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http://www.archive.org/details/cu31924000225361
BACTERIOLOGY
MANUAL
OF
BACTHRIOLOGY
for Practitioners and Students
WITH ESPECIAL REFERENCE TO PRACTICAL METHODS
BY Dr 8. L. SCHENK
PROFESSOR EXTRAORDINARY IN THE UNIVERSITY OF VIENNA
TRANSLATED FROM THE GERMAN
(BY THE AUTHOR’S PERMISSION)
WITH AN APPENDIX
By W. R. DAWSON, B.A., M.D. Univ. Dust.
LATE UNIVERSITY TRAVELLING PRIZEMAN IN MEDICINE
WITH 100 ILLUSTRATIONS, PARTLY COLOURED
LONDON
LONGMANS, GREEN, AND CO.
AND NEW YORK: 15 EAST 16" STREET
1893
All rights reserved
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TRANSLATOR’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
v1 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 formule 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.
Durum: June 1893.
AUTHOR’S PREFACE
+00
Tue 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
vill 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.
1
* * * * * *
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.
8. L. SCHENK.
! The words omitted have reference to the German edition.
CONTENTS
CHAPTER I
GENERAL MORPHOLOGY AND BIOLOGY OF
MICRO-ORGANISMS
‘ PAGE
IntRopucrory—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
—Alge—Protozoa—Examination of Micro-organisms . 1
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 REacenTs—Microscope—Steam Steriliser
—Incubator — Thermo-regulator — Schenk’s Thermo-regulator —
Meyer’s Thermo-regulator—Girtner’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 Cr cee aa Stains—Other Utensils
—Centrifugal Machines . 3 5 ‘ 5 15
CHAPTER III
NUTRIENT MATERIALS AND METHODS OF CULTIVATION
Nurrient Merpia—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
ve BACTERIOLOGY
w= PAGK
Gelatine and Agar, &c.—Blood Serum—Modifications of Serum—
Eggs of Birds—Plovers’ Egg Albumen—Hens’ Eggs—Potatoes—-
Rice, Bread, and Wafers. Mopzs or Cuntivation—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—Ot Spores—
DrcoLorisIneé AcENts—Koch and Ehrlich Method of Staining—
Ziehl and Neelsen’s Method—Ehrlich’s—Giinther’s—Weichsel-
baum’s— Fraenkel’s—Gabbet’s — Method of Pfiuhl 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
Paraffine. On Sramine or Szcrroxs—Unna’s Drying-on Process—
Combination of Staining Methods—Kiihne’s Methyl Blue Method
—Koch’s Method — Létiler’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.
ExprRiments on Livine Anrmats—Transmission of Micro-organisms
to Animals—Infection by the Air-passages—Infection by the Diges-
tive Canal—Subcutaneous Infection—Intrayenous Infection—In-
fection into the Anterior Chamber of the Eye : : . 65
CHAPTER V
THE BACTERIOLOGICAL ANALYSIS OF AIR
Micro-or¢anisms IN tHE Arr—Simple Methods of Examining Air—-
Pouchet’s Method — Miquel’s— Emmerich’s — Welz’s— Hesse’s —
Method of Strauss and Wurz—Petri’s Method—Tyndall’s Method
—Penicillium glaucum—Brown Mould—Yeast—Micrococcus ra-
diatus—Micrococcus versicolor —Micrococeus cinabareus—Micro-
coccus flavus tardigradus — Micrococcus candicans — Micrococcus
viticulosus—Micrococcus Urew—Micrococcus roseus—Diplococcus
citreus conglomeratus — Micrococcus flavus liquefaciens and
CONTENTS
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 fuscus; ruber; vulgatus—Bacillus liodermos —
Bacillus melochloros—Bacillus multipediculosus—Bacillus nea-
politanus—Atmospheric Spirilla
CHAPTER VI
THE BACTERIOLOGICAL ANALYSIS OF WATER
Mrcro-organismMs or Water—Filtration and Filters—Variations in
Water Depending on Source—Iixamination of Water—Pfuhl’s
Method—Kirchner’s Method —Other Methods —Micrococcus aqua-
tilis—Micrococcus agilis —Micrococcus fuscus—-Micrococcus luteus
—NMicrococcus aurantiacus—Micrococcus fervidosus—Micrococcus
carneus—Micrococcus concentricus—Diplococcus luteus—Bacillus
fluorescens liquefaciens and Bacillus nivalis (Glacier Bacillus)
—Bazcillus 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 Ziirnianum—Bacillus membranaceus ame-
