DEWITT BISHOP COLLEGE OF AGRICULTURE DAVIS, CALIFORNIA V BACTERIOLOGY MANUAL OF BACTERIOLOGY Jfar Ijrattiticrners antr WITH ESPECIAL REFERENCE TO PRACTICAL METHODS BY DK S. 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. DUBL. LATE UNIVERSITY TRAVELLING PRIZEMAN IN MEDICINE WITH 100 ILLUSTRATIONS, PARTLY COLOURED LONDON LONGMANS, GEEEN, AND CO. AND NEW YORK : 15 EAST 16th STREET UNIVERSITY OF CALIFORNIA DAVIS All rights reeved UNIVERSITY OF CAl)roRNlA I IRR ADV TRANSLATOR'S PREFACE THE present is practically a new edition in English of Professor Schenk's Gnuulris* der Baktcriologie, 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 * — Tn.' 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 72S33 VI BACTERIOLOGY % parasitism of protozoa, and on the action of light on bacteria, and this has been done in an appendix, for which the Translator alone is responsible. He has en- deavoured to comply with the Author's intention by describing practical details with considerable fulness, but has thought it best to depart from the rule of not giving references, the observations on these subjects being so recent that certainty has not yet been attained. These remarks apply also to the note at the end of Chapter X. on Sabouraud's recent researches. A few additional methods and formulae have also been given, the body of the work has been divided into chapters, and the index greatly extended. The Translator has to express his thanks for assistance on individual points from various sources, but especially to Professor Schenk for his unvarying courtesy and readi- ness in affording additional information ; and to acknow- ledge occasional obligations to many of the leading English and German text-books. W. E. DAWSON. DUBLIN : June 1893. AUTHORS PREFACE THE present work is intended to furnish the student and the practitioner with a guide to the science of Bacteriology ; consequently the methods of investigation have been dealt with as thoroughly as possible, special attention being paid to the elementary technique. The Author has thought it best to describe the micro-organisms according to the loca- lities in which they are met with, a plan which rendered it possible to go thoroughly into the respective methods of research in their proper places. Particular attention has been given to the pathogenic micro-organisms, and so much the more regard had to be paid to the chemical relations of the life of bacteria, and to their biology gene- rally, that recent events give us reason to hope for an ex- tension of our therapeutic powers from this direction. Engravings of the most important bacteria have been provided, showing their form and the appearances pre- sented by their growth. These are intended to serve the reader as models of the typical forms, to which he may be able to adhere in his own investigations. The Author has* endeavoured in the text to consider the views of all the different schools, and to this end viii BACTERIOLOGY he has consulted other manuals and bacteriological publi- cations. Conformably to the scope of a handbook like the present, however, all references to the literature have been omitted. It is his hope that this manual may contribute to preserve and promote the interest felt by practitioners and students in the science of Bacteriology. S. L. SCHENK. 1 The words omitted have reference to the German edition. CONTENTS CHAPTER I GENKKAL MORPHOLOGY AND BIOLOGY OF MICRO-ORGANISMS INTRODUCTORY — Varieties of Micro-organisms— Bacteria— Motility of Bacteria— Capsules of Bacteria -Multiplication of Bacteria — Pro- ducts of Metabolism in Bacteria — Influence of Bacteria on the Tissues— Toxines, Toxalbumins, and Ptomaines— Moulds— Yeasts Algae — Protozoa — Examination of Micro-organisms . CHAPTER II PRELIMINARY PROCESSES — APPARATUS AND REAGENTS STERILISATION — Sterilisation by Heat— By Steam— Fractional Sterilisa- tion— Sterilisation by Steam under Pressure — Chemical Disinfect- ants. Ai'i'Ai:\ns AND REAGENTS — Microscope — Steam Steriliser -Incubator — Thermo -regulator — Schenk's Thermo-regulator — Meyer's Thermo-regulator — Gartner's— Altmann's— Petroleum In- cubator—Miscellaneous Apparatus— Hot- water Filter— Apparatus for Plate Cultivations — Moist Chambers — Plates — Petri's Capsules — Soyka's Plates— Wire Crates— Reagents— Stains— Other Utensils — Centrifugal Machines . .15 CHAPTER III NUTRIENT MATERIALS AND METHODS OF CULTIVATION NUTRIENT MEDIA — Liquid Nutrient Media — Preparation of Meat Bouillon— Preparation of Meat-extract Bouillon — Solutions of White of Egg — Solid Nutrient Media— Preparation of Peptone Bouillon Gelatine — Of Meat-extract Peptone Gelatine — Additions to Nutrient Gelatine— Preparation of Urine Gelatine — Prepara- tion of Nutrient Agar — Of Peptone Bouillon Agar — Modifications of x BACTERIOLOGY Gelatine and Agar, &c — Blood Serum — Modifications of Serum Eggs of Birds— Plovers' Egg Albumen— Hens' Eggs— Potatoes— liice, Bread, and Wafers. MODES OF CULTIVATION — Slide Cultures — Koch's Plate Process — Roll Cultures— Modifications of the Plate Process — Plate Cultures on Serum and Plovers' Egg Albumen Cultivation of Anaerobic Micro-organisms — Permanent Cultures . 35 CHAPTER IV EXAMINATION OF MICRO-ORGANISMS UNDER THE MICRO- SCOPE AND BY EXPERIMENTS ON LIVING ANIMALS EXAMINATION in the Fresh State— In the Hanging Drop Staining of Micro-organisms — Simple Staining of Cover-glass Preparations- Preparation of Stain-Solutions—Staining of Flagella— Of Spores— DECOLORISING AGENTS— Koch and Ehrlich Method of Staining — Ziehl and Neelsen's Method— Ehrlich's Giinther's Weichsel- baum's— Fraenkel's — Gabbet's — Method of Pfuhl and Petri— Method of Pittion — Arens's Chloroform Method— Gram's De- colorising Method — Giinther's Modification of Grain'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 STAINING OF SECTIONS — Unna's Drying-on Process — Combination of Staining Methods — Kiihne's Methyl Blue Method — Koch's Method— Lomer'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. EXPEEIMENTS ON LIVING ANIMALS — Transmission of Micro-organisms to Animals — Infection by the Air-passages — Infection by the Diges- tive Canal— Subcutaneous Infection— Intravenous Infection— In- fection into the Anterior Chamber of the Eye .... 65 CHAPTER V THE BACTERIOLOGICAL ANALYSIS OF AIR MICRO-ORGANISMS IN THE AIR— Simple Methods of Examining Air-- Pouchet's Method — Miquel's — Emmerich's — WTelz's — Hesse's — Method of Strauss and Wurz — Petri's Method — Tyndall's Method — Penicillium glaucum— Brown Mould — Yeast— Micrococcus ra- diatus — Micrococcus versicolor —Micrococcus cinabareus — Micro- coccus flavus tardigradus — Micrococcus candicans — Micrococcus viticulosus — Micrococcus Urese — Micrococcus roseus — Diplococcus citreus conglomerate — Micrococcus flavus liquefaciens and CONTENTS xi Micrococcus desidens— Sarcina alba— Sarcina Candida — Sarcina aurantiaca — Sarcina rosea — Sarcina lutea — Staphylococci - Staphylococcus pyogenes aureus ; albus ; citreus — Streptococci — Bacillus subtilis — Bacillus prodigiosus —Potato Bacillus — Bacillus mesentericus f uscus ; ruber ; vulgatus — Bacillus liodermos - Bacillus melochloros — Bacillus multipediculosus — Bacillus nea- politanus— Atmospheric Spirilla ....... 90 CHAPTER VI THE BACTERIOLOGICAL ANALYSIS OF WATER MICRO-ORGANISMS OF WATER — Filtration and Filters —Variations in Water Depending on Source — Examination of Water .Pfuhl's Method — Kirchner's Method— Other Methods— Micrococcus aqua- tilis — Micrococcus agilis —Micrococcus fuscus — Micrococcus luteus — Micrococcus aurantiacus — Micrococcus fervidosus — Micrococcus carneus —Micrococcus concentricus— Diplococcus luteus— Bacillus tiuorescens liquefaciens and Bacillus nivalis (Glacier Bacillus) — Bacillus fluorescens non-liquefaciens — Bacillus Erythrosporus — Bacillus arborescens— Bacillus violaceus — Bacillus gasoformans — Bacillus phosphorescens (indigenus ; indicus)— Bacillus ramosus — Bacillus aurantiacus— Bacillus aureus— Bacillus bruneus — Ba- cillus aquatilis— Bacillus aquatilis sulcatus— Bacillus aquatilis radiatus — Bacterium Ziirnianum — Bacillus membranaceus ame- thystinus — Bacillus indigoferus — Bacillus ianthinus — Bacillus ochraceus — Bacillus gracilis — Bacillus sulphydrogenus — Bacillus of Asiatic Cholera and Allied Micro-organisms — Vibrio proteus — Vibrio Metschnikoffi— Bacillus of Typhoid Fever — Bacterium Coli commune — Spirilla in Water — Other Micro-organisms of Water . 122 CHAPTER VII BACTERIOLOGICAL ANALYSIS OF EARTH AND OF PUTRE- FYING SUBSTANCES MICRO-ORGANISMS IN THE SOIL — Method of Examination — Bacillus mycoides (Earth Bacillus) Bacterium mycoides roseum — Bacillus radiatus — Bacillus spinosus — Bacillus liquefaciens magnus — Bacillus scissus— Glostridium foetidum — Bacillus cedematis ma- ligni — Bacillus of Tetanus — Streptococcus septicus — Bacillus An- thracis — Plasmodium Malariae — Other Micro-organisms of the Soil. ANALYSIS OF PUTREFYING SUBSTANCES— Differences in Putre- factive Processes — Bacillus fuscus limbatus — Proteus — Proteus vulgaris — Zenkeri — Mirabilis — Hominis— Capsulatus — Bacillus saprogenes — Spirillum concentricum — Spirillum rubrurn . . 155 Xll BACTERIOLOGY CHAPTER VIII MICRO-ORGANISMS IN ARTICLES OF DIET PAGB Methods of Examining Different Foods. EXAMINATION OF MILK Methods — Bacillus lacticus (Bacillus acidi lactici)— Micrococcus acidi lactici — -Clostridium butyricum (Bacillus amylobacter) Micrococcus acidi lactici liquefaciens — Oidium Lactis — Bacillus butyricus — Bacillus Butyri viscosus ; fluorescens — Spirillum tyro- genum — Bacillus Lactis viscosus— Bacillus Lactis pituitosi— Bacil- lus actinobacter — Bacillus foetidus Lactis — Bacillus cyanogenus — Bacterium Lactis erythrogenes — Sarcina rosea— Micrococcus of Bo- vine Mastitis — Other Pathogenic Bacteria in Milk— Saccharomyces ruber — Bacillus caucasicus (Dispora caucasica, or Kephir Bacillus). EXAMINATION OF OTHER ARTICLES OF DIET — Bacillus megaterium Bacillus Aceti — Bacillus indigogenus— Pediococcus Cerevisiae — Sarcina Cerevisiae — Micrococcus viscosus — Bacillus viscosus Cere- visiae— Bacillus viscosus Sacchari — Moulds on Articles of Food — Aspergillus niger; albus; glaucus ; flavescens; fumigatus — Mucor Mucedo ; rhizopodiformis ; corymbifer ; ramosus .... 178 CHAPTER IX BACTERIOLOGIAL EXAMINATION OF PUS Properties and Composition of Pus — Actinomyces — Bacillus pyocy- aneus a and # — Staphylococcus cereus albus ; flavus ; aureus — Streptococcus pyogenes — Micrococcus of Gonorrhoea — Bacillus of Syphilis — Bacillus Tuberculosis — Bacillus of Glanders — Other Microbes of Pus CHAPTER X BACTERIOLOGICAL EXAMINATION OF THE ORGANS AND CAVITIES OF THE BODY AND THEIR CONTENTS MICRO-ORGANISMS OF THE LIVING BODY — I. THE SKIN — Micro-organisms of the Skin — Methods of Examination — Diplococcus subflavus — Micrococcus lacteus faviformis — Diplococcus albicans amplus— Diplococcus albicans tardus — Diplococcus citreus liquefaciens — Diplococcus flavus liquefaciens tardus — Micrococcus haematodes — Micrococcus of Trachoma — Diplococcus of Acute Pemphigus — Vaginal Bacillus — Bacillus of Symptomatic Anthrax — Lepra Bacil- lus— Bacillus sycosiferus foetidus — Ascobacillus citreus — Bacillus Xerosis — Trichophyton tonsurans— Fungus of Favus (Achorion Schcenleinii) — Microsporon furfur. NOTE : Trichophyton micro- sporon — Macrosporon .... ..... 222 CONTENTS xiii CHAPTER XI THE ORGANS AND CAVITIES OF THE BODY AND THEIR CONTENTS — (C0nt.) II. THE DIGESTIVE TRACT PAOR THE CAVITY OF THE MOUTH — Micro-organisms of the Mouth and their Examination — Leptothrix — Bacillus buccalismaximus — lodococcus — Micrococcus salivarius septicus and Bacillus salivarius septicus — Bacillus ulna— Bacillus Gingivre— Bacillus Diphtherias— Spirillum Miller — Spirochrete Dentium (Denticola)— Vibrio rugula— Fungus of Thrush— Other Bacteria of the Mouth. THE TYMPANUM. THE STOMACH — Micro-organisms of the Stomach— Sarcina Ventriculi— Micrococcus tetragenus mobilis Ventriculi — Bacterium Lactis aerogenes— Bacillus indicus— THE INTESTINE —Intestinal Micro- organisms— Micrococcus aerogenes — Bacillus putrificus Coli — Bacillus coprogenes foatidus — Bacterium Zopfi — Bacterium aero- genes, Helicobacterium aerogenes, and Bacillus aerogenes — Bacillus Dysenteriae — Bacillus of Fowl Cholera —Other Intestinal Bacteria . 237 CHAPTER XII THE ORGANS AND CAVITIES OF THE BODY AND THEIR CONTENTS — (C0nt.) III. THE F^CES AND URINE THE F^CES— Composition and Modes of Examining— Bacillus subti- lif ormis Bacillus albuminis — Bacillus cavicida — Micrococcus tetragenus concentricus. THE URINE— Micro-organisms of the Urine_Yeasts and Moulds in Urine— Urobacteria— Staphylococcus Ureaa candidus — Liquefaciens — Micrococcus Ureas liquefaciens — Bacilius Ureee— Urobacillus Freudenreichii— Madoxii — Micrococ- cus ochroleucus — Streptococcus giganteus Urethrae — Bacterium sulphureum Bacillus septicus Vesicaa— Urobacillus liquefaciens . 254 CHAPTER XIII THE ORGANS AND CAVITIES OF THE BODY AND THEIR CONTENTS — (cont}. IV. BACTERIOLOGICAL EXAMI- NATION OF THE RESPIRATORY TRACT, AND (v.) OF THE BLOOD THE NASAL SECRETION — Micro-organisms in the Nasal Secretion— Micrococcus cumulatustenuis— Micrococcus tetragenus subflavus — Micrococcus Nasalis— Diplococcus Coryzas — Staphylococcus cereus aureus— Bacillus foetidus ozcense — Bacillus striatus albus et flavus — Bacillus of Ehinoscleroma— Bacillus capsulatus mucosus — Vibrio Nasalis — Other Nasal Bacteria. THE KESPIRATORY PASSAGES — Micro-organisms of the Respiratory Passages — Sarcina Pulmonum XIV BACTERIOLOGY PAGE — Sarcina aurea — Diplococcus Pneumonias — Pneumobacillus Friedlaenderi — Micrococcus tetragenus — Bacillus aureus — Tubercle Bacillus and Actinomyces — Bacillus Tussis convulsivas— Bacillus pneumosepticus. V. BACTERIOLOGICAL EXAMINATION OF THE BLOOD — Micro-organisms in the Blood — Methods of Examination — Influ- enza Bacillus — Bacillus Endocarditis capsulatus — Bacillus of Swine Erysipelas — Bacillus murisepticus — Spirochaete Obermeieri — Protozoa in the Blood , 264 APPENDIX (by the Translator) A. VACCINATION AGAINST ASIATIC CHOLERA— Principle of Anti-cholera Vaccination— Preparation of Vaccine — Kesults of Vaccination. B. PARASITIC PROTOZOA — Pathogenesis in Protozoa — Coccidium oviforme— Amoeba Dysenteriae— Protozoa in Carcinoma— Protozoa in other New Growths, &c. C. THE ACTION OF LIGHT ON MICRO- ORGANISMS —Action of White Light — Action of Coloured Light — Mode of Action of Light — Applications in Nature. D. AD- DITIONAL METHODS AND FORMULA— Fixing Methods— The Gum Freezing Method — Staining Formulas 283 INDEX , 303 Errata Page 11, line 5 from bottom, for CRENOTIILE read CRENOTIIIIIX , 236, line 4, the words NOTE BY TRANSLATOR refer to what follows, not to what precedes. „ 288 line 4, from bottom (note 1), for Klemperei read Klemperer. „ 298, note 1,/or quoted without reference, &c., read Pfliiger'x ArcJiir, xxr, p. 95. „ 802. lines r>-3 from bottom should read as follows: minutes in Luffler's or Kiilme's methyl blue, washed, dipped for an instant in 10 per cent, tannin solution, slightly decolorised in feebly acid water, washed in water, XIV BACTEKIOLOGY PAGE — Sarcina aurea — Diplococcus Pneumonias — Pneumobacillus Friedlaenderi — Micrococcus tetragenus— Bacillus aureus — Tubercle Bacillus and Actinomyces— Bacillus Tussis convulsivas— Bacillus pneumosepticus. V. BACTERIOLOGICAL EXAMINATION OF THE BLOOD — Micro-organisms in the Blood — Methods of Examination — Influ- enza Bacillus — Bacillus Endocarditis capsulatus — Bacillus of Swine Erysipelas — Bacillus murisepticus — Spirochaete Obermeieri — Protozoa in the Blood , 264 APPENDIX (by the Translator) A. VACCINATION AGAINST ASIATIC CHOLERA — Principle of Anti-cholera Vaccination — Preparation of Vaccine — Kesults of Vaccination. B. PARASITIC PROTOZOA — Pathogenesis in Protozoa— Coccidium oviforme — Amoeba Dysenteriae — Protozoa in Carcinoma— Protozoa in other New Growths, &c. C. THE ACTION OF LIGHT ON MICRO- nf Coloured Light— BACTERIOLOGY CHAPTEE 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, mirro-nryanisms, which, alighting upon the surface, may effect an entrance in various ways into the interior ; they belong partly to the vegetable, partly to the animal kingdom, and have the property of developing on animal and vegetable bodies. According as they are capable of growth upon dead substances or living matter, we distinguish respectively saprophytes and parasites ; the latter of which are subdivided into obligate and facultative parasites — the obligate or strict parasites being those which grow exclusively in the living body and perish apart from it, whereas the facultative parasites have the power of adapting themselves to altered conditions of life, and of flourishing externally as well as internally. With fur- ther reference to their relation to the human or animal frame we speak also of ectoyenous micro-organisms, which occur outside the body, of endogenous, which exist in its interior, and of cmibigenov*, 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, alga, and protozoa. Bacteria, schizomycetes, or fission-fungi, are colourless cells of a glassy transparency, possessing an enveloping membrane with protoplasmic contents but no nucleus, and having a length which amounts in general to a few thousandths, and a breadth of some ten-thousandths, of a millimetre. The interior of the bacterial cell has usually a homogeneous appearance, but sometimes shows oil-like granules. Bacteria are distinguished according to their form as cocci, bacilli, and spirilla. Cocci are globular in shape, and are found either singly or united in groups. If they lie singly they are called mono- cocci; if grouped in masses, 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 sarcinte ; and if they are strung together in chains, streptococci. Bacilli are minute straight rods, the smallest discovered up to the present time being the influenza bacillus. Their ends are sometimes sharply cut across, sometimes rounded off, and the rodlets themselves are in some cases thin, in others stout and thick, in others again swollen in the centre, and so forth. Spirilla are spirally curved rods, and are subdivided into comma-bacilli or vibrios, spirilla in the more restricted sense, and spirochcetcz. The vibrios usually form strings of cells which strongly resemble spirilla ; the spirochsetse are distinguished by their flexibility. MOTI LIT Y— CAPSULES Motility of bacteria. — A large number of bacteria pos- sess the power of movement, accomplished by means of cilia or jluji-lla, 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 FIG. 1. — FORMS OF B.\< TKHIA. Magnified about 700 times. (After Baumgarteu.) 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 ' (Brown inn increment). Capsules of bacteria. — Capsules are formed by swelling of the membrane, and are best seen in those micro- organisms which are found in groups — viz. the diplococci, streptococci, tetracocci, and sarcinae ; but various bacilli also, such as Pfeiffer's capsule-bacillus and the Bacillus n 2 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 zooglcea, and may spread over the surface of fluids in the form of a pellicle. This pellicle sometimes serves to distinguish between micro-organisms which strongly resemble each other — for instance, cholera bacilli form a pellicle, whereas Finkler- Prior bacilli do not. Multiplication of bacteria takes place by fission, hence their name of 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 sarcinae. 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 trie 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 SPOKES— PRODUCTS OF METABOLISM 5 occupy one end, as in the Tetanus bacillus, the cells show some resemblance to a drum- stick. Formation of spores in the interior of the mother-cell is described as proper or endogenous, while arthrogenous is the term applied to that which takes place when single portions separate from the cell and develop gradually into independent individuals (arthrospores). Bacteria require for their growth a certain amount of moisture, many of them speedily perishing if d^ied. 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 Barilla* jinxliaiosus a smell resembling that of trimethylamine. Micro-organisms have also the property of producing changes in the medium on which they are cultivated by the products of their metabolism, so that albuminoid sub- stances are peptonised by some of them and gelatine liquefied. Many have the faculty of resolving organic bodies into their simplest elements ; but some possess the power of forming nitrates by the conversion of am- monia into nitrous and nitric acids. Certain microbes break up the chemical combination of albuminoid bodies, causing putrefaction, while others induce fermentation ; and a small number, again, have the property of becoming 6 BACTERIOLOGY luminous in the dark (phosphorescence), in consequence of the molecular activity of their protoplasm. Influence of bacteria on the tissues. — The influence which bacteria exert on the tissue of the bodies of men and animals depends both on the qualities of the bacteria and on the nature of the tissue. The action of the pathogenic bacteria is not alike in all animals, and those which are insusceptible to certain bacteria are said to be immune in their relation to those particular organisms. Animals, however, which are commonly immune towards a patho- genic micro-organism may, under altered conditions, lose their immunity ; for example, the frog, although usually immune to anthrax, becomes susceptible to it at a higher temperature. The virulence of pathogenic bacteria may be diminished by various factors, amongst which are included sojourn in the body of immune animals, increased atmospheric pres- sure, the action of higher degrees of temperature, the influence of sunlight, &c. This weakening is dependent on an alteration in the products of metabolism. Toxins, toxalbumins, and ptomains, — As products of the metabolism of those bacteria which effect an entrance into the body substances are formed, some of which exert violent toxic action, and which are divided into toxins and ptomains, or cadaveric alkaloids, and to these is ascribed the action of the pathogenic bacteria in originating morbid processes. The toxins are further subdivided into tox- albumins and proteins. Toxalbumins are albuminoid bodies formed during the growth of the bacteria upon culture-media, especially in bouillon. Their activity depends upon certain definite degrees of temperature, and they decompose at the boiling- point. Our knowledge of them is due to the researches of Eoux, Yersin, Brieger, and C. Fraenkel. TOXINS— TOXAUJr-M I NS-PTOMAINS 7 Protein* are albuminoid bodies which are contained in the actual substance of the bacteria, and whose chief point of difference from toxalbumins consists in their not being decomposed by boiling, even if kept up for hours. They were discovered by Nencki, and further studied by Buchner. Proteins can be abundantly obtained by boiling pure cultures of bacteria in their bouillon or mixed with water. Koch's tuberculin belongs to this group of substances. Toxins, when injected in small quantity into animals, have the property of affording immunity from infection with the corresponding bacterium. According to Brieger, the cultures or juice from the tissue should be filtered by means of earthenware cells, so as to free the liquid from living germs. The albuminoid substances are then precipitated with ten times the quantity of absolute alcohol, redissolved in dilute alcohol, and pre- cipitated a second time with an alcoholic solution of cor- rosive sublimate. The mercury is next removed by means of sulphuretted hydrogen, the residue dissolved in water, and again treated with sulphuretted hydrogen ; this process having been several times repeated, the toxins are finally precipitated from the aqueous solution by absolute alcohol. Scholl produced a toxopeptone from cultures of cholera- bacilli in hens' eggs, without filtration, by the following process : — The albumen liquefied by the bacteria was poured into ten times its quantity of absolute alcohol, and the precipitate washed with alcohol, digested with water, and filtered. The aqueous solution was then repeatedly added to ether and alcohol slightly acidulated with acetic acid, decanted each time from the residue, and the latter re- dissolved in water rendered alkaline, after which a final addition to pure ether, which was then evaporated off, resulted in the obtaining of the poisonous substance. To procure ptomains from the actual bacterial cells 8 BACTERIOLOGY after the method of Buchner potato cultures are prepared, and the mass of bacteria is then scraped from them, and rubbed up in a mortar with a little water, mixed with fifty times its volume of a half per cent, solution of caustic potash, and then digested in a water-bath until the greatest possible degree of fluidity is reached, after which it is filtered through several small filters. Dilute acetic or hydrochloric acid is next added to the filtrate until, while avoiding any excess of acidity, the reaction becomes dis- tinctly acid. The protein precipitated in this way is collected on a filter, washed, and dissolved in water which is feebly alkaline. Roemer procured his extracts of the Bacillus pyocyaneus and pneumobacillus in the following manner : — The masses of bacteria are carefully scraped from well- developed cultures on potato and rubbed to a fine emulsion with ten times their bulk of distilled water. The emulsion, having been sterilised by boiling for several hours, is left for about four weeks in the incubator, during which time it must fre- quently be boiled for an hour or two, so that in the course of the process it is boiled for from thirty to forty hours in all. When the four weeks have expired the emulsion is filtered through a tubular filter made of kaolin (Chamber- land's candle), or through one of infusorial earth, and the resulting filtrate is a clear brownish or yellowish fluid con- taining albuminoid substances. Koch obtained his tuberculin by extraction from pure cultures of tubercle bacilli, which he grew upon a feebly alkaline infusion of veal containing an addition of one per cent, peptone and four to five per cent, glycerine. The culture-vessels are inoculated by floating a fairly large piece of the seed-culture on the surface of the fluid, and are then kept at a temperature of 38° C. In from three to four weeks the surface is covered with a tolerably thick mem- KOCH'S TUBERCULIN— MOULDS 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 Mycelium' FIG. 2.— MUCOR MUCEDO. (After Baumgarten.) bottom. The cultures (which thus require from six to eight weeks for their growth), when fully ripe, are evapo- rated to a tenth of their bulk in a water-bath, and filtered through earthenware or infusorial earth. Sterigmata with •pom Fructification FIG. 3.— ASPKIIGJLLUS 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, Mycelium FIG. 4. — PENICILIUM G-LAUCUM. Magnified 400 times. (After Baumgarten.) spoken of as mycelium, and possessing special seed-bearing organs called hyphce or thallus, from which the moulds derive the name of Hyphomycetce. According to the form of the seed-organ they are divided into mucorinece, aspcr- giUinece, penicilliaccce, and oidiacccc. Conidia Hyphe Mycelium FIG. 5.— OJDIUM LACTIS. (After Baumgarteu.) In the mucorinece, or headed moulds, the ends of the hyphse 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 aspergillinece (knob-moulds) have the knobbed ends MOULDS 11 of the hyphae covered with a variable number of spore- carriers, or stcriymata, from the extremities of which the spores divide off in rows (fig. 3) . The hyphae of the pcmnlliacca (pencil-moulds) are branched, which is not the case with the mucor and asper- Gronp of buds (gemmation) Mother-cell — Vacuole FIG. 6.— YEAST CKLLS (Sacefiaromycet Cerevitut). 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 mnidia, are separated off in the form of chains (fig. 4). Arthrosporus 1.1 SinirN- segments sheath sur- rounding the sepa- rate spores FIG. 7.— CKKXOTJU.F. KDIXIAXA. MaLMiificd COO times. (After Zopf.) The oidiaccfc are distinguished by the fact that the hyphae form no special spore -bear ing organs, but become articulated at their extremities, and so divide off the spores in the form of segments. 12 BACTERIOLOGY Yeasts. — These possess neither spore-bearing organs nor spores, but multiply by gemmation, which consists in the budding out of daughter-cells in different places from the gradually enlarging mother-cell, these in their turn be- coming mother-cells, thus forming groups of buds. The individual yeast-cells are round or elliptical, and often display in their interior colourless lacunae, which are not spores, but may perhaps consist of minute drops of fat, and are called vacuoles. The yeasts play an important part in nature in causing fermentation. Several species of them form pigments. Algae. — Of these the cladothrix, crenothrix, and becj- giatoa varieties belong to the micro-organisms. They are jointed filaments, which multiply not by fission but by germination at their extremities (fig. 7). Protozoa. — Of the protozoa those important as regards bacteriological investigation are the sporozoa, which include the gregarince, psorospermii, and coccidia. They are unicellular organisms which can only live in a moist or liquid medium, and in the absence of water, nutrient material, or oxygen, are transformed into roundish durable cysts. They possess a sort of larval condition, consisting of irregular and roundish little masses of protoplasm, which move by means of processes projecting oat like limbs (pseudopodia) , or by flagella, and often, losing their mobility, take up a permanent residence in other cells. The contents of the cyst separate by division or gemmation into particles called sporocysts or psetidonawceUa, the contents of which, again, break up into a number of sickle- shaped germs. Pfeiffer considers the plasmodia of malaria to be also cysts of this nature containing crowds of spores. Examination of micro-organisms. — Microscopic examina- tion alone is not sufficient to establish fully the properties of micro-organisms in their morphological and biological EXAMINATION OF MICROORGANISMS 13 relations. The attempt must be made to obtain pure cultivations, which are then, on the one hand, to be sub- segmentation into rods Spirilla-like coils Branch resembling spirochaeta FIG. 8. — FORMS OF VEGETATION OF CLADOTHRIX DICHOTOMA. (After Zopf.) mitted to microscopic examination, and on the other transferred to substances liable to fermentation and putre- 14 BACTERIOLOGY faction, and used for experiments on animals. In order to procure pure cultures, however, the instruments and utensils employed must be freed from the micro-organisms adhering to them, or sterilised. It seems, therefore, advisable first to discuss the methods of sterilisation, and then the preparation of culture-media, passing on after- wards to microscopic examination, and, finally, the methods of transmission to living animals. 