. Threads resembling
mycelium appear on the plate even in a day, and in thrust-
cultures abundant filaments pass out from the needle track,
extending in a radial direction, but often crossing each
other. No growth takes place on serum. The bacilli thrive
best at a temperature between 20° and 30° C. ; higher than
this their vitality is reduced.

Bacterium aerogenes, Helicobacterium aerogenes, and Bacil-
lus aerogenes. — These micro-organisms were found by Miller

252 BACTERIOLOGY

in the intestinal tract. They are motile bacilli which do
not liquefy gelatine by their growth, possess the peculiarity
of offering resistance to moderately concentrated acids, and
hence are able to pass through the stomach without injury
to their vitality.

Bacillus dysenteriae. — Chantemesse andWidal discovered
short rods with rounded angles and but scanty power of
movement in the contents and walls of the intestines, as
well as in the spleen and abdominal glands, in cases of
dysentery. They take up the aniline colours badly, and
do not liquefy gelatine. On the plate there develop at first
small white specks, which assume a yellow colour and unite
with the neighbouring islets ; but in some days the yellow
colour vanishes, and the colonies become white and granular.
A dry yellow membrane develops on potatoes.1

Bacillus of fowl cholera. — Poultry typhoid or fowl cholera
is an epidemic disease accompanied by diarrhoea, often
occurring amongst poultry, and which Perroncito first in-
investigated more thoroughly. Pasteur describes it as
* cholera des ponies,' and more recently Marchiafava, Celli,
and Kitt have been occupied in bacteriological research
concerning it.

In the blood, in the capillaries of all the various organs,
particularly the spleen and liver, and in especial abundance
in the intestinal contents, there are found short plump rods
which are somewhat constricted in the centre. A typical
attack of the disease can be caused in hens by inoculation
with these or by feeding the fowl with them, and other
birds also, such as geese and pigeons, are very susceptible
of infection, as are, moreover, mice and rabbits. Guinea-
pigs, however, prove refractory. The growth of the bacilli

1 [Many cases of dysentery are, however, believed to be due to the action
of a protozoon, named by Losch Amceba coli, by Councilman and Lafleur
Amceba dysenteries, for an account of which the reader is referred to the
Appendix.] — TB.

BACILLUS OF FOWL CHOLERA 253

does not liquefy gelatine, and the colonies on the plate are
roundish with uneven edges. When grown in thrust-
cultures a delicate film appears on the surface, while a slight
whitish-grey coating develops on agar. A dirty yellow fur
appears on potato only at incubation temperature, and a
shining whitish deposit upon serum.

A characteristic point in the staining of these microbes
is that the poles only are coloured by aniline dyes, the
centre remaining unstained. They discharge the colour
when treated by Gram's process.

Other intestinal bacteria. — Besides the above, the Bacillus
butyricus, Vibrio rugula, and Bacterium coli commune are
met with in the intestinal canal, as well as the cholera
bacillus, typhoid bacilhis, Vibrio proteus (Bacillus Finkler-
Prior), Bacillus neapolitanus (Bacillus Emmerich), Proteus
rulgaris, Vibrio Metsehnikqffi, and in many cases the thrush-
fungus. [Various protozoa have also been described.]

254 BACTERIOLOGY

CHAPTEE XII

THE ORGANS AND CAVITIES OF THE BODY AND THEIR

CONTENTS (continued)

III. THE F^CES AND URINE
The Faces

Composition and modes of examining. — The fgeces must
at this point be dealt with as a supplement to the study of
the intestine, and they are very rich in micro-organisms.
When the stools are examined we find constituents derived
from the vegetable matters eaten, and remnants of un-
digested pieces of animal food, especially larger or smaller
pieces of striped muscle fibre, which show the transverse
striation distinctly. Fat often occurs in normal stools in
the form of droplets, and, in addition to these constituents,
epithelial cells are also found (usually cylindrical), red
and white blood-corpuscles, masses of detritus, crystals, and
under pathological conditions still other appearances. Cer-
tain varieties of bacteria can be detected in the meconium
of new-born infants, as well as in the evacuations of adults.
According to Nothnagel, micrococci are found in preponde-
rating numbers in firm, and bacilli in fluid, stools.

In the examination of faeces, forms are found almost
constantly which stain blue with tincture of iodine, and
which Nothnagel considers to be identical with the Clos-
tridium butyricum. Other micro-organisms also assume a
blue colour under application of tincture of iodine ; for
example, Von Jaksch found rods in the faeces which recall

KECAL MICRO-ORGANISMS 255

the appearance of Liptothrix buccalis. The number of
microbes which stain with iodine is found to be particularly
large in intestinal catarrhs. Fischer succeeded in growing
the Lsptothrix fibres artificially. Furthermore, the different
micro-organisms which livo. in the intestinal tract are also
to be found in the faeces.

When observing the faeces it is important to examine a
f-mall quantity in the hanging drop, when, besides the
micro-organisms, a copious quantity of desquamated
cylindrical epithelial cells are found. Even in such an
investigation as this it is very often seen that, in the case
of the majority of the different micro-organisms described
up to this point, pure cultures of one or other are present,
the other microbes either having been suppressed altogether
or being present in such small numbers that no attention
need be paid to them. Hence it happens, for example,
that, besides the cylindrical epithelium, nothing but actively
motile cholera bacilli are encountered on examining the
rice-water stools of that disease.

Yeast cells are constantly found in the faeces, especially
in the acid stools of children (summer diarrhoea), and in
acute catarrh of the small intestine in adults. These cells
stain brown with iodine, which Von Jaksch connects with
the glycogen they contain.

Bacillus subtilis appears with tolerable frequency in
normal as well as pathological alvine evacuations ; and
besides these, in diseased conditions of the intestines the
respective micro-organisms are met with in the faeces
— for instance, the bacilli of cholera, typhoid fever, and tuber-
culosis. Netter was also able to isolate the Staphylococcus
pyogenes from the faeces.1

1 [Lambl, Losch, Koch, Pfeiffer, Kartulis, and others have also described
protozoa occurring in the faeces in health as well as under various patho-
logical conditions.] — TB.

256 BACTERIOLOGY

Bacillus subtiliformis. — Bienstock found, as a constant
inhabitant of human faeces, a bacillus the morphological
characters of which strongly resemble those of Bacillus
siibtilis, except that motility is wanting and the rods are
always connected in long filaments. A luxuriant fatty
coating of a whitish-yellow colour develops upon agar.
When spores form, the rods are distended in the centre to
a spindle shape.

Bacillus albuminis. — The same observer very often en-
countered a micro-organism in faeces, which possesses the
power of energetically decomposing albumin, and to which
he gave, in consequence, the name Bacillus albuminis.
The rods are fairly long and show a marked motility,
that part of the bacillus from which the spore divides off
.being always foremost during movement. A whitish
layer, with the lustre of mother-of-pearl, develops on agar.

Bacillus cayicida. — Brieger isolated from faeces and putrid
substances a bacillus which has the property of de-
composing saccharine solutions and generating propionic
acid. The rods are short, being but twice as long as their
diameter, and liquefy gelatine into a viscid fluid. On
plates there develop white colonies with beautiful concentric
rings resembling the scales upon the back of a tortoise.
A dirty yellow deposit forms on potatoes and on serum.
Subcutaneous injections have an extremely poisonous
action upon guinea-pigs, often causing death even within
a few hoars.

Micrococcus tetragenus concentricus. — The author found
in the liquid evacuations of a person suffering from gastric
dilatation cocci which were commonly united in tetrad form.
The elements are small, round and motile, stain readily
with all the different aniline colours, and thrive upon the
various nutrient media at present used in bacteriological
research. Gelatine is not liquefied. On the plate several

MICROCOCCUS TETRAGENUS CONCENTRICUS 257

round islets appear, which when inoculation has been exten-
sive coalesce with one another, and by confluence of several

Concentrically stratified
superficial growth

Deep portion of the needle-track

Concentric stratification in
sure in the gelatine

FIG. 92.— THRUST-CULTURE IN GELATINE OF MICROCOCCUS
TETRAGENUS COXCEXTRICUS.

a larger roundish compound colony is formed, resembling a
holoblastic ovum in process of segmentation. Thrust-cul-

258 BACTERIOLOGY

tures show a superficial deposit possessing concentric rings,
which may be described as diurnal rings, since a difference
exists between the growth in the dark and that in daylight,
which expresses itself in this form (fig. 92). If the
culture be kept in the dark the con-
centric circles on the upper surface
disappear. On agar there appears a
deposit showing scroll-like tracings,
and on potato growth takes place
SSEST on the site of inoculation (fig. 93.)
The microbe cannot be found to

possess any pathogenic properties, subcutaneous inocula-
tions upon mice having produced no results.

The Urine.

Micro-organisms of the urine. — In the normal state no
micro-organisms are found in urine when freshly passed ;
those which appear in it reach it from without, or are de-
rived from diseased conditions of the urinary passages. If,
however, the urine stands for some time until alkaline fer-
mentation sets in, a multitude of yeasts, moulds, cocci,
and bacilli make their appearance, which, reaching the urine
probably from the air, and finding there a favourable environ-
ment, set up a fermentation of the urinary sugar and cause
a splitting up of the urea into ammonium carbonate ; in
fact, an entire series of processes taking place in the urine
are dependent on the vital activity of micro-organisms.

A true bacteriuria has also been described in different
quarters (Roberts, Schottelius, Fischer), which appears to
be of a morbid nature : in pathological processes in the
different organs, but especially when bacteria get into the
blood and therewith also into the renal circulation, bacilli
may penetrate into the urine during its secretion and be
expelled with it.

URINARY MICRO-ORGANISMS 259

In a case of endocarditis, Weichselbaum found the specific
micro-organisms in the urine, and Von Jaksch detected in
erysipelas abundant streptococci which he identified with
the Streptococcus pyogenes ; while in typhoid fever Neumann
found the corresponding micro-organisms in the urine, and
glanders bacilli and tubercle bacilli have also been dis-
covered in it. In other diseases, too, the respective bacteria
are found in the urine — for instance, the bacillus of anthrax
or the spirillum of relapsing fever. In gonorrhceal processes
gonococci are found in it, and in suppurative processes con-
siderable numbers of staphylococci ; and tubercle bacilli
appear in s-shaped groups (see fig. 81) in tubercular
ulcer ative disease of the urinary passages. The process
of centrifuging is especially to be recommended when ex-
amining the urine for micro-organisms.

Bacteriuria may also be caused by the conveyance of
micro-organisms into the urethra in the introduction of in-
struments, when they may set up a decomposition of the
urine in the interior of the bladder.

A number of different micrococci found in the air, in
water, and in the soil are also met with in the urine ; these
include the Micrococcus urea and various species of sarcina.

The majority of the micro-organisms occurring in urine
are bacilli.

Yeasts and moulds in urine, — The occurrence of any
considerable number of yeast-cells is in all probability de-
pendent upon the urine being rich in sugar. Moulds,
too, only appear in saccharine urine, and then not until
the alcoholic fermentation of the sugar has come to an end.

Urobacteria, — A number of bacteria have the property
of converting urea into ammonium carbonate. That most
frequently found in the normal urine during fermentation
is the Micrococcus ure<% (Pasteur, Van Tieghem, Leube),
which is a constant inhabitant of the atmosphere, and has

s 2

260 BACTERIOLOGY

consequently been already described in the chapter on
the 'Bacteriological Analysis of Air.' It is usually found in
longish chains upon the surface of fermenting urine (see
fig. 34, p. 107).

Lundstroem found that two kinds of staphylococcus are
also endowed with this property, viz. the Staphylococcus
ureae candidus and the Staphylococcus ureae liquefaciens. The
former develops shining white deposits upon the gelatine
without liquefying it, the latter liquefies it.

Miquel encountered amongst the water bacteria
numerous varieties possessing the same power. They are
for the most part bacilli, only a few being micrococci ;
amongst the latter are found three which liquefy gelatine.
According to Leube, a variety of sarcina is also to be included
with these micro-organisms.

The Micrococcus ureae liquefaciens described by the last-
named observer forms small chains and liquefies gelatine
slowly.

Leube isolated a Bacillus ureae from ammoniacal urine,
consisting of short stout rods, the growth of which does not
liquefy gelatine. The colonies on the gelatine plate are at
first very transparent ; their coalescence gives the gela-
tine on that spot the appearance of a slab of ground glass.
The margins are indented in consequence of irregularity of
growth. In thrust-cultures also grey processes appear, ex-
tending out from the needle-track, and these grow at room
temperature.

Of other micro-organisms which decompose urea the
TIrobacillus Freudenreichii and the Urobacillus Maddoxii
should also be mentioned. The former grows best on gela-
tine at 20° C., forming a milk-white coating on the surface,
while deeper a cavity, filled with a turbid stringy fluid
develops. Liquefaction of the gelatine progresses slowly,
and the liquefied region gradually clears, a white slimy

MICROCOCCUS OCHROLEUCUS 261

mass with the smell of ammonia settling down into the
deeper part. If some urine be added to the gelatine a
coronet of very fine crystals forms after some days around
the white colonies. It also grows excellently upon bouillon
and upon urine.

The Urobacillus Maddoxii renders urine viscid and
stringy. Gelatine is not liquefied, bouillon is very quickly
rendered turbid, and in this case also crystals form round
the cultures in gelatine mixed with urine.