thystinus — Bacillus indigoferus — Bacillus ianthinus — Bacillus
ochraceus—Bacillus gracilis—Bacillus sulphydrogenus—Bacillus
ot 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
CHAPTER VII
BACTERIOLOGICAL ANALYSIS OF EARTH AND OF PUTRE-
FYING SUBSTANCES
MICRO-ORGANISMS IN THE Sorr—Method of Examination—Bacillus
mycoides (Earth Bacillus) Bacterium mycoides roseum—Bacillus
radiatus — Bacillus spinosus— Bacillus liquefaciens magnus—
Bacillus scissus—Clostridium fcetidum—Bacillus edematis ma-
ligni—Bacillus of Tetanus—Streptococcus septicus—Bacillus An-
thracis—Plasmodium Malarie—Other Micro-organisms of the
Soil. Awnatysis or Purreryinc Susstances—Differences in Putre-
factive Processes—Bacillus fuscus limbatus—Proteus—Proteus
vulgaris — Zenkeri — Mirabilis — Hominis—Capsulatus— Bacillus
saprogenes—Spirillum concentricum—Spirillum rubrum
96
122
Xil BACTERIOLOGY
CHAPTER VIII
MICRO-ORGANISMS IN ARTICLES OF DIET
PAGE
Methods of Examining Different Foods. Examination or MiLnK—
Methods—Bacillus lacticus (Bacillus acidi lactici)—Micrococeus
acidi lactici—Clostridium butyricum (Bacillus amylobacter)—
Micrococcus acidi lactici liquefaciens—Oidium Lactis—Bacillus
butyricus—Bacillus Butyriviscosus; fluorescens—Spirillum tyro-
genum—Bacillus Lactis viscosus—Bacillus Lactis pituitosi—Bacil-
lus actinobacter—Bacillus fetidus Lactis—Bacillus cyanogenus—
Bacterium Lactis erythrogenes—Sarcina rosea —Micrococeus of Bo-
vine Mastitis—Other Pathogenic Bacteria in Milk—Saccharomyces
ruber—Bacillus caucasicus (Dispora caucasica, or Kephir Bacillus).
Examination or Oruer Artictes or Diet—Bacillus megaterium—
Bacillus Aceti—Bacillus indigogenus—Pediococcus Cerevisie—
Sarcina Cerevisie—Micrococcus viscosus—Bacillus viscosus Cere-
visiee—Bacillus viscosus Sacchari—Moulds on Articles of Food—
Aspergillus niger; albus; glaucus; flavescens; fumigatus—Mucor
Mucedo; rhizopodiformis ; corymbifer; ramosus . - 3 . 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 Gonorrhea—Bacillus of
Syphilis — Bacillus Tuberculosis — Bacillus of Glanders — Other
Microbes of Pus. F : 2 : é . . 196
CHAPTER X
BACTERIOLOGICAL EXAMINATION OF THE ORGANS AND
CAVITIES OF THE BODY AND THEIR CONTENTS
MIcro-orGANISMS OF THE Livinc Bopy—I. Tur Sxin-—Micro-organisms
of the Skin—Methods of Examination—Diplococcus subflavus—
Micrococcus lacteus faviformis—Diplococcus albicans amplus—
Diplococecus albicans tardus—Diplococcus citreus liquefaciens—
Diplococcus flavus liquefaciens tardus—Micrococcus hematodes
—Micrococeus of Trachoma—Diplococeus 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
Scheenleinii)—Microsporon furfur. Nore: Trichophyton micro-
sporon—Macrosporon . : : 292
CONTENTS Xill
: CHAPTER XI
THE ORGANS AND CAVITIES OF THE BODY AND THEIR
CONTENTS—(cont.) II. THE DIGESTIVE TRACT
PAGE
THE Caviry or THE MoutH—Micro-organisms of the Mouth and their
Examination—Leptothrix—Bacillus buccalis maximus—lIodococcus
—Micrococcus salivarius septicus and Bacillus salivarius septicus—
Bacillus ulna—Bacillus Gingive—Bacillus Diphtheria—Spirillum
Miller—Spirochete Dentium (Denticola)—Vibrio rugula—Fungus
of Thrush—Other Bacteria of the Mouth. Tar Tyaeanuu. Tue
Stomach—Micro-organisms of the Stomach—Sarcina Ventriculi—
Micrococcus tetragenus mobilis Ventriculi— Bacterium Lactis
aerogenes—Bacillus indicus—Tue Intestine —Intestinal Micro-
organisms —Micrococcus aerogenes—Bacillus putrificus Coli—
Bacillus coprogenes foetidus—Bacterium Zopfi—Bacterium aero-
genes, Helicobacterium aerogenes, and Bacillus aerogenes—Bacillus
Dysenterie—Bacillus of Fowl Cholera —Other Intestinal Bacteria. 237
CHAPTER XII
THE ORGANS AND CAVITIES OF THE BODY AND THEIR
CONTENTS—(cont.) III. THE FECES AND URINE
Tur Facrs— Composition and Modes of Examining—Bacillus subti-
liformis — Bacillus albuminis— Bacillus cavicida— Micrococcus
tetragenus concentricus. Tue Urntye—Micro-organisms of the
Urine—Yeasts and Moulds in Urine—Urobacteria— Staphylococcus
Ure candidus—Liquefaciens—Micrococcus Urex liquefaciens—