15 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 a very high temperature are best sterilised by being held in the flame of a Bunsen burner or spirit-lamp until a red or white heat is reached, a method which is especially applicable to small platinum wires or plates. Bodies which have no great power of resistance, and instruments which could not be exposed to so intense a heat without impairing their efficiency and sharpness, are subjected for a longer time to a temperature of 150° C., by which both the micro-organisms and also their spores are destroyed. For this purpose a box made of sheet iron, with double walls, is best employed, which has thus a layer of air between the walls (hot-air steriliser). It must be put together with rivets, not with solder. By means of a single powerful burner or a number of small ones placed beneath, the temperature of the interior is rapidly brought to 160°-170°, after which half an hour suffices for sterilisa- tion. In the top of the box are openings for two ther- mometers, one of which extends into the interior, while the other registers the temperature of the space between the inner and outer walls ; and there is also a valve for 16 BACTERIOLOGY the purpose of regulating the temperature. This arrange- ment is suited for the sterilisation of instruments, apparatus, glassware, &c. (fig. 9). To prevent flasks and test-glasses from becoming re- 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 long as the lower. Sterilisation by steam. — Articles which cannot be ex- posed to so high a temperature are sterilised by the vapour of boiling water at 100° C. For this purpose a cylindrical vessel of sheet copper is used, measuring nearly a metre in height and about 20 cm. in diameter, covered with felt or asbestos to prevent loss of heat, and capable of being closed with a lid similarly protected. The latter is provided with an opening for the introduction of a ther- mometer, and does not fit quite air-tight (fig. 10). The bottom of the vessel is double, the inner bottom consisting of a grating fixed about 30 cm. above the outer, and the FIG. 9. — HOT-AIR STERILISER. STERILISATION BY STKAM 17 space between the two is about half filled with water, the height of which is observed by a gauge-tube at the side (Koch's Steam Steriliser). The articles to be sterilised are placed in a tin vessel provided with a lid, and the bottom of which is also grated, and are left in the steriliser for from half an hour to an hour (from the moment when an abundance of steam is given off), which suffices for complete sterilisation. Water-gauge Ring of flames Fio. 10.— KOCH'S STEAM STEIULISKK. For laboratories which are not supplied with gas, Badenbercfs 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° C. for any length of time without undergoing changes; gelatine, for example, loses the power of solidifying, which alone renders it applicable to bacteriological purposes. Hence it is advisable to expose all culture-media to a current of steam for not more than a quarter of an hour daily on three successive days. The heating on the first day kills all the micro-organisms present and most of the spores ; but some of the latter still remain and develop by next day into micro-organisms, which perish on heating for the second time, Any that remain are destroyed on the third day. Fractional sterilisation, — Certain substances, however, particularly the serum of blood, undergo so many changes even during a short sterilisation in the steam-current, as to be no longer suitable for use as culture-media, and in such cases recourse must be had to the process of discontinuous or fractional sterilisation, introduced by Tyndall. This con- sists of heating to a temperature of 54° to 56° C., for three or four hours daily during one week, in a chest with double walls between which there is a layer of water, the tem- perature being kept at a constant height by means of a thermo-regulator ; or, according to Heim's method, the test-glasses can be placed in the warm water of a bath, the temperature of which is kept continually at the height mentioned above. Sterilisation by steam under pressure. — High-pressure steam, applied by means of autoclaves, acts with greater rapidity than ordinary steam at 100° C. Chemical disinfectants. — Besides high temperatures, CHEMICAL DISINFECTANTS 19 various chemical substances are employed for the purpose of sterilisation. Those possessing the greatest germicide power of all are carbolic acid in strong solutions, corruain- sultUnifit'- in 1 in 1,000 solutions, and f////r/,7//w; but next to these chlorine, iodine, and Iromuie icntcrx, 1 per cent, solutions of oxinic acid, 1 per cent, solutions of potassium jicrnian'inmitc, oil <>i turpentine, ir<>n jicrchlnride, &c., have a more or less energetic disinfectant action. Of the above disinfectants, corrosive sublimate in 1 in 1,000 solution is at present most in use. Heider states that the efficacy of a large number of disinfectants is very markedly increased by moderately raising the temperature. Chloroform is recommended by Kirchner as an excellent disinfectant, having the advantage of great activity com- bined with a low boiling-point, so that it can be driven off with certainty from other fluids by heating after sterilisa- tion is complete. It is particularly suitable for sterilising blood serum, which cannot be exposed to a high tempera- ture, and which, therefore, as Globig has shown, it is im- possible to free by the method of discontinuous sterilisation from the germs of such micro-organisms as do not grow below 50° C., and are capable of withstanding a tempera- ture of 70° C. To sterilise by this method the fluids under treatment are shaken up with excess of chloroform, and allowed to stand for some days, after which they are freed from the chloroform before use by heating for an hour at 62° C., the boiling-point of chloroform being 61*2°. In bacteriological work it is often necessary to com- bine several modes of sterilisation in order to secure com- plete destruction of germs, but the method chosen varies continually according to the bodies to be so treated. For the sterilisation of instruments, boiling for five minutes in water is sufficient, according to Davidsohn ; and plates, c 2 20 BACTERIOLOGY at all events, may be sterilised over a gas or spirit flame after being cleansed with alcohol and corrosive sublimate, after which they are laid, with the heated side uppermost, on a sheet of clean paper and merely protected with a glass cover which has been likewise cleaned by means of alcohol and corrosive sublimate, or even with a cleaned soup-plate. Cleanliness of all objects coming in contact with the bacteria is of particular importance ; and therefore it fol- lows that in the practical application of bacteriology to surgery there is need of the utmost care in the cleansing of hands, instruments, and dressings, in order to render an aseptic procedure possible. For this purpose a thorough brushing of the hands (which have first been carefully cleansed with soap), followed by rinsing with alcohol and ether and washing in a TVth per cent, solution of corrosive sublimate, is absolutely necessary. APPAEATUS 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 tlier mo-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.1 A glass gauge, 1 [The incubator chiefly used in this country differs slightly from that described in the text, as it opens at the side instead of above, and is closed IN( TBATOR 21 fixed to the outside, indicates the height of the water. The heating of the apparatus is carried on by small gas- flames (tnirrti-ltiirm'rx), 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- r emulator is employed, which (with the very slightest varia- Felt coating Thermometer Water-gauge FlG. 11.— iNCUBATcilt. 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. 1 [The terms ' room temperature,' ' ordinary temperature,' &c., which will be frequently met with in the following pages, denote a temperature of about 20° C., while by ' incubation temperature ' is meant one of about the heat of the human body, i.e. 37° C. (German, Zimmertemperatur and Brut- temperatnr 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 therrno-regulators have been constructed on this principle. Schenk's thermo-regulator, — A regulator in use for the incubator in the author's Institute is constructed as fol- lows (it can be procured from Siebert, 19 Alserstrasse, Vienna) : A piece of glass tubing is sealed into a vessel of glass shaped like a test-tube, in such a way that one end of the former, which is widened out, adheres air-tight to the sides of the latter vessel, while the other end reaches nearly to the bottom. If now mercury be poured into this apparatus a portion of it will sink through the narrow tube to the bottom of the wider vessel, while the rest fills the small tube and extends above it to such a height as to allow of the test-tube being closed with a cork which is perforated with one aperture. The air contained in the apparatus renders it more sensitive by causing the mercury to rise and fall more rapidly with the alterations in its volume produced by variations of temperature. Into the cork is fitted a second glass tube, which is expanded in its upper half, this ex- panded part being closed by a cork perforated twice and traversed by two glass tubes bent at right angles. One of these, which extends down as far as the constriction of the upper vessel, is ground away obliquely at the end, and has a minute safety aperture in one side; the other is simply a bent tube reaching to the lower surface of the cork only. In actual use the apparatus is interposed between the supply-pipe of the gas and the burner by attaching to each angular tube a piece of india-rubber pipe, on the one side from the burner, on the other from the gas-tap. The test- SCIIENK'S THKILMO-JKEGULATOR 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 Glass tube sealed in - Mercury KKJ. 12.— SCHENK'S THERMO-REGULATOU. Now, as soon as this opening is covered with mercury, the passage of gas would necessarily cease and the flame of the burner be extinguished, did not enough gas escape through the lateral aperture to keep it burning. Accordingly, the filling of the regulator with mercury must be done in such a way that this limit is reached at the incubating temperature. By pushing in or withdrawing the glass vessel above the test-tube the temperature can be raised or lowered a few degrees according to need. If the temperature of the water in which that part of the apparatus containing the mercury is placed rises, then 24 BACTERIOLOGY the opening for the passage of the gas must become con- tinually smaller, and this is followed by cooling of the water and sinking of the mercury, so that the aperture transmitting the gas again enlarges, and therewith the flame increases and the temperature ascends, but cannot pass beyond the limit for which the regulator is set. With good management the variations are only very small. Meyer's thermo-regulator. — Another thermo-regulator, constructed by Victor Meyer, which is extensively used and can be highly recommended on account of its sensitiveness, consists also of a glass vessel like a test-tube, and which can be closed with a rubber cork. It is furnished with a small side- tube in the upper part, and is divided into two sections by a capillary funnel of glass, the end of which is just above the bottom. The lower division is filled with mercury, the surface of which is only some three cm. distant from the edge of the funnel, and the interspace thus left is occupied by a mixture of alcohol and ether. In the upper part is fitted a glass tube, cut off obliquely below, and passing through the rubber cork; it ends a little above the capillary funnel, and is pierced in one side above the lower opening with a hole the size of a pin's head. In order to graduate the regulator it is immersed in a water-bath, the temperature of which is controlled by an accurate thermometer ; even a slight increase of heat volatilises the ether and drives the mercury up, so that it comes to stand above the capillary funnel. If now the water has reached the temperature fixed on, the obliquely- cut glass tube is so far introduced into the mercury that the lower opening is quite covered and only the safety aperture at the side remains pervious. The regulator is so connected with the flame under the incubator that this receives only such a quantity of gas as can traverse the regulator. When the water attains too high a temperature, ALTMAXN'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 Regulating screw- Mercury vessel — Flo. 13.— ALTM ANN'S THXBMO-BBQCLATOft. 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 Giirtner's principle, being pro- vided with a horizontal tube which can be closed by a tap. 26 BACTERIOLOGY On each side of this tap a lateral tube proceeds in an oblique direction from the horizontal one, to unite at an acute angle with a vertical tube, which contains a vessel drawn out to a capillary termination, and filled with mer- cury in such a way that the convex surface of the metal begins at once to close the lumen of the angle formed by the union of the oblique tubes. If the lower end of the regu- lator is placed in too warm a medium the mercury expands in the capillary, and so permits the gas to pass only through the horizontal tube, where the supply can be still Thermometers Bearing of lever 1 I 1 ft Wooden casing Rubber tube Petroleum lamp Fm. YL — BAUMEYER'S PETROLEUM IXCUBATOII. further reduced by means of the tap. The height of the mercury in the perpendicular tube can be regulated by a screw at the side. Petroleum incubator, — If the laboratory is not provided with a gas-supply, incubators heated by petroleum can be employed, which are also provided with contrivances arranged for regulating the temperature in the chamber. Such an incubator is made of wood, and contains within it a chest of sheet metal, to the bottom of which is fitted a heating canal furnished with a chimney which runs per- pendicularly up the outside wall (fig. 14). The flame of a petroleum lamp plays into the heating canal and warms IIOT-WA'IKK HLTKK 27 the water circulating in six rubber pipes connected in a water-tight manner with the rnetal 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- WTatei* filtering funnel is fre- FIG. 15.— H..T-WATKI: Ku/mi (HEATED BY A KING liL'KXKU). 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-wTater 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 _ RPi - Glass plate Levelling screw Levelling stand Fio. 16.