Micrococcus ochroleucus. — Prove found cocci in normal
urine which are possessed of a motility particularly
marked when they are united in chains, and Legrain
met with them also in the pus of urethritis. They are
distinguished by the formation of endogenous spores, which
progresses best at 36° C. The gelatine is not liquefied.
After even one day colonies with raised edges appear upon
the plate, and these later on become yellow and push out
processes. In thrust-cultures a sulphur-yellow pigment
forms upon the surface, which is soluble in alcohol and is
destroyed by acids. A dirty white, creamy layer develops
on agar, in which the inoculated streak is prominent as a
yellow stripe. On potato there forms a warty elevation of
a yellow colour. All cultures diffuse an intense sulphurous
odour.

Streptococcus giganteus urethrse. — Lustgarten and Manna-
berg have discovered in normal urine and in the human
urethra large cocci arranged in rows, which form wavy
lines and often dense coils. No growth takes place on
gelatine, and even on agar it is slow, and best, moreover,
at incuba,tion temperature.

Besides these, the same observers described four varieties
of bacilli and seven of cocci as constant inhabitants of the
urethra, amongst them the Staplnjlococcus pyoyenes aureus
and Micrococcus subftavus.

262 BACTERIOLOGY

Bacterium sulphureum. — Kosenheim found different
bacteria in urine which form sulphuretted hydrogen therein
and do not liquefy gelatine.

Holschewnikoff isolated a micro-organism from mud,
which generates the same gas, and is also, although rarely,
to be met with in urine. It consists of fine rods with
rounded ends, and endowed with a slow motility. Their
development causes liquefaction of the gelatine. Upon
plate-cultures there form small punctate colonies, which
sink inwards in funnel form during liquefaction. In
thrust-cultures the superficial colonies are white, the deep
reddish-brown. A viscid grey layer forms rapidly on agar ;
but on potato development takes place only when air is
excluded, the growth being reddish-brown.

Bacillus septicus vesicse. — In persons suffering from
cystitis and pyelonephritis Clado found, amongst numerous
other microbes, an organism showing motile rods, mostly
isolated, and which develop ovoid spores. They take up
aniline stains readily, and do not discharge their colour
under Gram's process. Gelatine is not liquefied. Upon the
plate there develop punctiform colonies, which, however, do
not go beyond a pin's head in size. In thrust-cultures the
deeply- seated colonies appear larger than the superficial,
and the latter unite to form a delicate opalescent film. On
agar there develops a delicate coating, and upon this minute
circular shining milk-white colonies grow. Both gelatine
and agar quickly become alkaline. Growth in bouillon is
particularly abundant, and a dry, light-brown layer develops
upon potato.

Urobacillus liquefaciens.— Schnitzler has found a bacillus
in cystitis to which the above name has been given, and
which is apparently identical with the Urobacillus liquefa-
ciens sej^ticus discovered by Krogius in cystitis and pyelo-
nephritis. It consists of short motile rods rounded at the

UROBACILLUS LIQUEJL<1AC1ENS 263

ends, which give up the stain when treated by Gram's
method. Gelatine is rapidly liquefied. The plate-culture
shows in the centre of the liquefied colony a nodule of the
size of hemp-seed, with fringed edges. There develops a
thick greyish -white un wrinkled coating upon agar even in
one day. On potato a brownish-yellow layer forms. The
cultures generate ammonia and smell like decomposed
urine. This bacillus seems to be nearly akin to the Proteus
vulgaris (see p. 175).

The Staphylococcus pyogenes albus and aureus are also
very often found in cystitis.

264 BACTERIOLOGY

CHAPTER XIII

THE ORGANS AND CAVITIES OF THE BODY AND THEIR

CONTENTS (continued)

IV. BACTERIOLOGICAL EXAMINATION OF THE RESPIRATORY
TRACT; AND (V.) OF THE BLOOD

The Nasal Secretion

Micro-organisms in the nasal secretion. — The micro-
organisms occurring in the vicinity of men and animals
may very easily penetrate into the respiratory passages
along with the current of air. They first reach the cavi-
ties of the nose, and find in the alkaline viscid mucus
there a medium possessing the conditions most suitable for
their development. The secretion of the nose contains
epithelial cells, both pavement and ciliated, leucocytes, and
even in the normal state a considerable number of micro-
organisms— cocci, bacilli, and spirilla. Under pathological
conditions the nasal mucus becomes thin, and more pus-
corpuscles are found, as well as the characteristic micro-
organisms in definite local morbid conditions, the kinds of
microbe depending upon the variety of disease.

Micrococcus cumulatus tennis. — Von Besser isolated from
the normal nasal mucus rather large non -motile cocci, the
growth of which does not liquefy gelatine. Upon agar a
development of raised colonies appears, showing a thin
zone with ragged edges around a central nucleus. Pota-
toes are not favourable for its growth.

Mirococcns tetragenns subflavns shows non -motile cocci

DIPLOCOCCUS CORYZ.E 265

arranged in fours, and was discovered by Von Besser in
the normal mucus of the nose. No growth takes place
upon gelatine, but it develops well on agar and potato.
The older cultures have a yellowish-brown colour.

Micrococcus nasalis. — Hack encountered in the naso-
pharyngeal cavity motile diplococci, the growth of which
does not liquefy gelatine. The islets on the gelatine plate
show a small undulating excavation in the centre, and in
individual colonies it is easy to recognise a radiating
arrangement or coiled form, frequently with a rayed
margin. A concentric stratification of the superficial layer
is often seen in thrust-cultures, together with development
of nodules along the thrust-canal. A greyish-white shining
deposit forms upon agar and potatoes.

Diplococcus coryzse. — This diplococcus was described by
Hajek, who found it in the nasal secretion. It consists of
cocci which are somewhat elongated, so as to look like
short bacilli. Gelatine is not liquefied by them. Flat
superficial-lying colonies develop on the gelatine plate, and
on thrust-cultures a deposit forms, which is at first raised
but becomes continually more and more flattened out, a
fact which serves to distinguish the culture of Diplococcus
coryzce even by the naked eye from Friedlander's Pneumo-
bacillm, since no alteration takes place in the nail-culture
formed by the latter. A coating develops upon agar.

This diplococcus was at first regarded as the exciting
cause of those changes in the mucous membrane which are
characteristic of coryza. Experiments on animals, however,
have yielded results which are negative in this respect.

Staphylococcus cereus aureus. — This microbe resembles
the Staphylococcus cereus flavus, only differing from it in the
orange-red colour of its colonies, which is particularly marked
in thrust-cultures. It was discovered by Von Schrotter and
F. Winkler in the author's Institute, in the thin secretion

266 BACTERIOLOGY

of a cold in the head, and in association with the Staphylo-
coccus cereus flarus (see fig. 76).

Bacillus fcetidus ozaense. — Hajek found in the secretion
of patients suffering from ozcena short actively motile rods,
during the growth of which the gelatine is liquefied. Eound-
ish colonies form at first upon the gelatine plate, which
appear sunken like pock-marks while liquefaction is be-
ginning, but later become confluent and liquefy the
entire gelatine. In thrust-cultures the liquefaction shows
itself both superficially and along the track of the needle,
and if the culture is kept at 37° C. a strong odour of
putrefaction is developed, which is not the case at the
ordinary temperature. Upon agar a superficial coating
forms, and in like manner becomes foul-smelling at incuba-
tion temperature. A brownish deposit forms on potato. If
animals be injected subcutaneously with the cultures a
violent inflammation results, in the course of which the
tissues become necrosed. The bacilli completely discharge
their colour when treated by Gram's method. Hajek
particularly recommends staining with alkaline methyl
blue to which some aniline water has been added. Diluted
alcoholic solutions stain the bacilli only very slightly.

Bacillus striatus albus et flavus. — Both bacilli were met
with by Von Besser in the normal nasal mucus, but the
Bacillus striatus albus is very rare. Gelatine is not liquefied.
A fairly good growth appears on the different nutrient media .
Upon potato a streaky layer forms. Bacillus striatus flavus
develops a sulphur-yellow pigment.

Bacillus of rhinoscleroma. — The bacilli of rhinoscleroma
are short rods rounded at the ends, which are devoid of
power of automatic movement and are enclosed in capsules.
They are found in rhinoscleroma in the tissue of the tumours
and in the juice from them, and are believed to be the cause
of this form of disease. Von Frisch, Paltauf, Von Eiselsberg,

BACILLUS OF RHINOSCLEROMA 267

Dittrich, Cornil, Alvarez and several other investigators
have made researches concerning this micro-organism.

The best solution to use for staining sections of
the rhinoscleroma tumours when it is desired to examine
them for the presence of the bacilli, is the methyl blue or
methyl violet solution of Loffler. The sections remain one
or two days in the stain, are then washed in water con-
taining iodine, and finally decolorised for a rather long time
(two or three days) in absolute alcohol. The bacilli do
not lose their colour under Gram's process. If they be
stained with gentian violet in aniline water, washed in
water containing acetic acid, and double-stained with
carbolic fuchsine, the capsules round the bacilli appear
coloured; safranine may be used with advantage for
double-staining instead of the carbolic fuchsine.

Gelatine is not liquefied. Eoundish colonies appear on
the gelatine plate, and thrust-cultures take the nail form,
but do not attain any very extensive development. Upon
agar a widespread milk-white coating appears even in half
a day, on potato there likewise forms a creamy mass which
gradually spreads out from the site of inoculation, and on
blood- serum also a white coat develops. In all these methods
of cultivation the capsule is retained even in the later stages
of development, but it is lost in bouillon cultures. Inocula-
tions do not produce rhinoscleroma, even when the injections
are made into the nasal mucous membrane, but according to
Stepanow tumours of granulation-tissue develop when one
is made into the eye of a guinea-pig, and these tumours are
rich in bacilli. Injections into the pleura of these animals
resulted in death.

The bacillus of rhinoscleroma seems to be at least closely
akin to Friedlander's Pneumobacillus, but is less virulent.
The points of difference with reference to their morphology
which might be cited would perhaps be the greater trans-

268 BACTERIOLOGY

parency of gelatine cultures of the former, and the persistence
of capsules on most of the culture media.

Bacillus capsulatus mucosus. — At the Institute of Professor
Klemensiewicz in Graz, Fasching discovered, in crusts
removed from the naso-pharynx of a case in which this
cavity was diseased, a micro-organism with short and rather
thick rods lying singly or in pairs or fours in a common
enveloping capsule. The bacilli give up the dye under
Gram's process. It is easy to bring out the capsule
successfully as a delicate rose-tinted area by protracted
staining of the prepared cover-glass infuchsine, or by slightly
warming after covering it with the stain. The bacilli are
destitute of motility, and do not liquefy gelatine. On the
plate colonies develop which have the appearance of drops
of mucus of the size of pins' heads. In thrust-cultures
there forms a typical nail-like figure, with active generation
of gas, and upon agar and serum a thick moist creamy layer
develops. A moist, viscid, white coating occurs on potato.
Subcutaneous injection causes a genuine septicaemia in
white mice. Fasching also found this micro-organism in
the sputum of a phthisical patient.

Vibrio nasalis. — Both in the nasal mucus and in the
buccal cavity rods of considerable size are found which
possess no power of automatic motion, and are arranged as
vibrios. They were cultivated pure by Weibel. Gelatine
is not liquefied, and the growth on the plate leads very
slowly to the formation of round islets. In thrust-cultures
a delicate white streak develops along the thrust-canal,
resembling a string of mucus. The culture on agar is less
transparent and thicker, and spirilla are found in it which
show a large number of bends (over thirty). No growth
takes place on potato. When treated by Gram's process
the spirilla lose their colour.

Other nasal bacteria. — Eeimann isolated a great number

MICROBES IN THE RESPIRATORY PASSAGES 269

of bacteria from the nasal mucus, amongst which also the
Spirochaete dentium is often to be met with.

In tubercular diseases of the cavity of the nose the
pathognomonic tubercle bacilli are found in the nasal
mucus, in glanders ulcers the Bacillus mallei, and thrush
fungus and moulds frequently occur in the secretion. [See

p. 195.]

The Respiratory Passages

Micro-organisms of the respiratory passages. — Micro-
organisms may also be very easily conveyed from the
air into the bronchi by respiration. Straus and Dubreuil
have ascertained that the expired air is almost free
from organisms, and even when germs do occur the
proportion of those in expired to those in inspired
air is very small (1 : 600). Tubercle bacilli produce
infection with greater ease, as Cadeac and Molet found,
when the particles of dust to which they adhere are damp
with aqueous vapour, than when they reach the lungs in a
dry state. Owing to the discovery in recent years of a
series of micro-organisms which excite morbid processes in
the lungs, it is of very great importance to acquire a more
intimate knowledge of the microbes occurring there, and
this is most conveniently done by a thorough examination
of the sputum. Certainly there are found in the sputum
admixtures of matter from the naso-pharynx and the buccal
and nasal cavities ; still, it is possible to exclude all the
elements coming from other cavities, and to retain only
those derived from the lungs.

Pansini constantly found different varieties of strepto-
cocci in the sputum both of healthy and diseased individuals,
and next to these in frequency, various kinds of sarcina.
A yellow, reddish, or green colour in sputum is caused
by chromogenic bacteria. Pansini derives the yellow and
reddish colour from the vital activity of Bacillus aureus,

270 BACTERIOLOGY

Sarcina lutea, and Sarcina aurantiaca, while the green
colour is caused by the Bacillus pyocyaneus and Bacillus
fluorescens non-liquefaciens. Virchow and Lichtheim not
rarely found moulds in the sputum, and amongst these
particularly Aspergillus fumigatus. The fungus of thrush
and yeast cells may also be frequently met with, and
moreover, Fasching found here his Bacillus capsulatus
mucosus.