Bacilius Uree—Urobacillus Freudenreichii—Madoxii—Micrococ-
cus ochroleucus—Streptococcus giganteus Urethre—Bacterium
sulphureum—Bacillus septicus Vesice—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
Tur Nasat SEecreTION—Micro-organisms in the Nasal Secretion—
Micrococcus cumulatus tenuis—Micrococcus tetragenus subflavus—
Micrococcus Nasalis—Diplococcus Coryz#—Staphylococcus cereus
aureus—Bacillus foetidus ozenze—Bacillus striatus albus et flavus
—Bacillus of Rhinoscleroma—Bacillus capsulatus mucosus— Vibrio
Nasalis — Other Nasal Bacteria. Ture Resprratory Passages —
Micro-organisms of the Respiratory Passages—Sarcina Pulmonum
XIV BACTERIOLOGY
PAGE
— Sarcina aurea — Diplococcus Pneumonie — Pneumobacillus
Friedlanderi—Micrococeus tetragenus—Bacillus aureus—Tubercle
Bacillus and Actinomyces—Bacillus Tussis convulsive—Bacillus
pneumosepticus. V. BacterirotoaircaL EXAMINATION oF THE BLoop—
Micro-organisms in the Blood—Methods of Examination—Influ-
enza Bacillus — Bacillus Endocarditis capsulatus — Bacillus of
Swine Erysipelas—Bacillus murisepticus—Spirochete Obermeieri
—Protozoa in the Blood . . ‘ . ‘ ‘ e » 264
APPENDIX (by the Translator)
A. VaccINaTION AGAINST AstaTIc CHoLERA—Principle of Anti-cholera
Vaccination—Preparation of Vaccine—Results of Vaccination.
B. Parastric Prorozoa— Pathogenesis in Protozoa—Coccidium
oviforme—Ameeba Dysenterisee-—Protozoa in Carcinoma —Protozoa
in other New Growths, &c. C. THe Action or Licut on Micro-
orGANisMs —Action of White Light—Action of Coloured Light —
Mode of Action of Light—Applications in Nature. D. Ap-
DITIONAL MrtTHops anpD FormuL#—Fixing Methods—The Gum
Freezing Method—Staining Formule . ‘ : . . 283
INDEX ‘ ‘ : - ; . . . 303
GENERAL MORPHOLOGY AND BIOLOGY OF MICRO-ORGANISMS
BACTERIOLOGY
CHAPTER I
Errata
Page 11, line 5 from bottom, for CRENOTHIE read CRENOTHRIX
236, line 4, the words Notr sy 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 Phliiger’s Archiv, xxx,
p. 95.
302, lines 5-3 from bottom should read as follows: minutes in Liffler’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,
the power of adapting themselves to altered conditions of life,
and of flourishing externally as wellas internally. With fur-
ther reference to their relation to the human or animal frame
we speak also of ectoyenous 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 acrobes; but a large number
B
X1V BACTERIOLOGY
PAGE
— Sarcina aurea — Diplococcus Pneumonise — Pneumobacillus
Friedlenderi—Micrococeus tetragenus—Bacillus aureus—Tubercle
Bacillus and Actinomyces—Bacillus Tussis convulsive—Bacillus
pneumosepticus. V. BacrertoLoaica, ExaMINaTION OF THE BLoop—
Micro-organisms in the Blood—Methods of Examination—Influ-
enza Bacillus — Bacillus Endocarditis capsulatus — Bacillus of
Swine Erysipelas—Bacillus murisepticus—Spirochete Obermeieri
—Protozoa in the Blood . 3 3 : ‘ ; i ‘ » 264
APPENDIX (by the Translator)
A. VAccINATION AGAINST AsiaTIC CHo~tERA—Principle of Anti-cholera
Vaccination—Preparation of Vaccine—Results of Vaccination.
B. Parasrric Prorozoa— Pathogenesis in Protozoa—Coccidium
oviforme—Ameeba Dysenterie-—Protozoa in Carcinoma — Protozoa
in other New Growths, &. C. Tue Action or Licut on Micro-
orcanismMs —Action of White Light—Action of Coloured Light—
Mode of Action of Light—Applications in Native TN 4n
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 wellas internally. With fur-
ther reference to their relation to the human or animal frame
we speak also of ectoyenous 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 acrobes; 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, alge, 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, staphylococci; 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 sarcine ; 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 wbrios, spirilla in the more restricted
sense, and spirochete. The vibrios usually form strings of
cells which strongly resemble spirilla; the spirochete are
distinguished by their flexibility.