— LEVELLING APPARATUS FOR MAKING PLATE-CULTIVATIONS. large glass dish filled with water and pieces of ice and covered with a thick glass plate or a sheet of iron. The latter having been brought into a horizontal position with the help of a spirit-level, glass plates to receive the gelatine can be laid upon it and protected with a bell-glass (fig. 16). Moist chambers are employed for the further carrying out of the cultures ; they are made of glass, and have a diameter of about 24 cm. and a height of 6 to 7 cm. (see p. 56). Instead of the ordinary glass plates, which are the size of a photographic quarter-plate, round glass dishes are also used. Petri's capsules consist of flat double dishes of glass, of which the lower has a diameter of 10 cm. REAGENTS USED IN BACTERIOLOGICAL RESEARCH 29 Soyka's plates are similar to Petri's capsules, but differ from them in having eight to ten depressions ground in the lower plate, which resemble the * wells ' in hollowed slides. In addition to the above, all articles employed in microscopic investigation are required. The slides used for examining micro-organisms in the ' A*////////// 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.— Hou.ow sum-:. Fio. 1*. VUKK CKATK. 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 carbolic acid are necessary reagents for the laboratory table. The oils employed are aniline, cedar, and those of bergamot and cloves, used partly for clearing the microscopic preparations, partly as solvents. For imbedding, a harder and a softer variety vi paraffine, and celloidine, are used. Canada balsam, and less frequently glycerine, are em- ployed in the preparation of permanent microscopic objects. The latter finds, however, its most extended application in preparing nutrient materials. Gelatine, agar-agar, blood-serum, albumen from the eggs of hens and of insessorial birds, potatoes, starch, paste, milk, rice, and bread are all used for making nutrient sub- stances. Their application will be gone into in detail in treating of culture-media. Stains. — The following is a list of the colouring matters which are indispensable in making bacteriological pre- parations : — Fuchsine, methyl blue, gentian violet, Bismarck brown, methyl violet, malachite green, cosine, safranine, and dahlia are aniline colours which are sufficient for nearly all investigations. Besides these, however, carmine, picm- carmine, picric acid, hcematoxyline, and Magdala red are re- quired for preparations of tissues ; and for certain methods of staining still other dyes are used, such as extract of logwood,1 &c. Distilled water, alcohol, ether, xylol, and oil of turpentine complete the equipment of reagents. Other utensils. — Platinum wires with loops sealed into 1 [Practically identical with Extractum Hcematoxyli, B. P.] — TK. MISCELLANEOUS APPARATUS 31 glass rods— best done over a Bunsen burner or with the aid of a gas blow-pipe. Of instrument*, 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 U'.il.lM-r ball FIG. 19. — KOCH'S IXJKCTIXCJ SYUIX<;K. With the instruments now specified, a laboratory is in a position to begin bacteriological work, and it need only be mentioned that the articles necessary for all scientific work must also be at hand, as, for example, working benches, cupboards for reagents, test-tube stands, corks, meat-presses, scales, different kinds of glass and metal vessels, bibulous paper, &c. Centrifugal machine. — In order to examine fluids which are poor in corpuscular elements, Stenbeck has introduced 32 BACTERIOLOGY a centrifugal machine, or centrifuge, to be driven by hand. This contrivance carries a metal frame or a disc with several apertures in which are fixed metal cases for the reception of small glass tubes. The fluid to be examined is poured FIG. 20. — STENBECK'S CKNTIUFUGE (after Jakscli). 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 — ---- Hole for the end of the catgut FIG. 21. — GARTNER'S CExnuKr«;i:. revolving with very little friction in sockets in a cast-iron frame, which serves to fasten the entire apparatus to a table or a window-sill. A hole is perforated in the lower part of the spindle, into which the end of a catgut string is introduced, the string itself being wound round the shaft. By pulling away the string the apparatus is put in rotation after the manner of a child's top, the number of revolutions at starting reaching over 3,000 in the minute, and the motion keeps up with gradual slackening for ten to fifteen minutes (fig. 21). For decanting the supernatant liquid 34 BACTERIOLOGY Gartner uses a little contrivance consisting of a cork with two glass tubes which is inserted into the mouth of the test-tubes, and converts them into miniature wash-bottles. The fluid is forced out by blowing and the sediment remains behind on the bottom. Less recently Csokor constructed a large centrifugal machine marked by its fixed and perfectly even rate of rotation. It is driven by water-power, and makes over 3,000 revolutions per minute. 35 CHAPTEK III NUTRIENT MATERIALS AND METHODS OF CULTIVATION Nutrient media. — In order to observe the growth of micro-organisms, it is absolutely necessary to provide a number of nutrient materials in which the individual microbes may multiply into larger masses, so that their peculiarities can be more thoroughly made out. Some of these culture media are so prepared as to approximate more or less closely to the natural soil of the micro-organisms, and others in such a manner as to render them suitable for use as general media on which the most widely differing varieties may be cultivated. They are divided generally into liquid and solid media. Liquid nutrient media. — Fluid media fall rather into the background in use compared with the solid, since the con- ditions of growth and characteristic pecu- liarities in the shape of the colonies come out less strongly on them than on the latter. They are employed either in sterilised test tubes closed with a plug of cotton- wool, or in little flasks, of which those . -n , ,. , , f , ,„ ERLEN-MEYEU'S FLASK. of Erlenmeyer are particularly useful (fig. 22). The media, especially bouillon or broth, after bein# distributed into such smaller vessels, must be carefully heated in the current of steam for 15 minutes daily on three to five successive days, in order to sterilise them. The D 2 36 BACTERIOLOGY inoculation and further cultivation of the micro-organisms depends on the particular kind under observation. Preparation of meat bouillon. — The following recipe gives Loffler's method of preparing a liquid medium (broth or bouillon) which has come into general use : — A half-kilogram weight of meat freed from fat is chopped fine in a mincing machine. (Such meat cannot, strictly speaking, be termed free from fat, because only the masses of fat are cleared away, whereas that which exists in the substance of all meat is not removed.) A litre of ordinary water is poured over the meat and the whole is allowed to stand in a cool place for twenty-four hours. In this way the albuminoid bodies and other substances soluble in water are dissolved out from the meat, the result being an aqueous extract, coloured in most cases with haemoglobin, and which is separated from the residue by squeezing it through a cloth. About a litre of fluid is thus obtained, which must now be freed from albuminoid bodies by heating in a water-bath or in the steam-steriliser. The heating must be kept up until a sample, when filtered and boiled, no longer shows any turbidity, which usually takes about half an hour. The fluid is then filtered, and to the filtrate are added one per cent, of dry colourless peptone and 0*5 per cent, common salt, which is equivalent to 10 grams peptone and 5 gramti 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 so if desired. The broth thus prepared is boiled once more and filtered after boiling ; it should not become turbid, either during sterilisation or on standing. The filtrate must be clear, pale yellow, and of neutral Ml-AT EXTRACT BOUILLON 37 or feebly alkaline reaction. Turbidities are either caused by the reaction being strongly alkaline, and are in that case removed when this is corrected, or are due to a finely floccu- lent precipitate of albuminates, which are cleared away by adding the white of a hen's egg and boiling for a quarter of an hour [with subsequent filtration]. Preparation of meat extract bouillon. — A second mode of making bouillon consists in the combination of meat-extract and sugar to obtain a liquid culture medium. The fol- lowing is Hueppe's process : — To a litre of water are added ^ per cent (5 grams) of extract of meat and 3 per cent. (30 grams) of dry peptone, or instead of extract of meat and peptone, 2 to 3 per cent. (20-30 grams) of peptone of meat. A further addition of five grams of grape or raw sugar is then made, and the liquid boiled, carefully neutralised with solution of sodium carbonate, and subse- quently sterilised. Admixture of glycerine with the bouillon is also advantageous, and tubercle bacilli grow excellently on the medium thus obtained. Solutions of white of egg. — Solutions of white of egg are well suited to form fluid culture- media after they have been completely freed from germs by the discontinuous method of sterilisation. The white of plovers' eggs lends itself well to this purpose, being clear and transparent, and capable of dilution with water, and of being filtered ; it admits also of the addition of dextrine, sugar, or other ngredients according to need. The albumen when sterilised affords for a considerable time a suitable medium for culti- vations in test tubes or on glass plates placed in the moist chamber. Solid nutrient media. — Owing to the introduction of the use of solid culture media in bacteriological research, a series of micro-organisms have been more thoroughly examined, and it has thus been possible to observe that 38 BACTERIOLOGY certain characteristics in the growth and multiplication of the elements in this way are more distinctly brought out and that the individual forms are thereby specially distinguished. Preparation of peptone bouillon gelatine. — The Koch- Loffler peptone bouillon gelatine is that most in use, and is prepared in the following manner : — 500 grms. of meat freed from fat are minced up fine, mixed with a litre of water, and allowed to stand for twenty-four hours, after which the mass of meat is squeezed out, the nitrate boiled in a water- bath for three quarters of an hour until all albuminoid bodies are precipitated, and then filtered again. Another method is to place the meat, over which a litre of water has been poured, at once upon the fire, between the flame of which and the vessel a plate of asbestos must be interposed. The broth is made to boil for several hours and then let cool, in order to separate the fat, after which it is filtered and sufficient water poured in to replace that lost by evaporation. 100 grms. gelatine, 10 grins, colourless pep- tone, and 5 grms. common salt are next added to the filtrate, and this mixture is allowed to stand for some time and then heated in the water-bath until all the gelatine is dissolved. In order that the gelatine may be colourless, the solution must not become concentrated while being heated in the water-bath, but from the very commencement as much water must be added, from time to time, as it ap- pears to have lost by evaporation. The reaction of gelatine is always acid, and this is also the case with broth, so that it must be neutralised with a concentrated solution of sodium carbonate or with solution of caustic soda. The entire mass is next filtered through a creased filter paper in the hot-water funnel.1 The creased paper must be moistened with warm water before filtering, as otherwise 1 [The paper is not folded in the manner usually adopted, but creased in folds radiating from the centre, somewhat like a circular fan ; the piece Nl TRIENT GELATINE 39 the pores would be clogged by solidification of the gelatine. To avoid using the hot-water funnel, Kirchner suggests allowing the gelatine to cool slowly in the steam steriliser, after turning out the flame ; it is then after a few hours perfectly clear and can be filtered with facility. Instead of the folded paper, a thin layer of cotton- or glass-wool may be used for filtering. If a sample of the filtrate is taken in a test-tube and heated until it boils, it must remain clear and should also not become cloudy while cooling. Any turbidity, if such occurs, may possibly be due to the gelatine having been rendered too strongly alkaline in neutralising ; as in heating such a solution the carbonic acid is driven off, and then compounds are thrown down which cause the turbidity. It need hardly be said that care must be taken in such cases to neutralise exactly in order to get an efficient gelatine. That perhaps other faults, such as inferior paper, dirty vessels, &c., may be to blame, is also evident, and such must be avoided in the preparation of nutrient media. Cloudiness is most easily dealt with by adding the white of a hen's egg to the lukewarm gelatine while it is still fluid, and shaking so as to divide it finely, after which the solution is again boiled and filtered in the hot-water filter. Indeed it is the rule to add the white of an egg to nutrient gelatine immediately after neutralising, so as to ensure the avoidance of all faults of turbidity. The gelatine when ready should be clear and of an amber-yellow colour, and should not become cloudy on heating. Carefully cleaned test-tubes are filled with about 10 cubic centimetres each and plugged with cotton wool, or Schill's double test-glasses may be used (see p. 16). The should be about eighteen inches square, and folding is begun by doubling it down the centre. The creased paper is finally gathered up, inserted into the funnel, and the superflous part cut off.] — TR. 40 BACTERIOLOGY test-tubes are sterilibed 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 30 grm. peptone have been added. As soon as the gelatine is dissolved in water the other ingre- dients are mixed in and the whole boiled, after which the solution is filtered off by means of the hot- water funnel and neutralised. Should clouding by any chance occur, recourse must be had to those measures described in speak- ing of the preparation of peptone bouillon gelatine. When this has been sterilised — which must be done with par- ticular care, as the extract of meat contains many germs— a culture-medium is obtained which can in most cases be used exactly like the preceding. The finished gelatine is stained a brownish colour, owing to pigments derived from the meat extract. Both these modes of preparation yield gelatines which are most extensively used in all bacteriological researches. Attempts are now made to alter the culture-media by adding various substances to the gelatine, such as grape- xiiynr (up to 2 per cent.) and dextrine or ////// v/v we (4 to 6 per cent.). By means of these different admixtures, the nutritive value of the gelatine for certain micro-organisms is said to be increased. In summer the gelatine has a tendency to liquefy, and its strength must accordingly be increased from 10 to 15 per cent. ; while for cultivating anaerobes a 7J per cent, gelatine is required. Additions to nutrient gelatine. — A modification deserving of special notice is litmus gelatine, which is prepared by mixing a tolerably concentrated solution of blue litmus with the gelatine, thus obtaining the substance to which this name is given. Its importance lies in the fact that the acids or alkalies formed by micro-organisms in their growth can thus be qualitatively demonstrated. It is advisable to add the most widely different sub- 42 BACTERIOLOGY stances to the gelatine, so as to meet individual require- ments in cultivating micro-organisms, and accordingly the greatest multiplicity of admixtures have been recommended by approved investigators. For example, Koch uses mixtures of gelatine with blood-serum', aqueous humour, infusion of hay and of 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 algae 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 NUTL'IKNT AGAR-A(;.