a ihe Git au J ay ade ye Bie eae a Taig LIBRARY NEW YORK STATE VETERINARY COLLEGE ITHACA, N. Y. BOUGHT OF ea - t ; oo. . ; a | _ ? cote ee - Bere, 3 inn ra « . ieee ile a ig bP’ te £L Se pf é — -) oye Ex SS f £# O20 CO (1t€al C5 ot A iadabied? ee tat ee ene Nees oy Conade Cornell University Library % ¥ 4 oy The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. 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 ” AVS JB4G aie Uy | woe 1843 OY EG ear ae DA be are ee lV, 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. 3) 8 3 is) /, N Be hae a at: BY Pee C3) fend . es 4 f ) gp 6% “4 B © ® |e % a a sel im 4 4 2 2 a og 8 3 8 iS $ 56 8 & 8 S oF 8 2 =) 3) o. 68 8 2 8 os B f=) 3 3 5 cs) is} fe) oO a ° cy) o +f 3 S B 8 = ££ € g @8 a a 2 S 2 a 8 BS a ed = & S na : es Z 5 # & 8 2 FE & £ n o 4 AQ & a ee es 2 e iS a ag 1s) g ze a Be a Oo MOTILITY—CAPSULES 3 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 = \ Short bacilli tt Bacilli in chains Vibrios (spirilla) ~ sf oa —S7" ___ Gomma-bacilli oe _ = Spirocheetee we a 8 o 3 z £ 3D o 3 ia N WS =S L (ie ( Fic. 1.—ForMs OF BACTERIA. Magnified about 700 times, (After Baumgarten.) the bacteria themselves), nor with such motion as occurs in inert particles suspended in a fluid medium, and which is known as the ‘molecular movement of Brown’ (Brownian movement). Capsules of bacteria.—Capsules are formed by swelling of the membrane, and are best seen in those micro- organisms which are found in groups—viz. the diplococci, streptococci, tetracocci, and sarcine; but various bacilli also, such as Pfeiffer’s capsule-bacillus and the Bacillus r2 Clostridia forms Knobbed bacteria with ~~ terminal spores 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 A G3 42% YA Hy ‘ “! = C, aN ae 7 A + H 4 aN \ NS : S Mi ko 4 Se, SU 73 4 ne 4 "9 Ui Py Z, Rai | yas & 7 My, th 4 4, & 17> ig’ VL. WSS Mm t 4ie seat vine NS. 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. The animal dies within forty-eight hours, when the spleen is found greatly enlarged, and abundant bacilli can be detected in it as well as in the blood. , IlL-—Afiscellaneous Affections of the Kidneys and Urine, price £1 Tos. sewed, £1 11s. 6d. cloth. 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