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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
Slender bacilli
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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 sarcine; but various bacilli
also, such as Pfeiffer’s capsule-bacillus and the Bacillus
r2
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Knobbed bacteria with
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4 BACTERIOLOGY
capsulatus mucosus of Fasching, are furnished with cap-
sules. By coalescence of the cell-capsules conglomerations
of cells are formed which are called zooglaa, 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 jisston, hence
their name of Schizomycetes, 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
sarcine.
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 3)
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 prodigiosus a smell resembling that
of trimethylamine.
Micro-organisms have also the property of producing
changes in the medium on which they are cultivated hy 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 toz-
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
Roux, 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 toxalbumains 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.
Roemer procured his extracts of the Bacillus pyocyaneus
and pnewmobacillus 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-
KOCIVS TUBERCULIN—MOULDS 9
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
-Acicular crystals of
calcium oxalate
Sporangium
—- Columella
Hyphe
A i
Fic. 2.—Mucor Mucrpo. (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
- Hyphe
Mycelium
Fic, 3.— ASPERGILLUS GLAUCUS.
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,
Conidia
Sterigmata
Basidia
Hyphe
aarss-— Mycelium
Fic. 4.—PENiciLiumM GLAUcUM. Magnified 400 times. (After Baumgarten.)
spoken of as myceliwm, and possessing special seed-bearing
organs called hyphe or thallus, from which the moulds
derive the name of Hyphomycete. According to the form
of the seed-organ they are divided into mucorince, asper-
gillinee, penicilliacee, and oidiacee.
Conidia
Hyphe
Fic. 5.—Oipium Lactis. (After Baumgarten.)
In the mucorinee, or headed moulds, the ends of the
hyphz 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 aspergillinee (knob-moulds) have the knobbed ends
MOULDS 11
of the hyphe covered with a variable number of spore-
carriers, or sterigmata, from the extremities of which the
spores divide off in rows (fig. 3).
The hyphe of the penicilliacee (pencil-moulds) are
branched, which is not the case with the mucor and asper-
Group of buds $20.
(gemmation )
@
Mother-cell ge” e e
—— Vacuole
Fic. 6.—YEAST CELLS (Saccharomyces Cerevisie). 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
Fic. 7.—CRENOTHLE KUnNIANA. Magnified 600 times. (After Zopf.)
The oidiacee are distinguished by the fact that the
hyphe 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 lacune, 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.
Alge.—Of these the cladothriz, crenothrix, and beg-
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 gregarine, psorospermti, 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 out 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 malania
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-
Portion ofa
branch
Branching
plant
Unsegmented
spiral
Unsegmented
spirocheeta
Segmentation ——+
into eect ge Fas
| rods
Segmentation
into short
rods
Segmentation pee ievae
into rods
— Branch resembling
Spirilla-like spirocheta
coils
Fie. 8.—FoRMS oF VEGETATION OF CLADOTHRIX DicnoroMa. (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
CHAPTER 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
_ avery 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 effiviency 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-
infected 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 Jong 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
Fic. 9.—Hor-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.
Thermometer
Lid
Water-gauge —
Ring of flames —
Fic. 10—Kocu’s STEAM STERTLISER.
For laboratories which are not supplied with gas,
Budenberg’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
C
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° ©. 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 perchloride, &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 ieee 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 méthod 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,
@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 +4,th per cent. solution of corrosive
sublimate, is absolutely necessary.
APPARATUS AND REAGENTS
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 thermo-regulator 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! 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-
Thermo- —
regulator
(i
Felt coating 8 Water-gauge
yg
Fic. 11,.—Incusaror.
tions) maintains the thermometer at a constant temperature.!
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.]—Tr.
! [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.]—Tr.
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—aA 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
Fic. 12.—ScuENk’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 com
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
em. 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 THERMO-REGULATOR 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 exit
~ Aperture of entrance
“he
Regulating screw
Mercury vessel —~~—
Fig, 13.—ALtMANN'’S THERMO-REGULATOR,
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
Fie. 14.-—BAUMEYER'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.—lf 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-WAIER 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- Fic.15—Hor-Warer Fivrmr (Heatep BY
A RING BURNER).
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
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 - Glass plate
glass
evel
Levelling screw ——__ Glass dish
Fic, 16.—LEVELLING APPARATUS FOR MAKING PLAYE-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
Tic. 17.—HoLiow SLIDE. Fic. 18.—Wme CRATE.
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-serum.