\i: 43 90° C. Hence this jelly is well adapted for use as a culture- medium for those micro-organisms which must be grown at the higher temperatures in the incubating chamber. Agar-agar appears commercially in the form of transparent strips, or four-cornered pieces, or as a white powder, and swells up in water. Preparation of peptone bouillon agar. — To make nutrient agar (peptone broth a;/ 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 Eichter's method the agar is dissolved in wine by two hours' mace- ration and subsequent boiling, and the solution is added to bouillon. Tischutkin allows the required quantity of agar to swell for 15 minutes in a very dilute solution of acetic acid, washes it in pure water, and thereupon adds it to the broth, in which it dissolves after only 3 to 5 minutes' boiling. When it has been neutralised and cooled, the whites of two hen's eggs are poured in, and the mixture kept in the steam apparatus for half to three-quarters of an hour. The sub- sequent filtration occupies only a short time, even without the hot-water funnel. The agar when ready is filled into sterilised test-tubes closed with cotton-wool, in such a way that about a third of the test-tube is occupied by the fluid. Care must next PEPTONE BOUILLON AGAR 45 be taken that the nutrient mass be sterilised, which is done by heating the filled test-tubes in the steam steriliser for twenty minutes daily on three successive days. The tubes when filled and sterilised are laid in a slanting position, for which purpose suitable contrivances of various kinds are employed, the result being that the surface of the agar after setting forms a very acute angle with the long axis of the tube. During solidification some water, the water of condensation, separates out and prevents the firm adherence of the agar to the vessel, so that the medium often turns if the test-tube is rotated, a phenomenon which does not disappear until the water of condensation has evaporated. Esmarch recommends the addition of some ;i um arable 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 grm. peptone to a litre of water. Sterilisation must be carried out with greater care than in the case of ordinary nutrient agar, and the prepared medium is of a brownish-yellow colour. The most varied modifications of nutrient agar can be produced by the addition of solution of litmus, grape- sugar, and other substances soluble in water. That most commonly used is the litmus aX KOll HOLDING THK PLATK.-. The plate a]>jntr«tits (see p. 27) consists of a triangle with feet formed by levelling screws, on which rests a glass vessel covered with a thick plate of glass and filled before use with iced water. It is rendered horizontal with the aid of a spirit-level and covered with a bell-glass. The sterilised plates having cooled under this bell, the first of the inocu- lated test-tubes is then unplugged, its upper edge is heated in order to sterilise that part over which the gelatine has to flow, and its contents are poured from a small height upon the plate in such a manner that the gelatine spreads out over it in a fairly thin layer. In a short time the gelatine sets under the bell-glass, and the plate is then brought into a moist chamber and laid upon either little glass benches 56 BACTERIOLOGY or pieces of glass which have been sterilised. The same process is gone through with the second and third inocula- tions, and the three plates can be laid on benches one above the other in a single moist chamber, or a separate one appropriated to each of them. The moist chamber consists of a large glass box, which is disinfected with corrosive sublimate solution and has a circular piece of blotting-paper moistened with a 1 per 1,000 solution of the same substance laid upon the bottom (fig. 25). The plates prepared as above are left at the temperature of an ordinary room until the individual cultures show them- selves on the surface. These appear in the form of islets Gelatine plate Glass bench FIG. 25.— MOIST CIIAMBEE. 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 57 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 FIG. 26. — GELATINE PLATE, COLONIES OP VAiimrs I--OU.MS 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 Unna, Fodor, and others have quite recently described contri- vances for facilitating the operation. Roll cultures. — A modification of the plate process which is known as roll culture has been invented by Von Esmarch, in which the gelatine, after being liquefied and inoculated, is kept rolling round the sides of a wide test-tube until it sets. Eoll cultures are prepared by inoculating the tube in the usual way, closing it with a cotton-wool plug which has first been singed in the flame, and drawing over the cotton an india-rubber cap sterilised in solution of corrosive subli- mate. The tube is then seized by its upper end with three fingers of the right hand and by its lower with three fingers of the left, laid horizontally in a vessel of iced water, and kept turning on its long axis until the gelatine has set in a layer of even thickness. The roll cultures when finished must at once be conveyed into a cool place. Modifications of the plate process. — A quicker and more convenient procedure is the introduction of portions of the gelatine into Petri's capsules. In using Soyka's plates a small quantity of liquefied gelatine is deposited in each hollow, and prepared with the seed-material by means of a platinum needle. By transferring the material from one hollow to another, it is possible to have all degrees of attenuation on the same plate. In order to economise gelatine Giinther places on a sterilised surface of glass a few drops of sterilised water or bouillon, lying isolated from one another. A sample of the material to be examined is mixed with the first drop by means of the platinum wire, and by the same means the inoculating matter is transferred to the remaining drops one after the other, the needle being continually sterilised at a red heat after each inoculation. From the last drop a MODIFICATIONS OF THE PLATE PROCESS -V.) 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 (Strichcultureri). In order, however, to render it available for plate cultivation, Hiippe mixes it with an equal quantity of a warm solution of agar ; and it may also be suspended in gelatine. Unna in- creased the coagulability of blood serum by rendering it strongly alkaline, in order to add it to gelatine or agar. This medium has special excellences for a number of micro- organisms. The albumen taken from plovers' eggs which have been previously sterilised with 1 per 1,000 corrosive sublimate may be inoculated and then dried over sulphuric acid under the receiver of an air-pump. Plates so inoculated can be kept for a considerable time if preserved in a sterile place, and when laid in a moist chamber there appear on the thin dry transparent film of albumen variously shaped areas of different sizes, which may be transferred to other culture media. Plover's egg albumen may also, like blood serum, be mixed with gelatine or agar, and so used for plate cultures. The micro-organisms can be transferred to other media from agar and serum plates in the same way as from gela- tine, and the appearances presented by them in their growth observed. Cultivation of anaerobic micro-organisms. — For the culti- vation of anaerobic microbes, that is, those which grow with a scanty supply of oxygen, or when it is totally excluded, an entire series of methods have been devised, of which the following are a few. The most direct plan is to introduce at once into the nutrient medium such substances as will extract the oxygen present, a result which is attained by adding to nutrient gelatine 2 per cent, of grape sugar or O'l per cent, of resorcine, or to liquefied nutrient agar J per cent, of formic acid or of sodium sulphindigotate. CULTIVATION <>I ANAEROBES Gl In order to prepare plate cultures of anaerobic iuicr<>- uryanisms 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 rcmnral <>f the oxyyen fnun 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 cm. from the bottom to a narrow neck. It is filled with 10 c.cm. of nutrient material by the aid of a funnel, closed with cotton- wool and sterilised. After inocu- lation, the wool plug is pressed down as low as the narrowed part and a tight-fitting rubber cork introduced into the mouth, through a hole in which passes a right-angled tube of glass connected with an air-pump. The air is then pumped out (the culture medium in the rarefied space being meanwhile kept in a water-bath at 30°-40° C.), after which the tube is sealed at the constricted part over the flame of 62 BACTERIOLOGY a Bunsen burner, and the gelatine rolled out by Esmarch's method. In order to displace the air in a test-tube by means of hydrogen, Fuchs recommends that the inoculated tube should be inverted and hydrogen conducted into it from below through a glass pipe, after which the test-tube is closed with a rubber cork. For test-tube cultivation, however, Liborius' method of preparing high cultures is specially adapted. A tube is filled high up with gelatine or agar, which is then freed from air and oxygen by thorough boiling and is cooled to 40° ; the matter to be inoculated is distributed in it as evenly as pos- sible with a platinum needle, and it is made to set rapidly in iced water. By this means the deeper layers of the nutrient mass are protected from the air by those higher up, while the superficial ones are exposed to the action of oxygen. When several varieties of bacteria develope, a means is hereby afforded of distinguishing the aerobes which grow on the surface, from the anaerobes growing in the deeper parts. High cultures are also used for obtaining thrust culti- vations (Stichculturen) of anaerobic micro-organisms, the platinum needle charged with infecting matter being thrust as deeply as possible into the stiff nutrient mass. Although at first • development only occurs in the deeper parts, the growth gradually mounts upwards as the gaseous products of its metabolism displace the air from the higher layers of the medium. Nikiforoff cultivates the anaerobes in the ' hanging drop.' A cover-glass is prepared with an inoculated drop of bouillon and sealed to a hollowed slide with a layer of vaseline. Between the edge of the cover-glass and that of the well in the slide, the contents of a platinum loop of strong solu- tion of pyrogallol are allowed to flow in on one side, and PEILMANKXT (TLTURES 63 on the opposite, after pushing aside the cover-glass, a similar quantity of caustic potash, so that the two fluids mix when the object has been brought into the right position. Hens' eggs seem to afford a suitable medium for anae- robic cultures. This kind of cultivation, which is recom- mended by Hiippe and Heim, has been described more in detail above (see p. 50). In cultivating ollif/ate anaerobes the materials used in the investigation must, according to Kitisato, be previously heated, in order to remove by this means the facultative anaerobes. Permanent cultures. — To preserve cultivations of bacteria so that they can be examined at any time, evaporation of the moisture contained in the nutrient medium must be prevented, as well as all possibility of contamination, and consequently the vessels must be hermetically closed. Krai punches cylinders out of boiled potatoes, cuts them into discs, and places them in round glass boxes, the covers of which are tightly ground on and the channels in them filled with glycerine. After inoculation they are closed germ-tight with paraffin and spirit varnish. Cultures may also be preserved in test-tubes hermetically sealed by melting the glass. Prausnitz pours an aqueous solution of gelatine con- taining 1 per cent, of carbolic or 5 per cent, of acetic acid over thrust cultures placed in iced water, and then closes them with corks and seals them. By Duclaux's method the cultures of bacteria are en- closed in the small tubes used to contain lymph. Jacobi first exposes the gelatine which contains the colonies, and which has been spread out in the thinnest possible layer, to the action of 1 per cent, bichromate of potash for from one to three days in the presence of light, 64 BACTERIOLOGY hardens it in alcohol, and cuts it into pieces which can then be strained like sections of tissue, and thus rendered permanent. To preserve small portions from agar plates, Giinther lays them in a drop of glycerine placed on a slide, deposits thereon a second drop, and finally the cover-glass. The superfluous glycerine having been absorbed out the prepara- tion is closed with cement. CHAPTEB 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 /*, = the thousandth part of a millimeter), or by comparison with other similar forms, especially red corpuscles. Examination in the hanging drop. — A very useful method of observing freshly-obtained micro-organisms is the examination in the ' hanging drop.' For this purpose the end of a platinum wire is bent with the aid of a pliers into a little loop. When this is dipped into a liquid containing bacteria, enough of the liquid remains adhering to it to 66 BACTERIOLOGY form a small drop, which is transferred to a cover-glass. A ' hollow ' slide is then taken, that is, one with an exca- vation or well ground in the centre ; this well is surrounded with vaseline by means of a fine hair pencil, and the cover- glass with the drop turned downwards is laid on the slide in such a way as to adhere firmly to the vaseline. If the substance, the microbic contents of which it is desired to examine, is not a liquid containing bacteria, but animal tissue or solid culture medium, a drop of sterile water or sterile salt solution is conveyed on to the cover-glass with the loop of the platinum needle, and a minute sample of the mass to be examined is transferred into the drop. In observing the hanging drop the edge must first be sought for with a low power, and then focussed with a higher ; since, as it appears bounded by a sharply- drawn line, the micro-organisms in the drop can in this way be more easily focussed, which very much facili- tates the examination for beginners ; and, morever, the elements are met with in a thinner layer at the border than in the centre. As the elements under observation are not stained, the narrowest possible aperture of the diaphragm must be used in the examination. With the hanging drop attention must in like manner be paid to the peculiarities which can be observed in bacteria examined in the fresh state, as detailed above, and their 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- STAIXIXfi o| MICROORGANISMS 67 cesses have been established in the course of the researches which have been made up to the present that we must describe them here more in detail, especially as they afford marks by which bacteria can be distinguished, and consequently