Sarcina pulmonum. — Even in earlier years different
authors have described sarcinae in the sputum which they
considered identical with Sarcina ventriculi. By the re-
searches of Hauser, however, it was brought out that a
special variety has here to be dealt with, distinguished
at once from the last-named by its smaller size. No
pathogenic properties can be ascribed to it, but it pos-
sesses the faculty of energetically decomposing urine, a
power shared by it with a sarcina discovered in that ex-
cretion by Leube. Gelatine is not liquefied, and on the
plate small white colonies develop which appear indented
at the margin and concentrically stratified during their
growth. A superficial moist coating forms in thrust-
cultures. Upon potatoes the growth is very scanty.

Sarcina aurea. — Mace isolated from the lung of a patient,
who had died of pneumonia with purulent pleuritis, a variety
of sarcina, the elements of which possess a very lively
oscillating motility. Some of them stain bluish-violet with
iodine and sulphuric acid, a reaction indicating the presence
of cellulose or starch in the sarcina. They develop a
beautiful golden-yellow pigment which is soluble in absolute
alcohol, and liquefy gelatine with tolerable rapidity. In
thrust-cultures a thick friable membrane, which very readily
falls apart into separate pieces, develops upon the surface
of the funnel-shaped area of liquefaction ; on agar there
forms a thick streak with warty surface and of a shining

Bffi

PNEUMOCOCCUS 271

golden-yellow colour, while a thick golden-yellow layer
grows on potato.

Diplococcus pneumonise, — Klebs and Eberth long ago
pointed out the presence of cocci in croupous pneumonia,
while A. Friinkel and Weichselbaum have studied them with
the help of the modern methods €>f research, and ascertained
that the cause of this affection is a micrococcus which Weich-
selbaum detected in ninety-one cases out of a hundred.

These cocci are round or sometimes elongated, and
possess a jelly-like envelope of varying thickness. They are
very often found arranged in pairs, and it is not unusual
for several such diplococci
to be connected in rows,
one behind the other, and
enclosed in a common cap-
sule (fig. 94). Motility is
absent.

These cocci occur in
croupous pneumonia not
only in the sputum and in Illl
the diseased lung, but also FlG> 94._MlCROBES OP PxEUMONIA.

in the blood, and they are

met with in different other diseases. According to Weich-
selbaum they are found in exudations in the cavum fcympani
and in the ethmoidal labyrinth, and Foa and Bordoni believe
them to be the sole cause of cerebro-spinal meningitis.
Klein, Biondi, A. Frarikel and Miller found them also
in the buccal and naso-pharyngeal cavities of healthy
individuals, so that they can exist as it were in the portals
of entrance to the respiratory system. They are iden-
tical with the microbes of sputum septiccsmia. Emmerich
found them in the dust of a room occupied by pneumonic
patients.

Their growth begins to make good progress at a tern-

272 BACTERIOLOGY

perature of 24° C., and gives the best results at incubation
heat. On the gelatine plate there form roundish colonies of
medium size, and in thrust-cultures little white granules
appear along the needle-track, and a slight transparent
prominence on the surface. The gelatine is not liquefied.
A thin transparent film forms upon agar, and a thin
transparent slimy coating upon serum. The cultures do
not last very long, and lose their virulence if kept at a
temperature of 42° C. even for one or two days. The same
result is also attained, according to Frankel, by cultivation
in milk. In order to preserve the virulence of the cocci,
they must be inoculated from time to time upon animals.

The diplococci cultivated upon artificial nutrient media
show no capsules, and after losing them they become regu-
larly round and range ^themselves in chains, in consequence
of which they have been described by Gamaleia as Strepto-
coccus lanceolatus. They exhibit capsules in the blood of
animals infected with the cultures.

The cocci stain in dilute alcoholic solutions of the
aniline dyes, and can easily be displayed in preparations
coloured by Gram's method, differing therein from the
Pneumobacillus of Friedlander. The capsules remain un-
stained by the ordinary methods : to colour them Kibbert
employs a hot saturated solution of the capsules of dahlia
violet in 100 parts water, 50 parts alcohol, and 12^ parts
acetic acid. Staining takes place very rapidly in this
solution, and moreover it is necessary to wash in water
only for a short time. The cocci appear dark blue, the
capsules light blue.

For transmission of pure cultures those on bouillon are
the most suitable, and of these one or two cubic centimeters
are used as a hypodermic injection. The cocci with their
capsules are found in the blood and organs, but subcuta-
neous injections fail to set up inflammatory symptoms in

FRIEBLANDER'S PNEUMOBACILLUS 273

the lungs. When the pleura is infected, however, inflam-
mations of it and the lungs do occur ; and when pure
cultures are introduced into the trachea of rabbits a pneu-
monia with all the characteristic symptoms follows.

Pneumobacillus Friedlaenderi. — Friedliinder found a
micro-organism in the expectoration and in the lung-
tissue, the elements of which are rods of different sizes,
lying singly or joined together in pairs or bands. They
possess a capsule in the form of a transparent surround-
ing area, but this is wanting in artificial cultures. The
bacilli are immotile. In contrast to the Diplococcus pneu-
monia, the pneumobacilli discharge the dye under Gram's
process. They also grow at a lower temperature than the
former.

Gelatine is not liquefied. Upon the plate there appear
roundish, sharply-defined colonies of granular texture, and
in thrust-cultures a thick porcelain-like prominence forms
on the surface, and the growth rapidly advances along the
thrust, so that there appears the distinct figure of a nail—
the ' nail-culture ' (fig. 95). Older cultures become brownish.
Upon agar a dense deposit forms, and a thick yellowish
moist coating upon potato. Bubbles of gas are often seen
in the cultures. Infections of mice by the hypodermic
method speedily result in death, but guinea-pigs are less
susceptible. Sub-pleural or mtra-pulmonary injections set
up a pneumonia. Croupous pneumonia seems, however, to
be due to the pneumobacillus only in a small proportion of
cases (9 times in 100 cases), according to Weichselbaum
and C. Frankel.

Micrococcus tetragenus. — Koch and Gaffky found in
the contents of a tubercular cavity small immotile cocci,
which were, as a rule, united with one another in fours
and surrounded by a capsule. They are also found in
the sputum, and are very frequent, according to Kar-

274

BACTERIOLOGY

linski, in dental abscesses. Gelatine is not liquefied, and
white punctate colonies form on the plate and display a

Superficial elevation

-Needle-track

Needle-track

FIG. 95.— THRUST-CULTURE IN GELATINE
OP FRIEDLANDER'S PNEUMOBACILLUS
(' NAIL-CULTURE ').

FIG. 96.— THRUST-CULTURE IN* GELATINE
• >K MICROCOCCUS TETRAGEXUS. (After
Mace.)

shining, porcelain-like appearance on the surface of the
medium. Isolated sharply-defined colonies appear along

BACILLUS TUSSIS CONVULSIVE 275

the canal in thrust-cultures, lying one above the other
like a pile of discs, the most superficial of which projects
in a hemispherical form above the surface (fig. 96). Upon
agar and serum, also, round well-defined colonies develop
along the inoculated strenk, and upon potato a slimy
coating forms. White mice die within four weeks after
infection.

Bacillus aureus. — The elements of this bacillus are short
rods showing but slight motility. They were found in water
by Adametz, and also upon the human skin in some
forms of eczema by Unna and Tommasoli. Gelatine is
not liquefied. Upon the plate there develop punctiform
colonies, which assume a yellow colour and become
uneven, and in the thrust-culture a dark yellow deposit
forms upon the surface. Their growth on potato takes the
form of shining hemispheres, which coalesce and assume
a colour varying from dark yellow to brownish- red.

Tubercle bacillus and Actinomyces, — The micro-organism
to which Koch gave the name of tubercle bacillus is con-
stantly found in the sputum of phthisical persons and in
the contents of cavities ; it has already been described in
the chapter on the ' Bacteriological Analysis of Pus ' (p. 206) .
The actinomyces fungus, which often occurs in sputum, has
also already been described in the same chapter (p. 196).

Bacillus tussis convulsive. — Afanassiew found a micro-
organism, which he described under this name, in the
sputum of persons suffering from whooping-cough. The
rods are short and actively motile, and best admit of
being cultivated at incubation temperature. Gelatine is
not liquefied by them. Bounded brown colonies form on
the plate and a superficial coating in thrust-cultures, while
there develops on agar a thick grey, and on potato a thick
brown, deposit.

Bacillus pneumosepticus. — In the respiratory organs, and

T 2

276 BACTERIOLOGY

in various other tissues of an individual who had died of
septic pneumonia, Babes found short immotile rods which
readily take up aniline dyes, but discharge them again
under Gram's process. The gelatine is not liquefied
by them. Extensive flat white islets develop on the
plate, and in thrust-cultures growth takes place along the
entire needle- track and diffuses an unpleasant, mawkish
odour. Upon agar indistinct colonies appear, which coalesce
and form a superficial film. A similar thick moist coating
is also found upon potato. Experimental inoculations on
mice, rabbits, and guinea-pigs speedily cause death. It is
not unusual for the bacilli to be found enclosed in the
leucocytes also. Virulence is lost after repeated trans-
missions to artificial nutrient media.1

v

V. BACTEKIOLOGICAL EXAMINATION OF THE .BLOOD

Micro-organisms in the blood. — In treating of the various
micro-organisms it has already been pointed out in a
general way that they are found not only in the several
organs, but also in the blood, and hence examination of
the latter must be undertaken both in microscopic prepara-
tions and by the methods of cultivation.

We find in the blood moulds, yeasts, and bacteria, and
it has been mentioned already in an earlier part, that
staphylococci, streptococci, tubercle bacilli (Weichselbaum),
and the bacilli of typhoid fever, anthrax, glanders, &c., may
be recognised here also. Besides these, however, there are
other organisms to be cited which occur par excellence in
the blood.

Methods of examination. — When it is desired to examine
human blood, the ball of the finger must first be care-
fully cleansed with sublimate, alcohol, and ether, after which

1 [The occurrence of AmosbfB in the sputum has also been described in
cases of dysenteric abscess. See Appendix.] — TE.

METHODS OF STAINING BLOOD 277

the blood is obtained by pricking it. The first drop having
been wiped away with a sterilised platinum needle, the
second is taken off with a disinfected cover-glass, spread
out by rubbing with another cover-glass (also disinfected),
dried, and heated to 120° 0. approximately. Disinfection
of the cover-glasses is best done in corrosive sublimate,
from which they are transferred to alcohol and ether with
a sterilised forceps, and then taken out and rapidly dried
in the air. The basic aniline dyes, commonly used for
bacteriological examination, are employed in aqueous solu-
tions, with the occasional addition of alcohol, glycerine, or
acetic acid to the fluid. The stains are allowed to act for
some minutes and then rinsed off with distilled water ; the
preparation is dried and Canada balsam applied to it, or if
it is not wished to keep the specimen for any length of time,
it is mounted temporarily in oil of cloves, origanum, or
cedar. Loffler's alkaline solution of methyl blue [pp. 86,
241], and Gram's method with its modifications (p. 76), are
also much used.

According to Giinther's method, the preparations of
blood after being dried and fixed are rinsed in dilute solution
of acetic acid (1 to 5 per cent.), by which means the
haemoglobin is extracted from the corpuscles and a great
part of the plasma washed from the glass, without thereby
impairing the adhesion of the bacteria. If now the sections
are again dried they may be stained after the ordinary
methods, and a tolerably isolated coloration of the bacteria
so effected. The blood-corpuscles are no longer seen save
as mere shadows, and do not now interfere with the appear-
ance of the stained bacteria.

The rinsing away of the plasma with acetic acid does
not succeed if the layer of blood be already too long dried
upon the cover-glass. Giinther accordingly treats dried-up
layers with a 2 to 5 per cent, aqueous solution of pepsine,

278 BACTERIOLOGY

when the plasma is peptonised in a short time, and the
bacteria remain well preserved.

Influenza bacillus. — Babes, Canon, Pfeiffer, and Kitasato
believe the exciting cause of influenza to be very diminutive
rods which can be detected in the blood of persons suffering
from the disease. They lie in part within the white cor-
puscles, and often appear ranked with one another in chains
of three or four. In order to detect them, Canon lays the
cover-glass preparation, after it has been dried by the air,
in alcohol for five minutes, and stains it for from three to
six hours in Chenzynski's solution of methyl blue and eosine,
rinses it in water, allows it to diy, and mounts in Canada
balsam. By this process the red blood-corpuscles are

Influenza bacillus completely
stained

Red corpuscles

Influenza bacillus stained at the extremities

FIG. 97. — INFLUENZA BACILLI IN HUMAN BLOOD. (From an original
preparation by Canon.)

stained red, the white corpuscles and bacilli blue (fig. 97).
Chenzynski's solution consists of 40 grams concentrated
aqueous solution of methyl blue, 20 grams of a half per
cent, solution of eosine in 70 per cent, alcohol, and 40 grams
water.

Upon glycerine agar solidified in a slanting position
there develop at incubation temperature very small droplets
of the transparency of water, — so small, in fact, as to be
scarcely perceptible unless magnified by a lens, — which
have no tendency to coalesce. Scanty white turbidities
form in bouillon in the first twenty-four hours, and sink to
the bottom, leaving the supernatant liquid clear.