Corrosive sublimate in solutions of 1 to 1,000 and
carboliec 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 parafine,
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, eosine, safranine, and
dahha are aniline colours which are sufficient for nearly all
investigations. Besides these, however, carmine, picro-
carmine, picric acid, hematoxyline, 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,' &c.
Distilled water, alcohol, ether, xylol, and oil of turpentine
complete the equipment of reagents.
Other utensils Platinum wires with loops sealed into
' [Practically identical with Hatractum Hematoxryli, B. P.]—Tr.
MISCELLANEOUS APPARATUS 31
glass rods—best done over a Bunsen 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.—Kocu's INJuCTING 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, «ec.
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
Fic. 20.—STENBECK’S CENTRIFUGE (after Jaksch).
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).
CENTRIFUGAL 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 —
Axis H——___-_—-—— Hole for the end
> of the catgut
Test-tube a
Bottom of Des
case
Fic. 21.—GAnTNER’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
D
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
CHAPTER TII
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 Fie. 22.
ERLENMEYER’S FLASK.
of Erlenmeyer are particularly useful (fig.
22). The media, especially bouillon or broth, after being
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
D2
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
Léffler’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 hemoglobin, 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 grams
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 slight 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 soif 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 4 per cent (5 grams) of extract of meat and 8 per
cent. (30 grams) of dry peptone, or instead of extract of
meat and peptone, 2 to 3 per cent. (20-80 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 grms. 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! 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.
Ifa 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.}—Tr. \
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 own 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 80 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 73 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 wheat, 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 alge 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 45
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 Richter’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 8 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 ina 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. Hsmarch recommends the addition of some
gum arabic to prevent slipping away from the surface of
the glass.
The mass of agar is clear and transparent while liquid,
but 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 orm.
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
erm. peptone, 90 grm. sugar, 18 grm. common salt, 9 grm.
sodium phosphate, 9 grm. ammonium sulphate, and 25
erm. 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 Heiler 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 com. 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 for a 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
placente.
Fic. 23.—APPARATUS FOR THE INSPISSATION OF BLOooD 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 propor-
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 Huppe 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 potash 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
E
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
ege 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 sterilisation 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 Huppe and Heim, hen’s eggs
may themselves be used with advantage as nutrient media.
POTATOES ol
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
micro-organisms, and one, moreover, which secures their
development in a quite characteristic way, is the potato.
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 4 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 dises which fit into small
sterilised boxes of glass, each individual disc can be used as
BE2
By BACTERIOLOGY
a medium upon which to cultivate (Esmarch’s potato dises) ;
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 Krlenmeyer’s flasks
(potato pap); and in this way a useful culture medium is
obtained after proper sterilisation. Hisenberg 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
must be rendered alkaline with sodium bicarbonate solu-
tion for certain micro-organisms, whose growth demands
alkalinity.
Rice, bread, and wafers.—Milk vice 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 mosi
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
café aw 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.
Moves 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.
TIE PLATE PROCESS 55
During these operations some plates should be under-
going sterilisation in the hot-air steriliser at a temperature
of 180°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.
Fis, 24,—CAsE OF SHEEI-IRON FOR HOLDING 'lHE PLATES.
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. Ina 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
Fie, 25.—Moisy 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 MICRO-ORGANISMS OT
micro-organisms 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
Fic, 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 an
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 Uniia,
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.
Roll 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 Ginther 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
serum is only used in the solid state, and is principally
adapted for surface or streak cultures (Strichculturen). In
order, however, to render it available for plate cultivation,
Huppe mixes it with an equal quantity of a warm solution of
agar; and it may also be suspended in gelatine. Unna in-
ereased 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.—F or 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 0-1 per cent. of
resorcine, or to liquefied nutrient agar 4 per cent. of formic
acid or of sodium sulphindigotate.
CULTIVATION OF ANAEROBES 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 the 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 at
about 15 em. 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 (Stichcultwren) 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-
robie cultures. This kind of cultivation, which is recom-
mended by Huppe 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. Kral
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, Gunther
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
CHAPTER 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 w, = 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
F
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, ag detailed above, and their
motility 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 heen 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.
Gunther 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
r2
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 itis 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 itis 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 Abbé’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 Abbé’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 SOLUTIONS 69
remove the diaphragms, and to examine with the full power
of the Abbé’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.—F or 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
jong, as it rapidly decomposes. To make it a test-tube
is filled with water which is shaken up with 1 to 2 ¢.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 veryslightly. 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 Zielhl’s solution,
and its composition is as follows :
Crystallised carbolic acid j : 5:0
Water . : 3 : , 4 100-0
Alcohol . F ‘ , : . . 100
Fuchsine * 3 < : : é 1:0
According to Kihne’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 ¢.cm. of a 10
per cent. potash solution, and Loffler 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.