staining is of the highest importance in determining the individual varieties. Staining of micro-organisms. — Staining constitutes an indispensable aid to the study of the finer structure of the micro-organisms, and of their relation to the cells of the body. In carrying it out a series of colouring matters are used which have been already detailed (p. 30), and solutions are prepared from these in different ways, which are employed both for isolated micro-organisms and also for those in the tissues. The basic aniline colours are for the most part kept in stock in alcoholic solutions which are mixed with water before use, so that we really employ a dilute alcoholic solution in staining. The dilution, however, must not be carried too far. Giinther has pointed out that absolute alcohol is not suitable for use in staining with basic aniline colours, just as it is incapable of extracting the dye from cells when once they have been stained. Simple staining of cover-glass preparations. — In this, the simplest kind of staining, the mode of procedure is as follows : A sample of the matter to be examined is con- veyed on to a cover-glass with the point of the sterilised platinum needle and is diluted, if needful, with water, after which the organisms suspended in the water are spread out over the surface of the glass with the flattened end of the needle (smear preparation) ; or a better way of managing this is to press another cover-glass upon the prepared one, and then slide it off, so that the mass -under examination appears equally distributed on both cover-glasses. The mass which contains the micro-organisms is frequently F 2 68 BACTERIOLOGY found to have so much moisture as to render the addition of water superfluous. If it is desired to examine the juice of organs, a piece of the organ is seized in a forceps and the cover-glass smeared with it, dried in the air, and passed three times through a flame to fix the micro-organisms to its surface, after which the staining is done by depositing a few drops of dye on the infected surface of the cover- glass, or by pouring some into a watch-glass and floating the cover-glass upon the solution with the prepared side downwards. After from one to five minutes it is freed from superfluous stain by washing in water, is dried in the air —a process facilitated by soaking up the drops with blotting- paper — and is mounted in fairly fluid Canada balsam ; or, if it is not wished to preserve the preparation, it may be examined in water, or in a very dilute solution of potassium acetate. Such objects are examined with ordinary or homogeneous immersion objectives by the aid of Abbe's illuminating apparatus without a diaphragm. Coloured preparations admit of being seen with distinctness, and their outlines can be accurately determined, such figures being spoken of as ' coloured images,' to distinguish them from the unstained * structural images,' which should only be examined with a diaphragm of narrow aperture. When Abbe's apparatus is used without a diaphragm, all the rays which enter the lower lens, and which form a very obtuse-angled pencil, are enabled to reach the object. Bacteria are difficult to observe in fluids and tissues, being only visible through the shadows caused by the differences in refractive power of the several structures. Hence but little light must be allowed to reach the prepara- tion, and consequently as small a diaphragm as possible used, and the result is an impairment of distinctness. If, however, the bacteria be stained, it becomes possible to PREPARATION <>l' STAINING SOLUTIONS 69 remove the diaphragms, and to examine with the full power of the Abbe's illuminator. The coloured image is best if the structural image be effaced by rendering the shadows of the unstained parts invisible in the broad cone of light. It must further be remarked regarding the structural image, that the diaphragm should have the narrowest possible aperture with a low power, but should increase in size as higher powers are employed. Preparation of staining solutions. — For the simplest kind of staining of bacteria solutions of fuchsine, methyl blue, gentian violet, Bismarck brown, vesuvine (in equal parts of water and glycerine), and methyl violet are used. Gentian violet and fuchsine stain quicker and more intensely than the others. In order to increase the staining power with the various micro-organisms, certain innococci, 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 rioJetj yt'nt'mn riolet, and Victoria blue). Gunther's modification of Gram's process. — Not only pure alcohol, but also alcohol to which 3 per cent, of hydrochloric acid has been added, is used for decolorising. The cover- glass or section of tissue is left for about two minutes in aniline water gentian violet, but in the case of 78 BACTERIOLOGY bacilli the dye is allowed to act for twelve hours, and in that of lepra bacilli for half a day. Superfluous stain is removed with blotting-paper, and the preparation is brought for two minutes into solution of iodine and potassium iodide, then for half a minute into pure alcohol, for exactly ten seconds into 3 per cent, hydrochloric acid in alcohol, and immediately afresh into plain alcohol for several minutes, changing the spirit as long as any colour is extracted from the preparation. The cover-glass is now dried and mounted in balsam, and sections of tissue are laid in xylol (which renders them transparent in half a minute), and then mounted on slides with Canada balsam dissolved in the same liquid. Weigert's modification of Grain's process. — The sections stained with gentian or methyl violet are not transferred to alcohol from the iodine solution, but laid upon slides and covered with aniline oil, which dehydrates and differentiates them. The aniline oil is then removed with blotting-paper, xylol is poured upon the preparation, and it is put up in Canada balsam in xylol. Impression preparations. — These are made for the purpose of rapidly gaining an idea, when examining plates, regarding the arrangement of the colonies and the microscopic peculiarities of the organism under investigation. A cover- glass is laid on the plate, pressed gently down, lifted care- fully with a forceps, and laid aside to dry. It can then be stained like an ordinary cover- glass preparation. Examination of micro-organisms in sections of tissues.— The examination of micro-organisms in the tissues, whether in the interior of the individual cells, or in the structures which are formed by them, is of pre-eminent importance in research directed to medical ends. Not only have the nature of the micro-organisms and their mode of entrance into the body to be discovered, but attempts must be made to EXAMINATION OF MICRO-ORGANISMS IN TISSUE 79 ascertain their bearing towards the elements of the tissues, and their exact situation in and between them. In par- ticular, their physiological action, in spite of very advanced methods of investigation, has not up to the present been fully explained. When 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 Abbe's condenser and a diaphragm). We cannot, however, ascertain by this method how the microbes are related to the tissues, nor their exact situation in the tissue and its elements. A small piece of it should there- fore be torn up with needles and treated with a drop of acid or caustic potash, so as to cause the connective tissue which forms the bulk of organs to swell up. In this way the bacteria, which exhibit greater power of resisting re- agents, are brought out distinctly. Through the introduc- tion of staining processes, however, methods have been discovered which render it possible to demonstrate the micro-organisms in uninjured tissue. Examination by the freezing method. — In order to be able rapidly to examine pieces of organs, recourse is had to the freezing microtome. The substance in the fresh state is laid upon a roughened metal plate and frozen by means of an ether spray apparatus. It is then cut into sections with a cooled knife, and these are laid on slides, allowed to thaw, and subjected to staining processes which will be described later on ; after which they are most conveniently examined in dilute glycerine. Hardening. — Owing to the frequent destruction of the tissue in using the freezing microtome, caused by the 80 BACTERIOLOGY crystals of ice which form, the organs are usually not cut until they have undergone a hardening process. In Histology an entire series of reagents is employed for hardening, the use of which is, however, impracticable in Bacteriology, because they deprive bacteria of their property of taking up aniline colours easily. The most convenient way is to harden the pieces, which should each be a cubic centi- meter in size, singly in absolute alcohol, which must be changed several times. The alcohol may be obtained as free from water as possible in the following way : Crystals of copper sulphate (blue vitriol) are heated in an iron capsule with frequent stirring until they have completely parted with their water of crystallisation and subsided to a white powder, which, after cooling, is introduced into a bottle and the alcohol is poured over it, when it greedily extracts the water therefrom, becoming again blue. As the piece of tissue contains water, it sinks to the bottom if thrown into absolute alcohol, and the hardening process goes on more slowly in the lower than in the upper half of the vessel, the alcohol above being less rich in water. Hence it is advisable to keep the organ in the upper part of the alcohol, either by means of a layer of cotton wool, or by suspending it with a thread fastened outside. A half per cent, chromic acid solution, with or without the addition of platinum chloride and acetic acid, has also been recommended, as in it the bacteria are well preserved. After eight days the pieces are rinsed in water until that which flows away shows no yellow coloration, and the hardening is then completed in alcohol. Instead of chromic acid, a concentrated aqueous solution of picric acid renders good service. The pieces are left in this for two days, washed for twenty-four hours in water, and transferred first to dilute, and from that to absolute, alcohol. 81 Portions of tissue so fresh as to be still warm are best hardened in mm w/rr n nil i mate. 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 stainer1 in use at the author's Institute is well adapted for this purpose. Several sections having been laid in serial succession upon a slide of larger size than usual, are covered with a nickel- plated grating and clamped in the section stainer. The whole is then passed through the various fluids and stains 1 Sold by Siebert in Vienna. IN RVRAI-TIM: 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 sfrial i>rr]m ration (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 FIG. 27.— SKCTIOX STAINKK K<>K I'KKI-AI: ATIONS IMI:KI>I>:.I> IN CKLLOIDIXK. 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. o 2 84 BACTERIOLOGY The sections are transferred one by one to xylol, in order to extract the paraffine from them, and are thence brought into alcohol and then into water. If they do not sink in the water the paraffine has not been completely removed, and in this case they must be returned to the alcohol and from that into xylol, and then transferred afresh to alcohol and water. After removal from the water they are sub- jected to suitable staining processes, cleared in xylol, and mounted in xylol Canada balsam. Oil of cloves should not be used for clearing the tissue, as it decolorises the micro-organisms. In the preparation of serial sections a softer paraffine is used for imbedding ,' the imbedding-block is otherwise prepared in the same way as before and cut to a square, and the microtome knife is fixed transversely. The sections, which adhere to each other, forming bands which resemble a tape-worm in outline, are laid one beside the other in corresponding order and fixed to the slide, usually with the white of a hen's egg diluted with water and glycerine. At ordinary temperatures the white of egg takes a long time to dry, but this may be expedited by gentle heating. A drop of creosote or carbolic acid should be added to the fluid to make it keep. Other fixing media are collodion, glycerine agar, or glycerine gelatine in a dilute condition. The adherent pieces are freed from paraffine with xylol, which is extracted in turn with alcohol; they are then washed in water, subjected to staining processes, rendered transparent with xylol in the same way as single sections, and put up in Canada balsam. ON THE STAINING OF SECTIONS The staining of sections is carried out after various nnthods, but a certain order of procedure is common to all. ON TTTK 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. X//l«l is employed because it behaves in a completely indifferent manner towards basic aniline colours, whether in nuclei or bacteria, wrhich 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, ]>hcnol, 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 xvlol. 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 - septiccemia a method may be used which differs from the foregoing only in the substitution of fuchsine for the methyl blue. Koch's method. — The sections after staining are trans- ferred to a saturated solution of potassium bicarbonate which has been diluted with an equal volume of water, and thence to alcohol, cedar oil, and Canada balsam. Lbffler's method. — Loffler stains the sections in an alka- line solution of methyl blue, decolorises in half per cent, acetic acid, and thence brings them into absolute alcohol, cedar oil, and Canada balsam. Chenzynsky's Method. — The sections are immersed in a methyl blue and eosine solution containing forty parts con- GRAM'S METHOD 87 centrated alcoholic solution of methyl blue, twenty parts of a •J per cent, eosine solution in 70 per cent, alcohol, and forty parts water, and after staining are rinsed in water, and the remainder of the treatment carried out in the usual way. Plehn recommends the addition of twelve drops of a 20 per- cent, caustic potash solution to the water. Grain's method. — This method is in a high degree suited for sections. They are stained in aniline water gentian violet, to the action of which they are exposed for from ten to thirty minutes ; but the time of staining may be shortened by heating. After staining they are rinsed in water and immersed for two to three minutes in a solution of iodine and potassium iodide, and are then kept moving to and fro in 90 per cent, alcohol until no more colouring matter comes away. The sections, which now appear of a slate-grey colour, are next transferred to alcohol, cedar oil, and Canada balsam. The bacteria are seen in violet on a yellowish ground. Double- staining with picrocarmine or Magdala red causes the violet tint of the micro-organisms to stand out distinctly against the red colour of the tissue. The method of Gram may also be reversed, and the sections first stained for fifteen minutes in picrocarmine or Magdala red, rinsed in 50 per cent, alcohol, and then laid in aniline water gentian violet. After decolorising in iodine solution, the preparation is treated with alcohol, oil, and Canada balsam. Giinther's modification, which is characterised by the exposure of the sections, after decolorising in alcohol, to the action of a 3 per cent, solution of hydrochloric acid, yields brilliant results (compare p. 54). Kuhne's modification of Gram's process. — Gram's method has undergone many further modifications in its use for sections, and Kiihne in particular has devised a number of processes, of which the following are the most important. 