Bacillus endocarditis capsulatus. — In a thrombus in the
cardiac auricle of a person who had died of endocarditis,

BACILLUS OF SWINE ERYSIPELAS 279

Weichselbaum found bacilli enclosed in a capsule and re-
sembling the Pneumobacillus. Gelatine is not liquefied, and
the thrust-culture shows a shining deposit resembling
stearine, while a greyish-white coat develops upon agar.
The bacilli discharge their colour when treated by Gram's
method. Subcutaneous injections cause the death of
the animals experimented on, and if the aortic valves be
injured, endocarditis sets in after infection.

Bacillus of swine erysipelas. — One of the most fatal diseases
affecting pigs is swine erysipelas, since the majority of the
animals attacked fall victims to this infectious disorder,
often even in a few hours. Feverish symptoms set in,
and at the same time there appear over the neck, abdomen,
and breast spots which are at first red, but later assume a
brown colour. The cause of this disease is considered by
Pasteur, Thuillier, Loffler, Schotte-
lius, and Schiitz to be a micro-
organism found in the blood and
juice of various organs, particularly
the spleen, and consisting of small
rods endowed with motility, which
show themselves in cultivation to
be facultative anaerobes. They take the aniline dyes
readily, and prove refractory to decolorising processes,
Gram's method included. The latter gives especially fine
results with sections. Gelatine is not liquefied, and colo-
nies form on the plate which possess a multiplicity of
radiating processes anastomosing with one another, so that
they look like bone-corpuscles (fig. 98). Thrust-cultures
grow slowly, development commencing beneath the free
surface of the gelatine and gradually advancing into the
deeper parts, while numerous ramifying fibres proceed out
from the thrust-canal, and fill the gelatine in such abund-
ance that it becomes cloudy. Upon agar a delicate layer

280

BACTERIOLOGY

forms along the inoculated streak. Morbid phenomena
can be set up in mice, pigeons, and rabbits by inoculation

Needle-track-

FlG. 99.— THIirST-CULTURE IN GELATINE OF BACILLUS MURISEPTICUS. (After Mac6.)

under the skin or into the abdominal cavity, but not in
guinea-pigs and poultry.

Bacillus murisepticus, — In putrefying blood and other

BACILLUS MURISEPTICUS 281

decomposing fluids, Koch found minute immotile rods
resembling the bacilli of swine erysipelas, but somewhat
thinner and smaller. They look at first sight like fine needle-
shaped crystals, and their true nature can only be recog-
nised by staining. They retain their colour when treated
by Gram's method. Gelatine is not liquefied, and indis-
tinctly defined colonies occur on the plate and spread out
over it in the form of delicate whitish nebulosities. In
thrust-cultures in like manner there appear bluish-grey
turbid clouds, which permeate the entire gelatine (fig. 99).
On agar round isolated yellowish-brown colonies develop

FIG. 100.— SPIRILLA OP RECURRENT FEVKR. (After Jakscli.)

along the needle-track. Inoculation causes death in house-
mice in two or three days, but field-mice and guinea-pigs
are immune. The bacilli are found in the vascular system,
and the white corpuscles are often found to be totally
destroyed. The blood of the dead animals proves very
virulent.

Spirochetae Obermeieri. — In recurrent fever Obermeier
found spirilla in the blood at the time of the attack. They
are about six times as long as the diameter of a red cor-
puscle, and move in a very lively manner (fig. 100). They
show themselves readily amenable to staining with the
ordinary basic aniline dyes, but give up the colour again

282 BACTERIOLOGY

under Grain's process. A nearly isolated staining may, how-
ever, be obtained by employing Giinther's method, in which
the air-dried cover-glasses are laid for ten seconds in 5 per
cent, acetic acid, to bleach the red corpuscles, before being
subjected to the action of the staining fluid, after which the
acid is removed by blowing, and finally, in order to free the
preparation from the last traces of adhering acid, it is laid
with the prepared side downwards over an open bottle con-
taining strong solution of ammonia which has just previously
been shaken. Staining is then done with gentian violet
in aniline water ; the preparation is rinsed in water and
put up in Canada balsam. Artificial cultivation has not as
yet succeeded. Transmission of blood containing spirilla is
stated to have produced results only in human beings and
monkeys.

Protozoa in the blood. — Protozoa are found in the blood
in several infectious diseases. The discovery of Laveran
that protozoa have to be dealt with in malarial disorders
has been brilliantly confirmed and extended by Celli, Golgi,
Saupelice, &c., and is to-day universally admitted. Since
malaria is closely connected with the soil, the plasmodia
of the disease have already been described in the chapter
on the ' Bacteriological Analysis of Earth ' (p. 169).

Danilewsky has detected similar forms in the red cor-
puscles of lizards, tortoises, and birds, and Smith has
proved that Texas fever in oxen is dependent on a like
cause. The hemoglobinurie bacterienne du bceuf has, accord-
ing to Babes, the same significance.

L. Pfeiffer also found protozoa in the blood in small-pox
and carcinoma. [See also Appendix.]

APPENDIX

BY THE TRANSLATOR

A. Vaccination against Asiatic Cholera

Principle of anti-cholera vaccination. — The idea of vac-
cination against cholera is not a new one, as it is now some
years since Ferran professed to have discovered a cholera
vaccine. Other observers were, however, unable to satisfy
themselves of the genuineness of his results, and it is only
recently that a process established on a scientific basis
has been brought forward by M. Haffkine, of the Pasteur
Institute.1

During an attack of cholera the specific bacillus is only
found in the intestinal tract (see p. 144), and experiments
show that it dies when injected subcutaneously. The mor-
bid process must therefore be due to the absorption of
toxines generated by it, and Haffkine's vaccination aims at
acclimatising the system by the injection of an 'exalted
virus,' much stronger than any which it is likely to en-
counter in the ordinary way of infection, so as to enable it
to bear such quantities of cholera poison as may be ab-
sorbed from the intestine while an attack of the disease is
running its course.

Preparation of the vaccine. — The ' exalted virus ' is pre-
pared as follows : 2 — A suspension, in about 3 c.cm. sterile
bouillon, of two or more standard cultures (twenty- four hours'

1 The experiments (on different lines from Haffkine's) of Klemperei, of
Vincenzi, and of Brieger and Wassermann should, however, be also men-
tioned.

2 Wright and Bruce in Brit. Med. Journ. March 4, 1893, p. 227.

284 BACTERIOLOGY

growth at 35° C. on an agar surface 10 cm. long) of cholera
bacillus having been drawn into a small pipette, the point
of the latter is bored through the abdominal wall of a guinea-
pig, at a spot previously sterilised by qauterisation after clip-
ping the hair short, and the contents are blown out into the
peritoneal cavity. Immediately after death (which takes place
in twenty-four hours) the abdomen is opened with the strictest
antiseptic precautions, and the fluid found in the iliac fossae
is transferred by means of a sterile pipette to a sterilised
test-tube, in which it is left in the incubator for 8 or 10
hours at 35° C., in such a position that the largest possible
surface of fluid is exposed to the air. The process of intra-
peritoneal injection, &c., is then repeated, and this is done
twenty to thirty times in succession, until the highest pos-
sible degree of virulence is attained, which may be over a
hundred times as great as that of the original growth.
Cultures of the exalted virus can then be made, but do not
retain their full virulence for many generations.

The vaccine actually injected may contain the living
bacilli (since these perish in the tissues), or may be 'car-
bolised,' so as to kill them. Carbolised vaccines are less dan-
gerous and liable to contamination, and may be kept in-
definitely in sealed tubes, but are not so powerful. The
vaccine is thus obtained : — A standard culture is made by
inoculating the whole surface of an agar tube and keeping
it at 35° C. for 24 hours, after which an emulsion is pre-
pared by mixing the growth with 2 or 3 c.cm. of bouillon,
and then diluting up to 8 c.cm. for living vaccines ; or with
6 c.cm. of sterile J per cent, carbolic acid solution for car-
bolised vaccines. The amount of either to be injected is
1 c.cm., and the injection should be made under the skin of
the flank.

Results of vaccination. — Injection of such a vaccine
causes a rise of temperature accompanied by malaise and

APPENDIX 285

other slight general symptoms, which soon pass off; locally,
however, severe inflammation and necrosis follow, unless
a preliminary injection has been made 3 to 5 days previously
with a weak vaccine prepared from cultures attenuated by
being grown in media kept continually aerated, and at 35° C.
The only local symptoms are then slight pain and osdema.
After the symptoms have passed off the animal is found to
be scarcely affected by many times larger intraperitoneal
injections than suffice to kill control animals not so pro-
tected.

These results have also been obtained with rabbits and
pigeons, and the symptoms following injection into human
beings, of whom about a hundred had been vaccinated up
to March last,1 are identical with those exhibited by all the
animals, subsequent hypodermic injections with the ex-
alted virus also producing the same results in both, so that
although, of course, human beings could not be directly
tested like the animals, there seems little room for doubt
that they are similarly protected. It may be added that
swallowing a draught of cholera bacilli in one case, and free
intentional exposure to contagion at Hamburg in another, pro-
duced no results in two observers who had been vaccinated.

More recently, however, some doubt has been cast on
the efficacy of the process to protect against the ordinary
infection of cholera by Klein,2 who found that precisely
similar results could be obtained with Vibrio proteus, the
typhoid bacillus, Bacillus coli, Bacillus prodigiostis, &c. Vac-
cination with an exalted virus prepared from any of these
conferred immunity against the cholera bacillus (even the
exalted virus) or any of the others, when injected intraperi-
toneally. He also found that guinea-pigs protected by Haff-
kine's method were killed by intraperitoneal injection of a

1 Haffkine, Fortnightly Beview, March 1893.

2 Brit. Med, Joum., March 25, 1893, p. 632.

286 BACTERIOLOGY

gelatine culture of the cholera bacillus liquefied by its growth.
From these observations he concludes that Haffkine's vac-
cination only affords protection from the intracellular poison,
which is the same in all the bacteria tried, but not from
the poison generated externally, which differs in all the
varieties, and is the cause of the distinctive disease.

It was, however, pointed out in reply 1 that the pro-
ducts of gelatine cultures would be more or less special,
and that Haffkine's method protects against cholera virus
introduced into the alimentary canal ; while the conclusion
regarding the identity of the intracellular poisons in all the
bacilli named is not regarded as unimpeachable.

B. Parasitic Protozoa

Pathogenesis of protozoa, — We have already seen that a
parasitic protozoon may originate morbid processes, in the
case of the Plasmodium malaria (pp. 169 and 282). There
are, however, a number of other protozoa which frequent
the bodies of men and animals, and some of these are be-
lieved, with greater or less degrees of probability, to be the
cause of certain diseases. The best known of these is the
Coccidium oviforme, which is important as throwing light
upon the habits of other less known protozoa, and more
especially as showing that the presence of such an organism
may cause proliferation of epithelial cells.

Coccidium oviforme, — The life-history of this parasite,
which causes a very fatal disease in young rabbits, has
quite recently been worked out by K. Pfeiffer,2 although its
existence was known as far back as 1839.

When discharged from the affected rabbit in the faeces,
it is a firm, translucent, oval cyst measuring about 36 x 22/*,
with granular contents. These contract into a ball, and by

1 Brit. Med. Journ., March 25, 1893, p. 639.

2 Beitrage zur Protozoen-Forschung, Heft i. Berlin, 1892.

APPENDIX 287

pushing out projections divide into four parts, each of
which subdivides into two crescentic germs and a nucleus,
and becomes surrounded by an inner capsule. In this
condition the coccidium may remain unaltered for months
unless swallowed, in which case the germs are set free by the
digestion of the capsules, become rounded and amoeboid, and
usually divide into crescentic germs, which also become free ;
the result being that the intestine, gall-bladder, and bile-
ducts are filled with sporozoa. The sporules finally pene-
trate into the epithelial cells of the mucous membrane, and
there change back into the encysted form, which is set free
by the bursting of the cell.

During their growth they produce great proliferation of
the neighbouring epithelium, causing the formation in the
bile-ducts of the liver of greyish-white adenomata, which
consist of epithelium and connective-tissue and are bounded
by a connective-tissue layer, the interstices between the
branching processes which grow from the mucous coat
being full of parasites in various stages of development. A
hypertrophy of the mucous membrane also takes place over
the affected areas of the small intestine. If the animal
recovers, the parasites are discharged or absorbed, being
in the latter case penetrated and digested by small-cell
infiltration,1 and a scar of connective tissue is left on the
site of the tumour.

Feeding with the ripe coccidia is the only method of
infection which has been attended with success. The
changes in the parasites may be observed by taking some
from recently killed animals, placing on a cover-glass, and
examining in drop cultivations.2

Amoeba dysenteriee. — The occasional occurrence of proto-

1 Buffer and Walker, Journ. of PathoL and Bacterial., October 1892.

2 The above account is mainly taken from the Morton lecture of this
year, by Dr. Galloway. See Brit. Med. Journ. February 4, 1893, p. 217, for
bibliography, &c.

288 BACTERIOLOGY

zoa in the stools is a fact that has long been known, but
Losch was the first to connect them with dysentery, in a
paper published in 1875 and describing a case of that
disease in which they were present. Many observations
have been since made, the most important communications
on the subject being those of Kartulis from Egypt, and of
Councilman and Lafleur in America (to the latter of which
the above name is due, Losch having called the protozoon
Amceba coli). The variety of the disease ascribed to its
agency is that known as tropical dysentery, which differs
both clinically and pathologically from other forms, and
the amoeba was found by Kartulis T in 500 cases of this
prior to the appearance of his second paper, as well as
in every case of dysenteric liver abscess examined, while it
was absent from ' ididpathic ' liver abscesses ; and Council-
man and Lafleur 2 found it in all the fourteen cases on
which their very elaborate monograph is based.