Incertain 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, Léffler 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 toa 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 4 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 (Ziehl'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 darkred. The hay bacillus, and especially the Bacillus
megathertum, 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, and then washed off.
The spores are dark red in the interior of blue or green
bacteria.
DeEcoLoriIsing 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-EHRLICH METHOD OF STAINING 73
reagents. These are known as decolorising agents, and
include amongst them water, alcohol, 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 83 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.
Zieh] 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 88 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.—-F or 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.
Giinther’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 20partsnitric 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
ina 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, 80 grms. water,
and 20 grms. nitric acid, in which it remains ? 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 fuchsine 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 8 ¢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 8 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 Friedlander shows no staining after going
through the process, and similarly the bacilli of Cholera
Asiatica, typhoid fever and glanders, gonococct, 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).
Giinther’s modification of Gram’s process.—Not only pure
alcohol, but algo 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 8 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 Gram’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 the 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 Abbé’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.
Ahalf 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 methods
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-
_G
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! 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
' Sold by Siebert 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
Fic. 27.--SECTION STAINER FUR PREPARATIONS IMBEDDED IN CELLOIDINE.
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.
G2
84 BACTERIOLOGY
The sections are transferred one by one to xylol, in order
to extract the paraffine from them, and are thence brought
into aleohol 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 toa 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 SraInING oF SEcTIONS
The staining of sections is carried out after various
mathods, 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.
Unna’s drying-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 are 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 ‘septicemia 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.
Liffler’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-
GRAMS 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.
Gram’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 stainig 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.
Gunther’s modification, which is characterised by the
exposure of the sections, after decolorising in alcohol, to
the action of a 8 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 Kithne 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
ce.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 secticns 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 aleohol 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 alcchol, 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 WEIGERTS METHODS 89
differentiate them, and after clearing with an etherial oil and
removal of this with xylol, are put up in Canada balsam.
In the 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 4 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 Kithne’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 bora 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 aleohol, 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 ' 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 method.—Noniewicz combined Loffler’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
tropeoline. 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.
EXPERIMENTS oN Living 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 pharmacopeia 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—!
Ist. That its presence can be detected with the micro-
scope in all cases of that disease.
Qnd. 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 Einfiihr. in das Stud. der Bakteriol.,
pp. 139 e¢ seq., 2nd ed.]—Tr.
INFECTION BY THE DIGESTIVE CANAL 03
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 esophageal 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 Rietsch 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.cm. 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 cesopha-
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
ee Opening
————eeee for the
admission
of air
Glycerine
plate
Aspirator -—————
to catch
the water
Ln nnn M00 UU TTT
CETTE FTCA ATTA UTC TTT
Fic. 28.—Poucuet’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 aéroscope 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
a
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 dust 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 caesar
Tube connected with —
the aspirator
Fic. 29.—MIQUEL’S APPARATUS FOR EXAMINING AIR.
from this the number spread over the whole plate is caleu-
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 80 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
Fic. 30.—EMMERICH’s APPARATUS FOR EXAMINING AIR.
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 8 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 Rubber cork
India. Ae
rubber a)
cap —-—_.—______ Aspirator
tube
'
Aspirator
Fic. 31—HEssh’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
¥ Lateral tubuluie
Vessel filled with gelatine
Fic. 32.—AU-TESTING APPARATUS OF SyRATss AND WURzZ.
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 Ksmarch’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 been 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
orain 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 ——— : = Wire netting
Receiving bottle
Fie. 33.—PErRi’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 uses glass wool instead of cotton.
With the aid of these methods, moulds, yeasts, micro-
cocet, 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 Penicilliwm 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 hyphe (Fruchthyphen) 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
hyphe 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 hyphe, 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,
thé 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
hyphe 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 IX 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 p to
3 » long, 2 » broad).
Micrococcus radiatus.—The JJicrococcus radiatus, iso-
lated by Fligge, 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 Fligge, 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
Fligge, 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 Flugge 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 (Flugge) 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 wilh 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-
MICROCOCC] IN THE AIR 107
work of fibres likewise runs into the substance of the gela-
tine without liquefying it.
Micrococcus uree.—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 ie Sal MGROSORSER TRE:
processes in urine, owing to its sina aderate te
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
gonorrheal 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 Fligge. The former has
larger, the latter smaller elements frequently arranged as
diplococei, 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. Italso 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
SARCINA IN THE AIR 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 yellow granules. A thickish
deposit of a fine yellow colour appears uponagar. Cultures
on potato are sulphur-yellow, and confined to the place of
inoculation. Gelatine is sometimes liquefied slowly.