88 BACTERIOLOGY A solution is prepared of 1 grm. Victoria blue in 50 c.cm. of alcohol diluted to half its strength, and this is again diluted to the same exten with a half per cent, aqueous solution of ammonium carbonate. Staining lasts from one to five minutes, and the sections are decolorised in iodine and potassium iodide, and further treated as directed by Gram, except that instead of alcohol a solution of fluoresceine (1 grm. fluoresceine to 50 c.cm. absolute alcohol) is used for extracting the colouring matter. A further process consists in adding some hydrochloric acid (1 drop to 50 grms. water) to a concentrated aqueous solution of violet and using this to stain the sections, which are otherwise treated as in Gram's method. In using carbolic methyl blue the sections are stained for from half an hour to two hours, rinsed in water mixed with hydrochloric acid, passed through a weak aqueous solution of lithium carbonate, and transferred from that to absolute alcohol and to aniline oil, in both of which a little methyl blue has been dissolved. After rinsing in pure aniline oil they are cleared in an etherial oil which is then removed with xylol, and are mounted in Canada balsam. Pragl recommends a modification of the carbolic methyl blue method, which consists in staining the sections, fixed to a slide or cover-glass, for from half to one minute in carbolic methyl blue, after which they are rinsed in water for a short time, decolorised in 50 per cent, alcohol, de- hydrated in absolute alcohol, cleared in xylol, and mounted in resin. Another method which is easily applied consists in stain- ing the sections for three to five minutes in carbolic fuchsine, rinsing in water, and passing through alcohol. They are then laid for a quarter of an hour to two hours in aniline oil containing some methyl green, in order to decolorise and KI'IINK'S AM) AYKIGKKTS MKTIKHiS 89 differentiate them, and after clearing with an etherial oil and removal of this with xylol, are put up in Canada balsam. In the fluorescent <>- 'natHs Kdlimis ] have been added, and then into pure alcohol, bergamot oil, and balsam. The cliroinir )n<'tJunl 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 tropaeoline. 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 flanie, 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 pharmacopoeia con- sists of 200 parts of potash soap and 100 parts of spirit of lavender, prepared from lavender flowers by maceration and distillation.]— TB. 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 — l 1st. That its presence can be detected with the micro- scope in all cases of that disease. 2nd. That it is never found in any other disease. 3rd. That when isolated and cultivated through many generations a culture inoculated on a susceptible animal invariably produces a disease identical with that in the animal from which the virus was taken, and 4th. That the same bacteria are found to be present in the animal so inoculated.] Transmission can easily be effected on the cutaneous surface, or on the mucous membranes of readily accessible 1 [See on this subject Giinther's Einfilhr. in das Stud, der BakterioL, pp. 139 et seq., 2nd ed.j — Tn. INFECTION JJV THE DIGESTIVE CANAL D3 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 maybe 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 oesophageal 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 oesophageal 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 Kietsch 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 oesopha- geal tube, and the experiment of introducing infection by the intestinal canal is complete. Subcutaneous infection. — Inoculation can also be per- formed subcutaneously by introducing the infecting matter beneath the skin with a Koch's syringe. In the case of small animals, such as white mice, the hair of the back in the neighbourhood of the tail is carefully removed, a minute incision is made into the skin with disinfected instruments (forceps and scissors), and the infecting matter introduced subcutaneously with the help of a sterilised platinum loop. Experiments of transmission into the peritoneal and pleural cavities, or into the organs themselves, are con- ducted after a similar fashion. INFKCTION INT<) 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 CHAPTEE V THE BACTEBIOLOGICAL 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 POUCIIET'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, Connoctinir ml). Aspirator FIG. 28.— POUCIIET'S AEKOSCOPE. it is possible to gain an approximate idea of the number of micro-organisms contained in the air. Pouchet's method. — Pouchet employed for the examina- tion of the dust of the air an aeroscope consisting of a glass cylinder, capable of being closed air-tight by means of a screw and clamps ; it is placed vertically upon a stand and perforated above and below. In the upper aperture is* a glass tube with a very narrow exit, the lower one communi- cates with an aspirator through an indiarubber pipe, and in the centre of the cylinder is a little table supporting a 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 Tube connected with — the aspirator — Glass flask FIG. 29. — MIQUEL'S APPARATUS FOR EXAMINING AIR. from this the number spread over the whole plate is calcu- lated, and thence the amount contained in a litre. Instead of the glycerine plate one of gelatine or agar may be laid on the little table, and an attempt thus made to isolate the micro-organisms (fig. 28) . Miguel's method. — Miquel constructed a flask with two lateral tubes (fig. 29) and another fitting by a ground joint into the aperture at the top, and supporting a cap or head of glass closed with a cotton-wool plug. One of the lateral tubes is connected with an aspirator, the other (by means of a piece of rubber piping) with a narrow glass tube sealed at one end. The flask is filled with 30 to 40 c.cm. water, and sterilised in the steam current ; the glass cap is then taken off and a given volume of air aspirated through, after which MI:TIIOI> 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) . Plug of cotton wool - Aspirator tube ( '<>\ led tulx; containing nutrient bouillon Fia. SO.—EMMEUICH'S APPARATUS FOR KXAMIMM; Ant. 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 100 BACTERIOLOGY may be able to flow into it ; and in this way a volume of air, corresponding to the quantity of water used, is aspirated into the receiving bottle. For the purpose of regulating the flow, two little glass tubes, drawn out to fine points, are fixed in the india-rubber tube which connects the two aspirating bottles. Cultivation is effected by conveying 1 c.cm. of the fluid in the receiving flask (after it has been thoroughly mixed) by means of a sterile pipette into 10 c.cm. gelatine, and pouring this out on plates. Hesse's method. — In this method the air is caused to pass by means of a small slowly-acting aspirator through a disinfected tube, the walls of which are coated with gelatine after the manner of Esmarch's roll cultures. This tube is 70 cm. long, and has a diameter of 3 to 4 cm. ; it is placed horizontally, and covered at one end with a tightly- stretched rubber cap having a round piece cut out of the centre, and over which a second cap, not perforated, can be drawn ; while the other end is closed with a caoutchouc cork, bored, and fitted with a small glass tube about 1 cm. wide and 10 cm. long, connected with the aspirator. While the air is being aspirated, the unperforated cap must be removed. Two bottles are used by way of aspirator, as in Welz's method, one filled with water, the other empty (fig. 31). The bacteria develop chiefly in the fore part of the tube, while the spores of moulds, being isolated and therefore lighter, are carried further and develop further on in the in- terior. When air is examined which presumably contains but few germs — for example, air out of doors during a calm— 10 to 20 litres are drawn through, but if it is probable that large numbers are present only 1 to 5 litres are aspirated. The process is concluded by replacing the unperforated rubber cap. In a few days the gelatine is seen to be covered with colonies which can be distinguished from one another by their form, their colour, and their action on the MKTHOD OF STRAUSS AND \VURZ 101 gelatine (liquefaction). The germs may then be isolated by further transference to culture plates, and submitted to microscopic examination. Cla-> tub.- 1! nbber cork Aspirator . M: \ i r- i .PI: i. \.\MI o 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 uir Vend niir.1 with -datiiu lateral tubuhnc Fiu. 32.— AIK-TKSTIXG APPARATUS OF SiRArss AXD Wcnz. the side and connected with an aspirator (fig. 32). A large volume of air, 100 to 200 litres, is thus tested, and when the aspiration is concluded the gelatine is poured out on 102 BACTERIOLOGY plates, or a roll-culture is made after Esmarch's method in the vessel itself. Petri's Method. — A sand-filter is prepared and carefully sterilised. This consists of a tube 8 or 9 cm. long and 1-5 cm. in diameter, into which sand is introduced after one end has 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 grain of which is £ to ^ mm. in size, is the best. About 50 to 100 litres of air are drawn through with a water aspirator Sand-filter -=^^ IPS^Win- netting Aspirator tube Receiving bottle — I FIG. 33.— PETRI'S SAND-FILTERING APPARATUS. at the rate of some 10 litres per minute, the quantity being determined by means of a gas meter. When aspiration of the air is concluded, each sand-filter is partitioned out separately into several glass capsules prepared with nutrient gelatine or some other solid culture medium. The second layer of the filter should be free from germs. Miquel uses powdered sodium sulphate to absorb the microbes instead of sand. Tyndall's method, &c. — Sterilised cotton wool is used for absorbing the micro-organisms, instead of air-filters con- sisting of substances in the form of powder, and is then transferred to gelatine, and plate cultures made therefrom. I'KMC'ILLIUM ULAUCT.M 103 Percy Frankland uses glass wool instead of cotton. With the aid of these methods, moulds, T/tv/x/x, mirm- cocci, laciUi, and xpirillu 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 Mimn-iH'rnn pn>- d'Kjiosus 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 PeninU'unn < -<>< •< us I-Yclatis. 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, Friinkel, and Von Eiselsberg, it is identical with the Streptococcus pyogenes (see p. 201). Bacillus subtilis. — This bacillus, also called the hay bacillus, was described by Ehrenberg, and is most easily obtained from an infusion of hay made by chopping up the hay, pouring water on it in a flask, and bringing it once to the boil. In this way all the other different micro-organisms are easily killed, the hay bacillus alone suffering no impairment of vitality. After two or three days a thick whitish pellicle forms on the surface, and consists of a pure culture of the BaciU/u* subtilis. The bacillus takes the formof 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 Yandervelde, 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 Kiibler, 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- faction Funnel-shaped area of lique- faction Fio. 39. — THRUST-CULTURE IN GELATINE OP BACILLUS, SUBTILIS (THIRD DAY). Needle-track in the unliquefied part FIG. 40.— THRUST-CULTURE IN GELATINE OP BACILLUS PRODIGIOSUS (FOURTH DAY). red colour. The thrust-culture liquefies from the surface down, and soon a funnel-shaped area of liquefaction is 116 BACTERIOLOGY formed, upon the surface of which the pigmentation takes place, owing to contact with the air, and then sinks gradually downwards (fig. 40). A beautiful purple-red colour develops on the surface of streak-cultures on agar, but the finest growth takes place upon slices of potato or wafers, on which, moreover, it progresses very rapidly at the temperature of the room, being less luxuriant at higher degrees of heat. The bacillus liquefies serum, and soon appears on plovers' egg albumen with a beautiful rose-red colour, which extends only as far as the coagulated mass has become liquid. The coating shows a punctiform appearance under a low power. The spectrum of the pigment, which is readily soluble in water, alcohol, and ether, has three absorption bands, one to- wards the violet end of the spectrum at [Fraunhofer's line] D, one at E, and another at F. The red colour becomes brown in the .air, owing to the action of ammonia, but recovers its raspberry-red colour if acetic acid be applied. Wyssokowitsch, and quite recently E. Ullmann, have proved that dead cultures of this bacillus are capable of exciting suppuration, and Grawitz and De Bary found that its pathogenesis is connected with the pigment. Potato bacillus. — Three varieties are distinguished: Bacil- lus mesentericus fuscus (Fliigge), Bacillus mesentericus ruber (Globig), and Bacillus mesentericus vulgatus. They show short filaments which are often connected together into chains, and have the power of active movement. They liquefy gelatine very quickly during their growth, whether on the plate or in thrust-cultures, and form round colonies which soon become yellowish, and in the case of the brown bacillus (Bacillus mesentericus fitscns) 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 ruler shows at a higher HACILLLUS 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 llnnUnx ni<'s<>ntcrirnx nilimtnx 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 ItanUns mcxt-nti-rii-ns /'//-sr/^.s have a yellowish colour, and those of the ntber variety a reddish, the membrane on the _ Peripheral radiating processes Liquefied part FIG. 41.