According to the latter observers, the amoebae when at
rest are round or slightly oblong bodies, consisting of an
outer pale homogeneous substance enclosing a somewhat
greenish highly-refractive mass, which contains vacuoles
of various sizes and a nucleus. Movement is their dis-
tinctive feature, however, and consists first of a progressive
motion, and secondly of a protrusion and withdrawal of
pseudopodia, both of which vary in activity. The pseudo-
podia are formed from the outer homogeneous part,
which may, however, be otherwise invisible both in the
resting and moving state. The amoebae often contain
foreign bodies, such as red corpuscles, pus-cells, blood-
pigment, micrococci, bacilli and their spores, &c.

Entering probably with the food, they pass on until the
large intestine is reached, where the alkalinity necessary

1 Virchoiv's Archiv, Ed. 118, 1889.

- Johns Hopkins Hosp. Eeports, vol. ii. Xos. 7, 8, 9.

APPENDIX 289

to their growth is obtained. Here they penetrate and
undermine the mucous membrane, producing their effects
by liquefying the tissues, and thus causing ulceration and
necrosis. In the mucous membrane they are found chiefly
in the submucosa, in the lymph-spaces and blood-vessels,
and in the gelatinous contents of the ulcers. They may
penetrate to the liver either by the portal vessels or through
the peritoneum (sometimes causing peritonitis), and set up
abscesses, which in the former case may be multiple, in the
latter lie close to the surface of the right lobe, the com-
monest position. According to Councilman and Lafleur,
the liver shows no inflammatory reaction, and the abscess-
cavity is filled, not with pus, but with debris of liver tissue,
or sometimes a necrosed mass ; the contents may, however,
be old pus, the suppuration being due to the action of
micro-organisms conveyed by the amoebae (Kartulis). The
liver abscess may extend directly so as to involve the lung,
or the amoebae may traverse the diaphragm and set up
an abscess by liquefying the tissue ; but here the cavity is
surrounded by an area of interstitial inflammation. That
the micro-organisms do not travel by the lymphatics is
shown by their absence from the mesenteric glands ; and
they have no special preference for the lymphoid follicles.

Antc-mortem they may be found in the stools, particu-
larly the gelatinous particles, in the pus from liver abscesses,
and in the sputum in cases of abscess of the lung, and may
be examined in the fresh state, best on the warm stage, or
in cover-glass preparations ; or sections may be cut from
portions of faeces hardened and imbedded in celloidine.
Councilman and Lafleur obtained the best results with
sections of tissue hardened in alcohol and stained in
Loffler's methyl blue, by which method the amoebae are
coloured dark blue, but unevenly. The nuclei are best
brought out by hardening in Flemming's solution (see

290 BACTERIOLOGY

Section D), and staining deeply with saffranine, and a
nucleolus can often be seen.

Kartulis succeeded in obtaining pure cultures by inocu-
lating alkaline infusion of straw with some of the contents
of a liver abscess in which no bacteria were present. In
twenty-four to forty-eight hours, at 35° to 38° C., a mem-
brane forms on the surface, consisting of young amoebae,
which are mixed with bacteria when the culture is not quite
pure. Injection of a pure culture into the rectum, followed
by tying of the anus, caused swelling and erosions of the
mucous membrane in cats. Eectal injections of dysenteric
stools have also been often, though not always, successful
in causing similar diseased conditions. No result followed
when amoebae were administered by the mouth.

Protozoa in carcinoma. — As far back as 1847 Virchow
observed certain inclusions in tumour cells, some of which
are judged from his plates to have been similar to the
bodies now regarded in many quarters as parasitic protozoa.
They were variously explained at the time, and it was not
until 1888-89 that their nature began to be seriously dis-
cussed in connection with a possible causative influence in
cancer. A number of observations were published in 1889,
perhaps the most important being that of Malassez and
Albarran (in the affirmative), and since then many researches
have been carried on, amongst the more recent of which the
observations of Euffer and Walker l in England especially
(which have been since confirmed by Burchardt and Foa),
and those of Soudakewitch and of Steinhaus on the
Continent, must be mentioned. Many of the observers seem
to have been misled by various other appearances, but some
of the earlier investigations and most of the later concur in
showing that a peculiar body is practically always present

1 Journ. of Pathol. and Bacterial, October 1892. This paper is beauti-
fully illustrated with chromo-lithographs, which see.

APPENDIX 291

in some of the cells of all varieties of carcinoma, although
the number is not constant.

These inclusions are found for the most part in the
body of the cell, usually singly, though as many as eight or
ten have been counted, and they appear as round or oval
figures, measuring from 2/^ to Wjj, or more in diameter,
surrounded by a capsule probably furnished by the epi-
thelial cell (Buffer and Walker), and having a large irregular
nucleus in the centre, from which processes extend out
radially. Lines are also visible, at regular intervals, running
in from the capsule towards the centre. Less frequently
the inclusions are seen in the cell nucleus, or sometimes
half in and half out, a condition in which the capsule is
slight or absent (Galloway),1 and sometimes the nucleus
contains a number of small ones, which eventually make
their way out. Still more rarely similar bodies are ob-
served in the intercellular spaces. The nuclei of the bodies
stain like the nucleoli of ordinary cells when treated by the
Ehrlich-Biondi method (see below).

Buffer states that reproduction takes place by repeated
division of the nucleus, followed by that of the capsule,
until a zooglcea-like mass of ' parasites ' is produced. He
has not observed formation of spores, but thinks that
some of the young ' parasites ' may be of this character.
Burchardt,2 however, on one occasion found an appearance
in one of the bodies (which he has figured), consisting of a
delicate oval outline, which contains a round, thick-walled
vesicle full of small round bodies, and these he believes to
be a spore, germ-capsule, and germs respectively.3

The nucleus of the cancer cell is pressed to one side, as

1 Morton Lecture, Brit. Med. Journ., February 4, 1893, p. 217

2 Virchow's Archiv, Band 131, p. 121, January 2, 1893.

3 Foa has described the division of the body into a number of highly-
refracting particles within the capsule, which he regards as spores, and which
eventually penetrate into fresh cancer cells.

u 2

292 BACTERIOLOGY

a rule, but the cell may otherwise remain normal, or it
may become vacuolated and finally converted into a cyst.
The cancer cells in the neighbourhood do not show greater
signs of activity than those elsewhere (Boyce), but on the
other hand the parasites are most numerous in rapidly-
growing cancers, in newly-started secondary tumours, and
at the growing edges and outlying parts.

The ' parasite ' itself does not seem to be very hardy, at
times appearing eaten away ; and Buffer and Walker have
also seen it attacked and destroyed by leucocytes, which
sometimes surround it and sometimes penetrate into its
interior.

The body, therefore, is treated as a foreign substance
in the tissues, looks like an organised structure, competent
zoologists (Balbiani and Metschnikoff) have pronounced for
its parasitic nature, and appearances resembling germination
have been observed. It also stains unlike normal cell-con-
tents, resembling in this, as well as in some other respects,
known sporozoa. On the other hand, it differs both in size,
durability, and some other particulars from the protozoa
with which it can best be compared, has never been seen in-
an amoeboid stage, and its sporulation is scarcely fully
established as yet. Finally, cultivation has not yet suc-
ceeded, although certain bodies of a doubtful nature have
been observed by Ballance and Shattock l to undergo multi-
plication in a piece of scirrhus of the breast kept at incuba-
tion temperature. The opponents of the parasitic theory
refer it to one of the following classes, with which it
certainly seems to have at times been confounded : 2 trans-
verse sections through two cells, one of which is invaginated
into the other ; leucocytes, or red corpuscles, enclosed in

1 Eeport of debate at London Pathological Society, Brit. Med. Journ.,
March 11, 1893, p. 520.

2 See Galloway, loc. cit.

APPENDIX 293

epithelial cells ; endogenous cell-formations, in which the
nucleus has divided, but not the cell ; asymmetrical mitoses ;
degenerations of the cancer cells, particularly if the nucleus
is affected. Upon the whole, it may be said that the weight
of probability is on the side of the parasitic view, but its
influence on the tissues is at present unknown.1

The pieces of tissue to be examined should, if possible,
be obtained fresh from the operating table, cut into very
small pieces, and immersed at once in absolute alcohol,
corrosive sublimate, or Flemming's solution (see Section D).
If either of the latter be used, hardening must be com-
pleted in alcohol. The pieces may then be imbedded in
paraffine or celloidine, and sections cut and stained.

Hsematoxyline stain alone, or better with eosine or rose
bengale double-staining, gives good results, especially after
hardening in Flemming's solution. The parasites show a
different tone of blue from the cell-nuclei.

By Eussell's method the sections are stained for ten
minutes in saturated solution of fuchsine in 20 per cent,
carbojic acid, washed for a few minutes in water and then
for half a minute in absolute alcohol, and transferred for
five minutes to a 1 per cent, solution of iodine green
(Griibler's) in 2 per cent, carbolic acid. The sections are
then rapidly dehydrated in absolute alcohol, cleared in oil
of cloves, and mounted in balsam. The ' parasites ' appear
purple or red, the tissue-cells blue ; but unfortunately some
other bodies also stain red and may lead to mistakes.

The Ehrlich-Biondi triple stain, which gave beautiful
results in the hands of Euffer and Walker, had better be
bought prepared by Griibler of Leipsic, as it is rather hard
to make properly. The following was the method em-
ployed by the observers just mentioned : — 1 grm. of

1 For review of the general arguments in favour of a parasitic origin of
cancer, see Ann. of Surg., vol. xvii. pt. 4 (April 1893), p. 478.

294 BACTERIOLOGY

Griibler's powder is dissolved in 80 c.cin. of water, and 15
c.cm. of a 0*5 per cent, solution of acid fuchsine is added.
The sections are stained for at least an hour, washed in
water for half a minute, placed in 95 per cent, alcohol for
one minute and then for two to five minutes in absolute
alcohol, and finally transferred to xylol and xylol balsam.

Sims Woodhead gives the following method of prepar-
ing and using this stain : — 5 c.cm. saturated solution of
methyl green, 10 c.cm. saturated solution of methyl orange,
and 2 c.cm. saturated solution of acid fuchsine are mixed,
after having first been diluted with about 40 volumes of
water each, to avoid the formation of a precipitate. Stain
for fifteen minutes to twelve hours, rinse in 1 per 1,000
acetic acid, wash in dilute and then for one minute in abso-
lute alcohol, and finally immerse in xylol and benzol, and
mount in xylol balsam.

The protoplasm of the epithelial cell is orange-red, the
nucleus green, the nucleoli brown or red ; the protoplasm
of the 'parasite' is pale blue and the nucleus red. Con-
nective tissue is red, and leucocytes and all other cells are
orange-red with green nuclei. The protoplasm of the
' parasite ' is, however, sometimes orange-red, and the
nucleus a little darker. Lastly, the living parasites may
sometimes be distinctly seen, according to Soudakewitch,
by examining scraped-off cancer cells in 0'6 per cent, salt
solution.

(For bibliography, refer to the papers by Buffer and
Walker, and by Galloway.)

Protozoa, in other forms of new growth. — Whether bodies
similar to those just described occur in sarcoma is still a
matter of doubt. Jackson Clarke, indeed, claims to have
found such, but the Morbid Growths Committee of the
London Pathological Society has decided ] that his supposed

1 See Report, Brit. Med. Journ., May 20, 1893, p. 1056.

APPENDIX 295

parasites are really giant cells which have undergone
necrotic changes.

Peculiar bright bodies which are visible in the masses of
molluscum contagiosum, lying partly free and partly in the
interior of horny cells, are held by many to be coccidia, and
to be the exciting cause of the diseased growth. Others,
however, believe them to be merely degenerated epithelial
elements.

Bodies of a similar nature have also been described (by
Darier, Wickham, and others) in the form of eczema of the
nipple known as Paget's disease, which is associated with
cancer of the mammary ducts, but Buffer and Walker 1 are
of opinion that many at least of the appearances are
degenerating epithelial cells, or due to endogenous cell
formation.

Wernicke has described coccidia in the granulomatous
tumours of mycosis fungoides. These are light yellow,
round, 3 to 30 ^ in diameter, and are contained in a
hyaline sheath. As many as ten have been observed in a
giant cell. They reproduce by segmentation, and the
young coccidia are set free by the bursting of the parent
cells.2

C. The Action of Light upon Micro-organisms

The action of white light. — The fact that certain effects
are produced upon micro-organisms by the sun's rays has
been known for some time ; thus certain pathogenic bacteria
show less marked activity after exposure to light, although
on the other hand some bacteria, probably only the aquatic
varieties (Buchner), grow better under these circumstances ;
and others belonging to the chromogenic species— Bacillus
prodigiosus, for example— cannot produce their pigment in

1 Loc. cit. 2 Centralb. f. Bakt. u. Parasitenk., Dec. 28, 1892.

296 BACTERIOLOGY

the dark. The first definite researches on the subject,
however, were made in this country by Downes and Blunt
in 1877, since when many investigations have been carried
on, with the result that the power of light to inhibit the
growth of many micro-organisms has now been placed
beyond a doubt.