Staphylococei——-The Staphylococcus pyogenes was fully
described by Rosenbach, by Ogston, and by Passet. Its
distinctive characteristic is its power of causing suppuration,
and it may consequently be described as a specific pus-
coceus, being constantly found in suppurative’ processes.
There are distinguished, according to colour, a Staphylo-
coccus pyogenes aureus, a Staphylococcus 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. 85). Those
cells, however, which are not in-
cluded in the clusters possess the
. § Be ots , power of moving with tolerable
eos ~ “@.. ° activity. The individual cells take
re aoe up all the different aniline dyes,
Fig, 35.—SraruyLococ grow even at ordinary tempera-
Parra ae tures, though more energetically at
degrees of heat approaching that of the human body, and,
if added to sterilised milk, precipitate the caseine.
The Staphylococcus pyogenes aureus grows very quickly
on the gelatine plate at the temperature of an ordinary
Smallest islets
An older
islet, lique-
fying in the
centre
Fie. 36, Fie. 37.
ISLETS OF STAPHYLOCOCCUS 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 AUREUS Ii
and this gradually advances deeper along the thrust-canal,
while, as the funnel-shaped liquid area enlarges, the cocci
; " __. Liquefied part of the
Liquefied part gelatine
Collection of bacteria
Needle-track in the __Il
non-liquefied part
Mass of bacteria collected
at the bottom of the
liquid funnel
iFic, 38.—THRUST-CULTURES IN GELATINE OF STAPHYLOCOCCUS PYOGENES AUREUS;
—TWO DIFFERENT FORMS OF GROWTH,
sink to the bottom and begin to take on colour (fig. 88).
When, however, the culture is exposed, not to ordinary tem-
112 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 cireu-
lation cause inflammation of joints and abscesses in the
kidneys and myocardium. According to Orth, Wyssoko-
witsch and Ribbert, they set up an ulcerative 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 ERYSIPELATIS 115
Leber isolated the active principle from the cultures in
the form of a crystalline body, to which he gave the name
of phlogosin, and which, if injected in small quantity, leads
to suppuration without the presence of micro-organisms.
Christmas obtained a pyogenic 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 arabbit’seye. 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 colonies 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, Frankel, and Von EHiselsberg, 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 sets
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 was
formerly classed with the micrococci. The individual rods
are motile. On acid nutrient media, however, they expand,
according to Kubler, into larger bacilli, which also possess
the power of motion. They form spores. On gelatine plates
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-
| —— Funnel-shaped faction
area of lique-
faction
Needle-track in
the unliquefied
part
Fic. 39,—Tarust-CULTURE IN GELATINE Fie. 40.—THnRust-CULTURE IN GELATINE
OF BACILLUS SUBTILIS (THIRD DAY). OF BACILLUS PRODIGIOSUS (FOURTH DAY).
red colour. The thrust-culture liquefies from the surface
down, and soon a funnel-shaped area of liquefaction is
12
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
erowth 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 £, and another at Fr. 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 (Flugge), 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. They
liquefy gelatine very quickly during their growth, whether
on the plate or in thrttst-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 rulgatus
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
Fic. 41.—IsLeT or BACILLUS MESENTERICUS VULGATUS ON a GELATINE 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.—F lugge 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 Von Schrétter, 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
srowth 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’ ege albumen it grows with a splendid emerald
ereen 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 Flugge,
shows small thin immotile rods. The colonies on a gela-
BACILLUS MULTIPEDICULOSUS 119
tine plate appear under a low power as circular, sharply-
defined discs with radiating processes, resembling insects
Hour-glass
depression
Superficial
Liquefied
coating
portion
a7; —- Needle-track
Needle-track
Fic. 42.—THRust-CULTURE IN GELATINE Fic. 43.--THrust-CULTURE IN GELATINE
or BACILLUS MELOCHLOROS (SECOND OF IMMEnICH’s BACILLUS (Bac. Nea-
DAY). 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 ‘ mualti-
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 feces. 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
CHAPTER VI
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 which stand the cholera bacillus de-
seribed 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
by 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 diatomaceous 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 filters 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
. aL Porcelain tube
= ~
“~ Enameled discharge-pipe
Fic. 44.—KaoLin FILTER, ON 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
septicemia 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
aérated 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 Rubner, the natural filtration through the
soil under which the spring water les purifies it so
thoroughly that it comes to the light of day in an almost
WOLFFHUGEL'S APPARATUS 125
sterile condition, only containing two or three germs per
cubic centimeter.
Examination of water—For purposes of examination
3 to 1c¢.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
Fic. 45.—WoLFFHUGEL's COUNTING-PLATE.