Thus Koch l found that tubercle bacilli are killed on
exposure to direct sunlight, varying from some minutes
to a few hours, according to the thickness of the layer,
while diffuse daylight required from 5 to 7 days to produce
the same effect. Similar results have been obtained with
the typhoid bacillus by Janowski,2 who exposed to diffuse
daylight three culture tubes, one uncovered, one wrapped
in white, and one in black paper, and also a culture in a
U tube one limb of which was covered, and found that the
uncovered culture or portion of a culture was greatly re-
tarded in its development, while 4 to 7 hours of direct
sunlight killed it altogether. Buchner2 exposed a plate
culture of the same bacillus in a Petri's capsule, to which
a cross of black paper had been attached, and then kept it
for 24 hours in the dark, when it was found that after 1 to
1^ hour of sunlight, or 5 of diffuse daylight, no colonies
developed except on the part of the plate protected by the
paper cross, all the rest having been destroyed. He also
obtained confirmatory results with Bacillus coli commune,
Bacillus pyocyaueus, and Vibrio cholera Asiatics, finding
that some water which contained 100,000 elements of the
first-named per c.cm. yielded no growth after an hour in

1 Vcrhandlung. d. 10. medic. Congress. Berlin, 1890, Bd. I. p. 42 (quoted
by Giinther).

2 For bibliography up to date, see P. Frankland and H. M. Ward,
* Report on the Bacteriology of Water,' Roy. Soc. Proc., vol. li. No. 310,
pp. 199 n. and 237-9 ; also the other papers by the latter (to be cited pre-
sently). It may be mentioned, however, that Janowski's paper appeared in
Centralb. f. Bakt. u. Parasitenk., Nos. 6-8, 1890 ; and Buchner's ibid.
Bd. xii. 1892, and Bd. xi. No. 25.

APPENDIX 297

direct sunlight. The most recent and conclusive experi-
ments of all, however, are those of Professor H. Marshall
Ward J with the very resistent and virulent spores of Bacillus
anthracis, confirming and extending the results obtained by
some previous observers with the same bacterium. Having
found that repeated exposure to sunlight destroyed the
spores in a few c.cm. of Thames water containing a very
large number, while a few weeks of bright daylight greatly
lessened them, he proceeded to make a series of accurate
experiments as follows :— Agar plates of anthrax were made
in Petri's capsules, using the virulent and resistent spores
obtained by keeping some c.cm. of distilled water, saturated
with material from an old agar culture, at 56° C. for 24
hours. To the bottom of each plate was attached a zinc
stencil having a letter cut out, the rest of the capsule was
covered with dull black paper, and the letter surface ex-
posed to direct or reflected sunlight for 2 to 6 hours, after
which the plate was kept in the incubator at 20° C. for 48
hours. The agar was then found to be grey and cloudy
from the development of a multitude of colonies, but the
space exposed to light remained quite clear, showing the
form of the letter, and the development of the colonies in
its neighbourhood \vere greatly retarded, owing to the action
of reflected light, when the letter was large. These results
were also obtained with other bacteria, as well as with the
spores of fungi ; and similar, though less marked, effects
were produced by the electric arc light, so much so that
Marshall Ward thinks that it may yet prove to be an
efficient disinfecting agent.

The action of coloured light, — When a plate culture of
anthrax spores is exposed to the solar spectrum the bacteri-
cidal action is found to be strongest at the blue-violet end
(Ward), and certain chromogenic bacteria have been

1 Royal Soc. Proc., vol. Hi. No. 318, p. 393.

298 BACTERIOLOG Y

seen,1 when examined in water by the micro-spectral objec-
tive, to make their way to the part furthest from this end.
Janowski exposed cultures under screens of various chemical
solutions and aniline dyes, and found that no action took
place under brown or yellow, whereas solutions of fuchsine
(which transmits violet rays), gentian violet, and methyl
blue had little more effect than colourless fluids. Confirm-
atory results have also been obtained quite recently by
Marshall Ward,2 who repeated his experiments with the
anthrax plates, covering the stencil letter with pieces of
glass of various colours, as well as with screens of saturated
solutions of potassium bichromate and of ammoniated
cupric oxide (which cut off respectively the violet and red of
the spectrum as far as the line 6), and found that no effect
was produced on the spores by exposure to sunlight behind
ruby, olive, orange, or green glass, nor behind the bi-
chromate solution (when sufficiently concentrated), while
4 hours' exposure behind violet and blue glass, and a much
thicker cupric oxide screen, was usually sufficient to kill
them. Less pronounced effects were produced by diffuse
daylight. The bactericidal action of light, therefore, seems
to depend on the more refrangible rays in the violet half of
the spectrum, which produce their effects whether the red-
yellow rays are transmitted or not.3

The mode of action of light. — That the effects produced
are really due to the rays of light and not to heat is shown
by the fact that they also take place under water (Buchner)
and behind a screen of alum solution (Janowski), that a ther-
mometer at the surface of the glass never registered above

1 Engelmann, quoted without reference by Woodhead (Bacteria and their
Products, 1892, p. 201).

2 Boy Soc. Proc., vol. liii.No. 321, p. 23.

3 Prof. "Ward states (May 25) that he has made great advances in his
experiments with chemical screens and with the spectrum, but has elicited
no contradictions to his earlier results, as already published.

APPENDIX 2991

18° C., and that a gelatine plate with a melting-point of
29° C. remained solid during 5 or 6 hours' exposure, while
the spores in the exposed area were killed (Ward). An
alternative, explanation, that the death of the spores may
be due to changes brought about in the nutrient medium,
has also been excluded by Marshall Ward, who proved that
no colonies develop (provided insolation has been complete)
if a piece of the clear letter on the agar is placed in fresh
nutrient material, while it is still capable of supporting a
growth of the bacillus if sown with fresh spores. He also
exposed a plate of dry spores and one of sterile agar under
stencil letters side by side, and then covered them respec-
tively with gelatine or agar, and with fresh spores, when
the former showed a clear area under the letter, whereas
the latter was covered by a uniform growth. The effects
are therefore due to the direct action of the rays of light
upon the micro-organisms themselves.

It is probable that ripe spores contain a reserve store of
fatty material, which would partly explain their resistance
to staining and to the ordinary methods of sterilisation, and
Duclaux has shown that vegetable oils are rapidly oxidised
by light. From these considerations, together with the
facts that the effect is stronger on spores than on bacteria
and has the character of chemical action, Ward has deduced
the theory that the bactericidal action of light may be due
to its destructive influence, in the presence of oxygen, on
fatty matters or other reserve material.

Applications in Nature. — The above theory §eems to.
explain and be supported by many facts in nature. Thus
chromogenic bacteria often produce their pigment only in
the presence of light, that is, when it would be required
to act as a screen, and fungi which grow in open places and
on the upper surface of leaves are nearly always protected
by having a dark, or a yellow-red, or an orange colour. Ward

oOO BACTERIOLOGY

could not obtain any positive results with fungus spores
which were of a deep colour, or dull yellow- brown, although
colourless and faintly tinged spores were readily killed ; and
Bachmann found that in the spectra of 42 pigments of
fungi the extreme blue-violet (i.e. the most active part) is
cut out from the line G on in all but two.

Further, the sporangia and spores of ferns are mostly
orange or reddish, pollen and its spores, which contain oils,
are almost always deep orange, and lastly Ward suggests
that one of the functions of chlorophyll may be to act as a
colour-screen.

This observer therefore puts forth the provisional hy-
pothesis that : — ' No plant exposes a reserve store of fatty
material to the danger of prolonged or intense insolation
without a protective colour screen calculated to cut out at
least the blue-violet rays, as these rays would otherwise
destroy the reserve substance by promoting its rapid
oxidation.'

D. Additional Methods and Formula

Fixing methods. — For fixing coccidia, Flemming's solu-
tion, corrosive sublimate, or absolute alcohol may be used.
The last-named was dealt with on p. 80.

By Flemming's method pieces of perfectly fresh tissue are
immersed in a mixture of 4 parts of 2 per cent, aqueous
solution of osmic acid, 15 parts of 1 per cent, chromic acid,
and 1 part of glacial acetic acid, for from one to three days,
•washed in water for three to six hours, and then finally
hardened by successive immersion for twenty-four hours
each in 30, 60, and 96 per cent, alcohol. This method
gives great sharpness of definition, but the sections do not
stain readily.

By the second method the fresh pieces are immersed for
six to twenty-four hours in a solution of 7-J- grms. corrosive

APPENDIX 301

sublimate in 100 parts of 0*7 per cent, salt solution, washed:
first in water, then in 30 per cent, alcohol to which a few
drops of tincture of iodine have been added, and then in
weak potassium iodide solution, and hardened by immersion
in 50, 75, and 90 per cent, alcohol for twenty-four hours-
each, and finally in absolute alcohol. Imbedding may then
be done in paraffin or celloidine.

The gum freezing method. — A very convenient method of
cutting sections, which is much used in this country, is that
of freezing in gum. The piece of tissue, which should be
as small as possible, is soaked for twenty-four hours in
water to free it from spirit. It is then left in thick gum
mucilage until completely saturated, which requires from a
few hours to some days, according to the density of the
tissue, after which it is frozen on the plate of the micro-
tome, and in that state has exactly the right consistence
for cutting. The sections are transferred to tepid water to
free them from gum, and can then be stained, &c., as usual,.
or kept in spirit. Fresh or imperfectly hardened tissues
can be cut with ease by this method.

Staining formulae. — Von Kahlden recommends the follow-
ing method of staining sections of tissue fixed in Flemming's
solution in order to bring out the nuclei: — The sections are
stained for half an hour to twenty-four hours in 1 per cent,
aqueous solution 'of saffranine, rinsed for a short time in
water, washed in absolute alcohol slightly acidulated with
a few drops (5 to 10 drops to a medium watch-glass) of spirit
containing 1 per cent, of hydrochloric acid, and finally in
absolute alcohol, cleared in xylol, and mounted in Canada
balsam.

The simplest hamatoxyline stain is made by adding
sufficient of a saturated alcoholic solution of the crystals to
a 1 per cent, aqueous solution of alum to render it pale blue
or pale violet, and exposing to light for a few days. Filter,.

302 BACTERIOLOGY

and stain for two to three minutes. Ehrlich's acid licema-
toxyline, which has been much used for staining coccidia, is
prepared by adding 6 parts of glycerine and 60 of water,
both saturated with alum, and 3 parts of glacial acetic acid,
to a solution of 2 parts haimatoxyline in 60 parts absolute
alcohol. Filter, stain for 3 to 5 minutes.

Eosine is used as a double stain in a y1^ per cent, aqueous
solution in which the sections, after being stained in haema-
toxyline and washed, are immersed for one or two minutes.

Picro-carmin'e is made by dissolving 1 grin, carmine in
5 grms. strong ammonia and 50 grms. water, and adding 50
grms. of saturated aqueous solution of picric acid, after
which the stain is left standing in a wide open vessel until
all the ammonia has evaporated, and is then filtered. Stain
for an hour, soak for half an hour in glycerine containing
1 per cent, of hydrochloric acid and coloured light yellow
with picric acid, wash for five minutes in water, and de-
hydrate in alcohol, both of which are similarly tinted.

Alum carmine solution is made by boiling 2 to 5 grms.
carmine with 100 grms. 5 per cent, alum solution for a half
to one hour, and filtering. Stain for ten minutes to two
hours.

Nicolle claims to have discovered a method of fixing the
colour of bacteria, useful for those which bleach under
Gram's process : — The sections are stained for two or three
minutes in Lofner's or Kiihne's methyl blue, slightly
decolorised in feebly acid water, washed, dipped for an
instant in 10 per cent, tannin solution, washed in water,
dehydrated, cleared in oil of cloves, and mounted in xylol
Canada balsam.

INDEX

ABSCESS, hepatic, in dysentery, 289

— pulmonary, 289

Abscesses in skin, staining micro-or-
ganisms in, 90

Aceti, Bac., 191

Achorion actaton, dicroon, euthythrix,
Schcenleinii, 234

Acidi lactici, Bac., 179 ; Mic., 180
— liquefaciens, Mic., 181

Actinobacter, Bac., 185

Actinomyces, 196, 275

Aerobes, 1

Acrogencs, Mic., 250 ; Bacter., Helico-
bacter, Bac., 251

Aeroscope, Pouchet's, 97

Agar, glycerine, 45 ; Kowalski's, 46 ;
litmus, 45 ; meat extract peptone,
45 ; peptone bouillon, 43 ; serum,
49 ; surface cultures on, 57 ; urine,
46

Agar-agar, 42

Agilis, Mic., 128

Air, analysis of, 96 ; methods of ex-
amining, 96 ; method of Pouchet,
97; of Miquel,98; Emmerich, Welz,
99 ; Hesse, 100 ; Strauss and Wiirz,
101 ; Petri, Tyndall, 102 ; Frank-
land, 103 ; micro-organisms in, 96

Alba, Sarcina, 108

Albicans amphis, Diploc., 225

— tardus, Diploc., 226
Albumen, plover's egg, 49

— solutions of, 37

Albuminate, Lieberkuhn's potash, 49
Albuminis, Bac., 256
Ambigenous organisms, 1
Amceba coli, v. dysenteric, 252, 288
Amylobacter, Bac., 181
Anaerobes, 2 ; cultivation of, 60

! Aniline colours, 30 ; A. oil, A. water, 69
! Animals, experiments on, 91 ; most

useful, 92
Anthracis, Bac., 165 ; effect of light

on, 297

Anthrax, symptomatic, Bac. of, 228
Apparatus, list of, 20 ; counting, 125 ;

plate, 28, 55 ; spray, 93
Aquatilis, Bac., 136 ; Mic., 128

— radiatus, Bac., 137

— sulcatus, Bac., 136
Arborescens, Bac., 131
Arthrospores, 5
Ascobacillus citreus, 231
Aspergillineae, 10

Aspergillus albus, flavescens, fumiga-

tus, glaucus, niger, 193
Attenuated vaccine, Haffkine's, 285
Attenuation, 6
Aurantiaca, Sarc., 108
Aurantiacus, Bac., 135
- Mic., 129
Aurea, Sarc., 270
1 Aureus, Bac., 135, 275

i BACILLI, 2

Bacteria, varieties of, 2 ; multiplica-
tion of, 4
Bacteriuria, 258
Basidia, 11, 103

BAUJIEYER, petroleum incubator, 26
j BAUMGAKTEN, staining of tubercle ba-
cilli, 215
; Beggiatoa, 12

Birds, tuberculosis in, 209
j Biskra, Mic., 221

. Blood, micro-organisms in, 276 ; modes
of examining, 276 ; staining of, 277
I — serum. See Serum

304

BACTERIOLOGY

Borax methyl blue, method, 90

Bougies, cesophageal, 94

Bouillon, 35 ; meat, 36 ; meat extract,

37

Bread, 53 ; moulds on, 193
BKIEGEK, extraction of toxines, 7
' Brownian movement,' 3
Bruneus, Bac., 135
Buccalis maximus, Bac., 238
Butyri fluorescens, Bac., 183

— viscosus, Bac., 183
Butyricus, Bac., 182

CADAVERINE, 148

Candicans, Mic., 106

Candida, sarc., 108

Capsulatus, Bac., 3, 221

— mucosus, Bac., 3, 268

Capsules, 3

Carcinoma, protozoa in, 282, 290

Carmine, alum, 302

Carneus, Mic., 129

Carragheen moss, nutrient jelly from,

47

Caucasicus, Bac., 188
Cavicida, Bac., 256 v

Celloidine, imbedding in, 82
Cells, inclusions in, 290

— epithelial, proliferation of, caused
by coccidia, 287

Centrifuges. See Machines, centri-
fugal

Cereus, Stapli., albus, &n.dflavus, 199 ;
aureus, 201, 264

Cerevisice, Sarc., 192

' Chamberland's candles,' 123

' Chameleon phenomenon,' 199

CHENZYNSKY'S solution, 86, 278

Cholera, inoculation of, 94, 144 ; vacci-
nation against, 283 ; reaction, 144,
147

Cholera Nostras, Bac. of, 144, 145

Cholera-red, 147

Cholera Asiatic CE, Bac., Vibrio, or Spi-
rillum, 139 ; effect of light on, 296 ;
toxopeptone from, 7

Choline, 148

Cilia. See Flagella

Cinabareus, Mic., 106

Citreus conglomeratus, Dipl., 107

— liquefaciens, Dipl., 226
Cladothrix, 12 ; C. dichotoma, 13 ; C.

canis, 197
Clearing agents, 85
Clostridium, 4, 159 ; C. butyricum,

181, 254 ; C. fcetidum, 159
Cocci, 2

Coccidia, 12, 295

Coccidium oviforme, 286

Coli commune, Bac., 152 ; effect of

light on, 296

Colonies on gelatine plate, 56
Coloured images, 68

— light, action of, on micro-organ-
isms, 297

Columella, 10
Comma-bacilli, 2, 140, 146
Concentricum, Spir., 176
Concentricus, Mic., 129
Condensation, water of, 45
Conidia, 11, 103

Conjugation, reproduction by, 194
! Coprogenes fcetidus, Bac., 251
Cory zee, Dipl., 265

— equorum contagiostx, Mic., 221
Counting apparatus, Wolffhugel's, 125
Crates, wire, for test-tubes, 29
Crenoihrix Kuliniana, 11
Cultivation, methods of, 53-64
Cultures, anaerobic, 60 ; ' hanging

drop,' 62 ; high, 62 ; permanent, 63 ;

plate, Koch's, 54 ; plate, serum and

albumen, 60 ; roll, Esmarch's, 58 ;

slide, 53 ; streak or surface, 57, 60 ;

thrust, 57, 62

Cumulatus tennis, Mic., 264
Cyanogenus, Bac., 185
Cystitis, Bac. in, 262

DECOLORISING agents, 72

Decomposition, 173

Dehydration, 85

DENEKE, Bac., 146, 183

Denticola, 242

Dentium, Spiroclitzte, 242

Diet, articles of, 178, 189

Digestive tract, the, 237; infection

by, 93

Diphtheria, Bac., 240
Diplococc'i, 2, 4
Discontinuous sterilisation, 18
. Disinfectants, chemical, 18
Dispora Caucasica, 188
Double test-glasses, Schill's, 16
i Drum-stick bacteria, 5
Drying-on processes, Unna's, 85 ;

Ktihne's, Weigert's, 89 ; Winkler's,

203

Dust, window and room, 156
Dysenteries, Amceba, 252, 288
j — Bac., 252

EABTH, analysis of, 155

— bacillus, 156

INDEX

305

E BERTH, bacillus of typhoid fever, 148
Ectogenous organisms, 1
Ectoplasm, 171
Eczema marginatum, 233
Eggs, birds', bens', 49, 50 ';' plover's, 49
EHKLICH-BIONDI triple stain, 293
EHRLICH, acid hasmatoxyline, 302
Emmerich, Bac., 120
EMMERICH, method of air-analysis, 99
Endocarditis capsulatus, Bac., 278
Endogenous organisms, 1
Enteric fever, 148. See Typhoid
Entoplasm, 171
Eosine, double-stain, 302
ERLENMEYER'S flasks, 35
Erysipelatis, Strept., 113
Eri/throsporus, B., 131
Eye, infection into anterior chamber
of, 9o

FACULTATIVE anaerobes, 2

— parasites, 1

Fasces, composition of, 254 ; protozoa
in, 255

Favus, 234 ; fungus of, 234, 235

Febris tertiana, quartana, quotidiana,
171

Fermentation, 12 ; acetic, 191 ; alco-
holic, 12, 188; alkaline, 258; bu-
tyric, 182 ; lactic acid, 180, 188, 192 ;
slimy, 184

Fervidosus, Mic., 129

Figurans, Bac., 175

Filters, hot-water, 27 ; creased paper,
38 ; sand, charcoal, asbestos, porce-
lain, clay, 122 ; kaolin (Chamber-
land-Pasteur), 123; micro-mem-
brane (Breier), 123

Filtration, 122 ; of media, 38, 43

Finkler- Prior, Bac., 145

' Fishing,' 57

Fission-fungi, 2, 4. See Bacteria

Fission, multiplication by, 4

Fixing tissues, methods of, 300 ; serial
preparations, media for, 84

Flagella, 3 ; staining of, 71

Flavus desidens, Mic., 108

— liquefaciens, Mic., 108
— tardus, Dipl., 226

— tardigradus, Mic., 106
FLEMMING'S solution, 293, 300
Flies, diffusion of ferment by, 191
Fluoresceine staining method, 89
Fluorescens liquefaciens, Bac., 130

— non-liquefaciens, Bac., 130
Foztidus lactis, Bac., 185

— ozcence, Bac., 266

Foods, methods of examining, 178,

189 ; moulds on, 193
Fowl cholera, Bac. of, 252
Fractional sterilisation, 18
FRANKKL, A., pneumococcus, 271
- B., stain, 214

FRANKLAND, water- bacteria, 122
Freezing method, 79 ; in gum, 301
FRIEDLANDER, pneumobacillus, 273 ;

stain, 213
Fruchthyphen, 103
Furuncles, staining of, 90
Fuscus limbatus, Bac., 174
— Mic., 128

GABBET, method of staining tubercle

bacilli, 75, 214
Gasoformans, Bac., 132
Gastric bacteria, 245
Gelatine, glycerine, 41 ; litmus, 41 ;

meat extract peptone, 40 ; Miquel's,

42 ; peptone bouillon, 38 ; potato

(Holtz), 42 ; serum, 49 ; urine, 42
Gemmation, multiplication by, 12
Generative organs, the, 222
Germs, sickle-shaped, 12
GIBBES, stain for tubercle bacilli. 215
Giganteus urethrce, Strept., 261
Gingivce, Bac., 239
' Glacier Bacillus,' 130
Glanders, Bac. of, 218
' Glanders-nodules,' 218
Glycerine jelly, imbedding in, 81
Gonorrhoea, Mic. of, 201
Gracilis, Bac., 138
' La graisse,' 192
GRAM, decolorising method of, 76,

87 ; Giinther's modification of, 77 ;

Kiihne's, 87 ; Weigert's, 78 ; dyes

suited for, 77
Gregarinffi, 12
Ground-water, 155

Gum arabic, freezing in, 301 ; im-
bedding in, 81

GUNTHER, methods of staining, 74,
87, 212, 277

Hcematodes, Mic., 227
Hasmatoxyline, extract of, 30 ; stain,

293, 301

Hasmoglobinuria of oxen, 282
HAFFKINE, anti-cholera vaccination,

283

Hanging drop, examination in, 65
Hardening, methods of, 79 ; in alcohol,

80 ; chromic acid, 80 ; corrosive

306

BACTERIOLOGY

sublimate, 81, 300; Flemming's
solution, 300 ; picric acid, 80

' Hay bacillus.' See Bac. subtilis

Helicobacterium aerogencs, 251

' Hemoglobinurie bact6rienne du
boeuf,' 282

Hens' eggs, 50 ; white of, 37, 49

Herpes tonsurans, 232

HESSE, method of air- analysis, 100

High cultures, 62

Hollowed slides, 29

Hot-air steriliser, 15

Hot-water filtering funnel, 27

Hyphse, 10

Hyphomyceta?, 10

lanthinus, Bac., 138

Imbedding, 81 ; in celloidine, 82 ; in
glycerine jelly, 81 ; in gum arabic,
81 ; in parafnne, 83

Immunity, 6

Impetigo contagiosa, 233

Impression preparations, 78

Incubators, 20 ; petroleum, Bau-
meyer's, 26 x

Indicus, Bac., 248

Indigoferus, Bac., 137

Indigogenus, Bac., 191

Infection, artificial, 92; by air-pas-
sages, 93 ; by digestive canal, 93 ;
into eye, 95 ; intraperitoneal, 94,
284 ; intravenous, 94 ; subcutaneous,
94

Influenza, Bac. of, 2, 278

Inoculation. See Infection

Inspissator, serum, Koch's, 47, 48

Intestine, micro-organisms of, 249, 253

Iodine method, 89

lodococcus magnus, I. parvus, I. vagi-
natus, 238

Islets, on plate cultures, 56

KAATZER, staining of tubercle bacilli,
211

Kephir, 188

Knob-moulds, 10

KOCH, comma-bacillus, 139 ; plate
process, 54 ; serum-inspissator, 47 ;
stain, 86 ; steam steriliser, 16 ; sy-
ringe, 31 ; tubercle bacillus, 206 ; tu-
berculine, 7, 8, 218

KOCH-EHRLICH method of staining
tubercle bacilli, 73

KUHNE, method of staining tubercle
bacilli in sputum, 213 ; methyl blue,
86

Lacteas faviformis, Mic., 225
Lacticus, Bac., 179
Lactis aerogenes, Bac., 248

— erythrogenes, Bacter., 186

— fcetidus, Bac., 185

— pituitosi, Bac., 185

— viscosus, Bac., 184

' Laveran's sickles,' 171
Lepra, Bac. of, 229

— cells, 229

Leptothrix buccalis, 238, 255
Levelling stand, for plate cultures, 28
Light, action of, upon micro-organisms,

295 ; coloured, 297 ; white, 295 ;

electric, 297 ; mode of action of, 298 ;

natural precautions against, 299
Liodermos, Bac., 117
Liquefaciens magnus, Bac., 159
Litmus agar, 45 ; gelatine, 41
LOFFLEB, diphtheria bacillus, '240 :

methyl blue, 86, 241, 277
LOSCH, amoeba of, 288
Lutca, sarc., 109
Luteus, DipL, 130
- Mic., 128

MACHINES, centrifugal, 31 ; Csokor's,

34 ; Gartner's, 33 ; Stenbeck's, 31
MalaricB, Plasmodium, 169
Malignant pustule, 169
Malleus, 218. See Glanders
Mastitis, bovine, Mic. of, 187
Measurement, standards of, 65
Media, fixing, 84

— nutrient, 35 ; liquid, 35 ; solid, 37 ;
turbidity in, 39

Megaterium, Bac., 189
Melanaemia, 170
Melanine, 170
Melochloros, Bac., 118
Membranaceus ametliystinus, Bac., 137
Meningitidis intracellularis, Mic., 221

— purulent fs, Mic., 221

I Meningitis, pneumococcus in, 271

Merismopedia, 4

i Mesentericus fuscus, ruber, vulgatiis,
Bac., 116

Metabolism in bacteria, products of,
5 ; methods of extracting products
(Brieger, Scholl, Koemer, Buchner,
Koch), 7, 8, 218

Methyl blue, borax, Unna's, 90 ; car-
bolic, 88 ; and eosine (Chenzynsky's),
86, 278 ; Kiihne's, 86 ; Loffler's, 86,
241, 277 ; Plehn's, 87

Metschnikcffi, Vibrio, 147

INDEX

307

Micra. Sec Micro-millimeters

Micro-burners, 21

Micro-membrane filter, 128

Micro-millimeters, 65

Micro-organisms, classification of, 1,2;
examination of, 12 ; examination in
fresh state and hanging drop, 65 ; in
sections of tissue, 78; M. in disease,
92; isolation of, 57; by Cohen's me-
thod, 127 ; staining of, 67 et seq. ;
transmission of, to animals, 91