The counting apparatus (Wolfthugel’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
126 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 le 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
class 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 86 cm. length of glass tube,
of the diameter commonly used for making connections
METHODS OF EXAMINATION 127
between apparatus, is bent toa 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 Girtner’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, Ali
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 Ali 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 arose-
red deposit both on agar and potato.
Micrococcus fuscus, described by Maschek, consists of
immotile cocci which frequently have an elliptical form.
Round 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 zooglea. It appears in irregular colonies on the
gelatine plate, while in thrust-cultures a yellow deposit is
MICROCOCCI IN WATER 129
seen on the surface, and granules 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 the
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. Round 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.
Gani
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Fic, 55.—TYPHOID BACILLI (‘ SPIDER-
Fic. ea eee ee PURE Gas hhe 1,100 times.
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 cadaverine,
putrescine, and choline. Pouchet also extracted toxines
from the actual cholera stools.
Bacillus of typhoid fever.—The bacilli of typhoid or en-
teric fever (the Typhus abdominalis of the Germans) are met
' [For an account of recent researches on cholera vaccination, see
Appendix ]—Tr.
BACILLUS OF TYPHOID FEVER 149
with in water as well as in the feces and organs of patients
suffering from the disease. They were described by Eberth
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 Léffler 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 ‘the 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
!
|
Needle-track
Fic, 56.—THRUS1T-CULTURE IN GELATINE OF THE BACILLUS OF TYPHOID FEVER.
NintH 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. H. Frankel and Ali 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.
Rodet 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 erms. 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 feetus.
[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.—Rodet and Roux 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
SPIRILLA 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
erape-sugar, and then generates a gas consisting of hydrogen
and carbon dioxide. Rabbits succumb to a subcutaneous
injection in from one to three days, with the symptoms of
diarrhea 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 sufticient point of distinction
between these two is, that the growth of Bacterium colt
we
PTw
~
Fic. 57.—SPIRILLUM UNDULA, WITH FLAGELLA. Magnified 800 times, (After Loftler.)
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 viticulosus, Staphylococcus pyogenes awreus, 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.
156
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 Reimers, 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 brown 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).—Fligee 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
-Fic. 58.—THRust-CULTURE IN GELATINE OF BACILLUS MYCOIDES (FourrH 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 diminish in size from above down-
wards (fig. 58). Ramifications 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.—Liuderitz 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 Liuderitz 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,
Lideritz 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 fetidum.—By the name Clostridia 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. 1).
The Clostridium fotidwm 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 edematis maligni—As long ago as the year
1872, Coze and Feltz found in their researches on septicemia
a micro-organism which was more fully described by Pasteur,
and which obtained the name of vibrion septique, or Bacillus
septicus. Koch called it the Bacillus adematis 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 cedema 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 edematous fluid and on the
surface of the organs, but not in the blood-vessels, whereas
BACILLUS OF MALIGNANT GiDEMA 161
the bacilli of anthrae 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 R. 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 cither 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 cedema 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
ou 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 89° C.; under 16° C. no development takes place. The
‘spores lie at one end of the rods, and ere very resistent.
%
Gas bubbles in the colonies =f jan
:
Fluid globules (colonies) -
Fie, 59—ANAEROBIC CULTURE OF THE BACILLUS OF MALIGNANT CEDEMA IN
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 cedema 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 ease of the mouse the bacilli effect an entrance
from the tissues into the circulation, probably by penetrat-
ing the walls of the blood-vessels.
Fic. 60.—IsLer or THE BACILLUS OF MALIG- Fie. 61—Teranus Bacinnl wirH Ter-
NAnr CipEMA (Bacillus septicus) oN A MINAL SPORES.
GELATINE PLATE. (After Macé.)
According to Penzo the bacilli of malignant cedema,
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 vulgaris.
The cedema 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
Rattone had already established the fact that tetanus is
M 2
164 BACTERIOLOGY
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
v1@.62—AxarnopicGua. 20 hour to an hour to a water-bath
Fear enone rue heated to 80° C.,in order to kill the micro-
Aft Fraenkel and c é 3
Pioiifer.) organisms which have grown along with
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 when 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 antiirax bacilli
go
TN 4
Solitary sina ee
f, ~ Spaces between the
rods of a chain
Bacilli containing spores
Fic. 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 well brought out by double staining with
Bismarck brown and methy! 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 which becomes visible in
BACILLUS 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
= the margin
G
Hair-like arrange-
ment of fibres ‘i (Ny Ke ip:
Fig. 64.—CoLoNY OF THE ANTHRAX BACILLUS ON A GELATINE PLATE, RESEMBLING
TressEs OF Hamm (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
Fic. 65.—THRUSYT-CULLURE 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 MALARIA 169
higher than their normal temperature. If the bacilli alight
upon a spot invested with epithelium, they 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 Hiselsberg,
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.
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