Microsporon furfur, 235

Milk, methods of examining, 179 ;
micro-organisms in, 188

Milk-rice, 52-3

Miller, Spirillum, 242

MIQUEL, method of air-analysis, 98

Moist chambers, 28, 56

Molecular movement, 3

Molluscum contagiosum, protozoa in,
295

Monilia Candida, 243

Monococci, 2

Mordants, 69

' Mother-of-vinegar,' 191

Mould, brown, 104, 156

Moulds, 9, 193 ; pathogenesis of , 194-5

Mouse septicaemia, 280

Mouth, the, micro-organisms of, 237,
244

Mucor corymbifer, 194 ; M. mucedo,
9, 194 ; M. ramosus, M. rhizopodi-^
formis, 194

Mucorineae, 10, 193

Multipcdiculosus, Bac., 118

Multiplication, modes of, 4, 194

Murisepticus, Bac., 280

Mycelium, 10, 103, 233

Mycoderma albicans, 243

Mycoides, Bac., 156

Mycosis fungoides, coccidia in, 295

NAIL, finger, 223
' Nail-culture,' 186, 248, 273
Nasal bacteria, 264, 268 ; secretion, 264
Nasalis, Mic., 265
- Vibrio, 268

Neapolitanus, Bac., 120, 147
NICOLLK, method of staining, 302
Nivalis, Bac., 130

NONIEWICZ, method of staining glan-
ders bacilli, 220

Ochraceus, Bac., 138
Ochroleucus, Mic., 261
(Edematis maligni, Bac., 160
Oidiacese, 11

O'idium albicans, 243
— lactis, 10, 181
Onychomycosis, 223, 233
Organs and cavities of body, 222
Osteomyelitidis, Mic., 221
Otomycosis, 195
Ozama, 266

PACKET-COCCI, 4

PAGET'S disease, protozoa in, 295

Pap, bread, 53 ; potato, 52

Paraffine, imbedding in, 83

Parasites, 1, 92, 286

Pediococcus cerevisice, 191

Pellicles, 4

Pemphigus acutus, Dipl., 227

Pencil mould, 11

' Pende's ulcer,' 221

Penicilliaceaa, 11, 103, 193

Penicillium glaucum, 10, 103

' Perlsucht,' 178

Permanent cultures, 63

PETKI, method of air-analysis, 102 ;

capsules, 28, 58
I Phlogosine, 113
| Phosphorescence, 6, 133

Phosphor escens, Bac., 132 ; indigenus,

132 ; indicus, 133
j Picrocarmine, 302
i Pigment-forming bacteria, 5 ; effect of

light on, 295, 299
' Plasmodia, 12, 169, 282

Plate process, Koch's, 54; apparatus
for, 28, 55 ; modifications of, 58, 59, 61

Platinum wires, 30

Plugs, cotton-wool, 16

Pneumobacillus Friedlccnderi, 8, 273

Pneumonice, Diploc., 271

Pneumosepticus, Bac., 275

Polymorphic bacteria, 197

Potato bacillus, 116

Potatoes, preparation of, for culture-
media, 51

! Poultry typhoid, 252
| Prodigiosus, Bac., 114

Proteines, 7

Proteus, 174 ; P. capsulatus, P. ho-
minis,P. mirabilis,P. Zenkeri, 175 ;
P. vulgaris, 175, 181

Protozoa, 12; in blood, 282; in car-
cinoma, 290 ; in molluscum conta-
giosum, 295 ; in mycosis fungoides,
295; in Paget's disease, 295; in
sarcoma, 294 ; parasitic, 286 ; patho-
genesis of, 286 ; staining of, 293.
See also under Amoeba, Coccidium,
Plasmodium, &c.

x 2

308

BACTERIOLOGY

Pseudodiphtheritic Bac., 242

Pseudonavieella, 12

Pseudopodia, 12

Psorosperms, 12

Ptomaines, 6, 173

Pulmonum, Sarc., 270

Pus, 196 ; microbes of, 196, 220 ;

staining of, 221 et passim
Putrefactive processes, differences in,

173

Putrefying substances, analysis of, 173
Putrescine, 148
Putrificus coli, Bac., 251
Pyocijaneus, Bac., 8 ; a and &, 198-9,

223 ; effect of light on, 296
Pyocyanine, 199
Pyogenes, Staph., 109 ; aureus, 110 ;

albus, citreus, 112
- Strept., 113, 201 •

Radiatus, Bac., 158 ; Mic., 105

' Kag-picker's disease,' 169

Ramosus, Bac., 133

' Kauschbrand,' 228

' Bay fungus,' 196. See Acfinomyces

Keagents, bacteriological, 29

Respiratory passages, micro organisms

of, 269; E. tract, 264; infection

through, 93

Ehinoscleroma, Bac. of, 266
Bice, milk, 52
Ringworm, 233
Boll cultures, Esmarch's, 58
Boot bacillus, 133
Rosea, Sarc., 109, 187
Rosens, Mic., 107
Rubrum, Spir., 176
Rugula, Vibrio, 242
BUSSELL, method of staining, 293

Saccharomyces cerevisice, 11, 105; S.
ruber, 188

Saffranine, staining with, 301

Saliva, 237, 238

Salivarius septicus, Bac., 238; Mic.,
238

Saprogenes, Bac., I., II., III., 175

Saprophytes, 1, 92

Sarcina, 2 ; S. alba, S. aurantiaca,
108 ; S. aurea, 270 ; S. Candida,
108 ; S. cerevisia, 192 ; S. lutea,
109 ; S. pulmonum, 270 ; S. rosea,
109, 187 ; S. ventriculi, 246

Sarcoma, protozoa in, 294

Schizomycetes, 2, 4

Scissus, Bac., 159

Section -stainer, 82

Sections, cutting of, 79 et seq., 301 ;
examination of micro-organisms in,
68 ; staining of, 84

Sedimentation, methods of, 127, 216

Seed-organ, in moulds, 10

Segmentation, 170

Septicaemia, mouse, 280 ; puerperal,
238

Septicus, Bac., 160

— vesicce, Bac., 262

Serial preparations, 83, 84

Serum, blood, 47 ; modifications of,
48 ; sterilisation of, 19, 47

Skin, abscesses in, staining of, 90 ;
micro-organisms of, 222 ; modes of
staining, 223-4

Small-pox, protozoa in, 282

Smear preparations, 67

Smegma Bac., 206

Soil, examination of, 156 ; filtering
action of, 156 ; micro-organisms of,
155, 172

' Soorpilz,' 243. See' Thrush-fungus

SOYKA'S plates, 29

Spectrum, experiments with, 297

Sphcerococcus acidi lactici, 181

' Spider cells,' 148-9

Spinosus, Bac., 159

Spirilla, 2 ; atmospheric, 121 ; water,
153

Spiritus saponatus kalinus, 91

Spirochsetse, 2

Spirochc&te dentium, 242

- Obermeieri, 281

Sporangium, 10

Spores, 4, 9, 103, 189, 291 ; effect of
light on, 297, 299 ; staining of, 71 ;
swarm, 12 ; terminal, 5, 163

Sporocysts, 12

Sporozoa, 12, 287

Sporulation, 4, 9, 291 ; arthrogenous,
5 ; endogenous, 5

Spring-water, 124

Sputum, 269 ; staining of, for tubercle
bacilli, 210 ; pure cultures of tu-
bercle bacilli from, 207

Staining, 67, 301 ; combination, 86 ;
cover-glass preparations, 67; sec-
tions, 84. See also under Tubercle
bacillus, Gonococcus, &c. &c.
— processes, Arens, 76, 214 ; Ehrlich,
74; Ehrlich-Biondi, 293; Frankel,
Gabbet, 75 ; Gibbes, 215 ; Gram, 76 ;
Giinther, 74, 212, 277 ; Koch and
Ehrlich, 73, 210 ; Loffler, 86, 241,

INDEX

309

277; Nicolle, 302; Pittion, Pfuhl
and Petri, 75; Weichselbaum, 75;
Weigert, 78, 89 ; Ziehl-Neelsen, 74,
213

Stains, 30 ; preparation of, 69

Staphylococci, 2, 4, 109

Staphylococcus cereus, 199 ; S. pyo-
genes, 109

Steam generator (Budenberg's), 17

— steriliser, Koch's, 16

Sterigmata, 11, 103

Sterilisation, 15 ; by chemical agents,
18 ; by combined methods, 19 ; by
heat, 15 ; by steam, 16, 18 ; discon-
tinuous or fractional, 18 ; of hands,
20 ; of instruments, 19

Steriliser, hot-air, 15 ; Koch's steam,
16

Stomach, micro-organisms of the, 245

STRAUSS and WURZ, method of ana-
lysing air, 101

Streptococci, 2, 4, 113

Streptococcus erysipelatis, 113 ; S. gi-
ganteus urethra, 261 ; S. pyogenes,
113, 201 ; S. septicus, 165

Striatus albus, Bac., S. flavifs, Bac.,
266

Structural images, 68

Styrone method, 224

Subflavus, Dipl, 224

SuUiliformis, Bac., 256

Subtilis, Bac., 113

Subungual space, micro-organisms in,
223

Sulphureum, Bact., 262

Sulphydrogenus, Bac., 138

Sweat, 223

Swine erysipelas, Bac. of, 279

Sycosis, 231, 233

Sycosiferus fcetidus, Bac., 231

Symbiosis, 164

Symptomatic anthrax, Bac. of, 228

Syncy anus, Bac., 185

Syphilis, Bac., 205

Syringes, hypodermic, 31

TABLET-COCCI, 4
Temperatures, 21
Test-tubes, charging of, 40
Tetanus Bac., 163
Tetracocci, 2, 4
Tctragenus, Mic., 273
- concentricus, Mic., 256

— mobilis ventriculi, Mic., 248

— subflavus, Mic., 264
Texas fever, 282

Thallus, 10

Thermo-regulators, 21 ; Altmann's,
25 ; Bunsen's, 21 ; Gartner's, 25 ;
Meyer's, 24 ; Schenk's, 22

Thrush-fungus, 243

Tinea circinata, T. sycosis, 233; T.
tonsurans, 232 ; T. versicolor, 234,
235

Tissues, the, influence of bacteria on, 6

Toxalbumins, 6

Toxines, 6

Toxopeptone, 7

Trachoma, Mic. of, 227

Transmission, 91 ; by air-passages,
93 ; digestive canal, 93 ; skin, 94 ;
peritoneum, 94

Trichophyton macrosporon (megalo-
sporon), T.microsporon, 236 ; T. ton-
surans, 223, 232, 236

* Trouser-leg culture,' 145

Tuberculine, Koch's, 7, 8, 218

Tuberculocidine, 218

Tuberculosis, Bac., 206, 275 ; action
of light on, 296 ; in birds, 209 ; pre-
paring pure cultures of, 207-9 ;
staining of, 73, 210 ; in sections,
217

Tubular buds, in moulds, 10

Tussis convulsive, Bac., 275

Tympanum, micro-organisms of, 244

TYNDALL, method of air-analysis, 102 ;
of sterilisation, 18

Typhoid fever, Bac. of, 148 ; action of
light on, 296

Typhus abdominalis, 148. See Typhoid

Tyrogenum, Spirillum, 183

Ulna, Bac., 239

Undula, Spirillum, 153

UNNA, examination of skin for micro-
organisms, 90, 224

Urea, Bac., 260-; Mic., 107

— candidus, Staph., U. liquefaciens,
Staph., 260

Urine, micro-organisms of, 258 ; yeasts
and moulds in, 259

Urobacillus Freudenreichii, U. Mad-
doxii, 260

Urobacteria, 259

VACCINATION against cholera, Haff-
kine's, 283 ; principle of, 283 ; re-
sults of, 284

Vaccines, anti-cholera, preparation of,
283 ; attenuated, 285 ; exalted, 284

no

BACTERIOLOGY

Vacuoles in cells, 12, 288
Vagina, micro-organisms of, 222-3
Vaginalis, Bac., 223, 228
Ventriculi, Sarcina, 246
Versicolor, Mic., 105
' Vibrion septique,' 160
Vibrios, 2, 4

— aureus, 121 ; V. cholera, 139 ; V.
flavescens, V. flavus, 121 ; V. Met-
schnikoffi, 147 ; V. proteus, 145

Vienna, water-supply of, 136
Vinegar ferment, 191
Violaceus, Bac., 132
Virulence, 6
Virus, exalted, 283
Viscosus, Mic., 192

— butyri, Bac., 183 ; V. cerevisice,
Bac., 192 ; V. lactis, Bac., 184 ; V.
sacchari, Bac., 193

Viticulosus, Mic., 106

WAFERS, cultivation on, 53

WARD, researches on the action of

light on bacteria, 297-300
Warm chamber, 20. See Incubator
Water, analysis of, 122 ; W. bacteria,

122; W., drinking, 124; ground, 155;

methods of examining, 125 ; Kirch-

ner's, Pfuhl's, 126 ; other methods,
127 ; micro-organisms in, 122, 124,
153 ; spirilla in, 153 ; spring, 124 ;
variations in, depending on source,
124

WEICHSELBAUM, method of staining
tubercle bacilli, 75 ; pneumococcus,
271

WEIGERT, modification of Gram's me-
thod, 78, 89

WELZ, method of air-analysis, 99

Whooping cough, 275

WOLFFHUGEL, counting apparatus, 125

' Wurzelbacillus,' 133

Xerosis, Bac., 232

YEAST, 12, 104 ; black y., 105 ; y.-cells,
12 ; pink y., white y., 105

ZIEHL'S solution, 70

— and NEELSEN'S method, 74

Zooglraa, 4

Zopfi, Bact., 251

Ziiernianum, Bact., 137

Zygospores, 194

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