— ISLET OF BACILLUS irasBNTERicus VULGATUS ox A GELATINE PLATE. potato shows no pigmentation at all in the case of Bacillus mesentericus nilijatn$. The potato bacillus develops with particular readiness on pieces of potato which are not completely sterilised, often destroying the cultures of other micro-organisms. Bacillus liodermos. — Fliigge found very widely distributed in the atmosphere, and often as a guest upon our nutrient materials, short, exceedingly motile rods, the growth of which on gelatine causes it to liquefy with great rapidity, a white pellicle floating on the surface. In thrust-cultures dirty grey flakes swim about in the fluid mass. A smooth, glossy coat resembling thin mucilage develops on potato, changing as the spores form into a thick and much- wrinkled membrane. The mucilaginous mass is soluble in 118 BACTERIOLOGY water. The Bacillim liodrnnos 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 Schrotter, in the caterpillars' excreta found in worm-eaten apples. It is at times a constant inhabitant of the air of the author's laboratory, and often appears as a guest upon cultures of other micro-organisms ; and it is possibly identical with the Bacillus butyri fluorescens, found by Lafar in butter. It consists of slender, fairly long rods, with smoothly rounded ends and actively motile, and is distinguished by its unusually rapid growth, so rapid that even in four hours there appear on the plate greyish-white colonies, in which darker and more closely-packed masses are to be seen ; while as early as the second day the gela- tine is liquefied with development of a greenish-yellow colour. In thrust-cultures also, on the second day, an hourglass-shaped depression shows itself, around which there is very rapid liquefaction (fig. 42). The speedy growth and greenish-yellow colour, are also seen in super- ficial cultures on agar, the surface of which very soon becomes overspread with a thick yellowish coating, while all the rest of the medium acquires a green tinge. On plovers' egg albumen it grows with a splendid emerald green colour, and on potato it forms a dirty reddish-yellow layer. The pigment developed by the Bacillus melochloros is very readily soluble in water, but not at all in alcohol or chloroform. It is destroyed by acids, but restored again by alkalies. Older cultures acquire an exceedingly unpleasant odour. When the pure culture is injected into the veins or peritoneal cavity of rabbits the animals perish in a week at furthest. Bacillus multipediculosus, which was discovered by Fliigge, shows small thin immotile rods. The colonies on a gela- ] \\ ( ' 1 1 , LUS MULTIPE J )ICULOSUS 119 tine plate appear under a low power as circular, sharply- defined discs with radiating processes, resembling insects Hour-glass depression • Liquefied portion . Superficial coating Xeedle-track .Needle-track Fi<;. 42.— THUUST-CULTUKE ix GKLATIXE OP BACH. i, rs MKi.nriiT.iiuiis < DAY). FIG. 43.— Tunt'sT-Ctri/rriiK ix GELATIXE "i IlMMEliiCH's BAfiLU's (Rac. Nea- politan //>•». with numerous radially arranged feet. In thrust-cultures also the processes appear extending from the needle -track 120 BACTERIOLOGY in all possible directions, and these peculiar projections have procured for this micro-organism its name of ' multi- pediculosus.' Gelatine is not liquefied. Bacillus neapolitanus. — This microbe was first discovered by Emmerich in the blood and in evacuations from the corpses of cholera patients in Naples, and it was subsequently ascertained to be present in normal faeces. Pathogenic powers were ascribed to it, because a disease resembling the cholera in human beings develops after the introduction of considerable quantities of it into the bodies of guinea-pigs, dogs, cats, and monkeys ; the introduction may be effected subcutaneously, or into the abdominal cavity or the lungs. Microscopic examination demonstrates the bacilli in all the organs. There have, however, been objections made by Weiser to ascribing pathogenic properties to it, and he has shown that it is present in the air also. The bacillus appears as a short rodlet with rounded ends and destitute of motile power, which forms on the gelatine plate colonies resembling porcelain and lying at a greater or less depth, of which the superficial ones spread as a coating over the surface of the gelatine, and the deep have a figure like that of a whetstone. In thrust-cultures the more vigorous growth takes place on the surface (fig. 43). Gelatine is not liquefied, but loses its alkalinity, which causes a clouding of the transparent jelly and a simultaneous separation out of crystals of salt. If tincture of litmus is added to the gelatine the blue colour disappears and becomes changed to a red. A dirty white mass forms on agar and potatoes. With regard to staining processes, it is a special characteristic of this micro-organism that it does not colour by Gram's method. Its resistance to external influences is so great that it retains its vitality after being frozen for twelve days and then thawed again. ATMosi'III.llIC SPIRILLA 121 Atmospheric spirilla. — The spirilla occurring in the air have been described by "VYeibel. 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 slmrp-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- scribed by Koch, which was discovered in drinking water in the neighbourhood of Calcutta, and the bacillus of typhoid fever ; but besides these, others also occur as a contamination of water. Some micro-organisms cannot grow in water alone, as it does not afford sufficient pabulum for their development, but large numbers also perish from being overwhelmed by the growth of the water bacteria. Very many of the micro-organisms met with in water generate pigment, often in such quantity that considerable volumes appear coloured or fluorescent owing to it, and a few exhibit a brilliant phosphorescence. Filtration and filters. — Microbes are removed from water by filtration, for which purpose use is made of sand filters constructed with sand and gravel, charcoal filters of plastic carbon, niters 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 sanurntn* (Wolffhiigel's counting-plate, fig. 45) consists of a black slab upon which the plate with the gelatine culture is laid, and over this is arranged a pane of glass on which squares of uniform size have been engraved. The islets in the individual squares are then counted with the help of a lens, and an average struck, when the number so obtained multiplied by the total number of squares on the plate gives approximately the total number of colonies for a certain area, a number which varies with different kinds of water. The water to be used in this experiment must not be kept, but must be examined imme- diately after collection. In examining water presumably rich in germs — for example, that from rivers or ponds — the L26 BACTERIOLOGY volume of water used for observation must be diluted with sterilised distilled water (generally in equal parts or in the proportion of one to nine), as otherwise the colonies lie so close together that they cannot be counted, or else they liquefy the gelatine too speedily. The counting apparatus can be rendered complete by cutting a piece of some size from the upper part of a square box, and placing a plane mirror obliquely in the interior. If the gelatine plate be now laid upon the box, the number of islets on each square can easily be ascertained by the transmitted light. Pfuhl's method. — If the examination can be carried out immediately at the spring, the water to be analysed is poured into sterilised vessels, which are at once closed with a sterilised plug of cotton-wool. To obtain the water without catching extraneous germs, Pfuhl uses flat-bottomed glass tubes partially emptied of air, and having the ends drawn out into capillaries, bent at a right angle, and sealed. The points are broken off actually at the spring, and the tubes filled with water and again sealed. For the purpose of transport small cylindrical glass bottles, provided with ground-glass stoppers, are used, which have been sterilised and covered with india-rubber caps. To collect the water from a delivery-pipe the cap and glass stopper are removed, the bottle completely filled, and carefully closed again. The water which first flows away, however, must not be used for examination. To obtain water from a spring the rubber cap is taken off, but the stopper is only removed under the surface of the water, which is allowed to flow into the flask for about a minute, and then the bottle is closed again and lifted out and the rubber cap drawn over the glass stopper. Kirchner's method* — About 36 cm. length of glass tube, of the diameter commonly used for making connections OF EXAMINATION \'2i between apparatus, is bent to a U form in the flame, and both ends arc 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 sonie preliminary information as to what micro-organisms are present, a few drops of the water to be examined are evaporated on a cover-glass, which is drawn several times through the gas flame to fix the dry residue and covered with one or two drops of a staining solution. In washing off the stain, the stream from the wash-bottle should not be directed on the actual deposit from the water. It is advisable that every examination by culture should be preceded by sedimentation, for which purpose Finkelnburg has contrived an apparatus consisting of a cylindrical vessel with a bottom capable of being lifted out. Water being allowed to drop in through an opening in the bottom which can be closed by a glass tap, the floating particles gather on the movable bottom of the glass, and in this way a de- position of the organised impurities can rapidly be obtained. Csokor's or Gartner's centrifugal machine serves this pur- pose still better. In order to isolate the bacilli of cholera and typhoid and other bacteria endowed with motility, 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 aqiiatilis. — 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 a rose- red deposit both on agar and potato. Micrococcus fuscus, described by Maschek, consists of immotile cocci which frequently have an elliptical form. Bound light- or dark-brown colonies appear on the gelatine plate and speedily liquefy, and in the canal of a thrust- culture liquefaction also progresses with tolerable rapidity, a sepia-brown pellicle forming on the surface of the fluid. The slimy deposit on potatoes is also distinguished by a brown colour. Micrococcus luteus. — This, which was described by Cohen, consists of small immotile elements, forming a rather flocculent zoogloea. It appears in irregular colonies on the gelatine plate, while in thrust-cultures a yellow deposit is 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 Mi< rococcus fn-r'ulnxiis, 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. A dirty grey deposit forms on potato. Diplococcus luteus. — This diplococcus, found by Adametz, forms rather long chains, whole pieces of which move about in a lively manner in the hanging drop. It liquefies gela- tine slowly and forms round brownish -yellow colonies which spread with tolerable rapidity. In thrust-cultures the growth is more active on the surface, showing lemon - yellow deposits in concentric layers. It appears on agar as a yellow or brownish-red coating, while that on potato is dirty yellow and exhales a mouldy odour. Caseine is precipi- tated from milk by cultures of the micro-organism. Bacillus fluorescens liquefaciens and Bacillus nivalis (glacier-bacillus). — These both display short, thick rods possessing the power of easy movement. Pick found the Bacillus fluorescens liquefaciens also in the conjunctival secretion, and Schmolk found the Bacillus nivalis in glacier ice. Both form on the gelatine plate colonies which have a funnel-shaped hollow in the centre, and exhibit a greenish- yellow fluorescence. Thrust-cultures grow slowly in the deeper part, but somewhat more rapidly in the case of Bacillus fluorescens liquefaciens than in that of Bacillus nivalis, and the former shows on the surface a depression resembling an air-bubble, owing to evaporation, whereas with the latter the liquefaction extends over the gelatine. The non-liquefied portion of the medium shows a greenish- yellow fluorescence. A whitish layer appears on agar along the inoculated streak, and the mass becomes fluorescent. The deposit on potato is yellowish-brown. Bacillus fluorescens non-liquefaciens. — A bacillus closely BACILLI IX WATER 131 related to the above-described fluorescent but liquefactive bacilli has been discovered in the HarMus ///^/v.sw//* )H)n-li<{tt<'fa<-i<>ns, small rods destitute of motility which form on the gelatine plate shimmering colonies with indented edges, having a darker spot in the centre, and a lighter coloured leaf-liki- figure all round. In thrust-cultures there is a superficial growth of considerable vigour, but nothing can be observed along the needle-track ; the shimmer, how- ever, pervades the whole of the gelatine. This bacterium is distinguished from the Barilla* erytkrotponu by the fact that the latter shows red-coloured spores. Bacillus erythrosporus was found by Eidam in drinking- water and in various putrefying albuminous fluids. The rods are slender, have rounded corners, and are actively motile, and the cultures are characterised by the develop- ment of a dichromatic pigment, appearing orange-yellow by direct, green by transmitted light. The colonies on the gelatine plate are circular, and show in the centre a darker spot around which spreads a wider light zone. Round every colony fluorescence appears, and soon spreads over the gelatine, so that the entire plate exhibits the phenome- non. Thrust-cultures show a growth upon the surface from which the fluorescence advances deeper until it extends along the whole- track of inoculation. A reddish colour, becoming later nut-brown, develops on potato. The spores are characterised by a red gleam, which gives them the ap- pearance of being stained with fuchsine. Bacillus arborescens has been frequently detected by P. Frankland in the water-supply of London. Its rodlets are thin and motile, and it appears on gelatine plates in iridescent colonies resembling a trunk with its branches, the latter being arranged in sheaves. A superficial irides- cence is visible likewise in the thrust -cultures, and slow liquefaction soon sets in ; while on agar and potato a yellow K 2 132 BACTERIOLOGY or orange-coloured growth develops, with an iridescent margin. Bacillus violaceus, — In water of a violet tint, in that from rivers and water-works — in the Thames and the Spree, for in- stance— there are found motile rods with rounded ends, whose colonies on the gelatine plate are at first small, resembling air-bubbles enclosed in the gelatine, but later on liquefy it and form granular islets of a bluish-violet colour, which are distinctly visible as early as the fourth or fifth day. In the thrust-culture a funnel-shaped area is formed by the liquefaction, to the bottom of which the blue-coloured masses of bacilli sink. A dark blue coating develops on agar, and the bacillus grows very well on potato, spreading out radially from the place of inoculation until the disc is nearly covered. Blood serum and plovers' egg albumen are liquefied with the formation of a violet colour. Bacillus gasoformans. — The gas- forming bacillus consists of small highly motile rods, and forms islets on the gelatine plate, which, though at first small, rapidly liquefy the medium and spread into its substance as well as over the surface. In this way a capsule-like figure is formed, in which bubbles of gas are visible ; but the formation of these bubbles is especially characteristic in the clear gelatine along a thrust-canal. The gelatine is speedily liquefied. Growth only takes place at the temperature of an ordinary room. Bacillus phosphorescens. — Fischer found the Bacillus phosphorescens indigenus, or native luminous bacillus, in phosphorescent water from Kiel harbour. Its rods are short, rounded at the ends, and actively motile, and will not grow on serum nor on potato. Small round colonies form on the gelatine plate, which liquefy rapidly, and in about eight days present the appearance of holes cut with a punch in the gelatine, and evidently containing air. The young colonies are sea-green, the older of a dirty greyish- BACILLUS PIIOSPHORESCEXS 133 yellow, forming variously shaped flakes. The thrust-culture is of a slightly conical or sand-glass form, with a thin de- posit on the edge of the gelatine ; but in old cultivations the colonies become heaped on the bottom without fluid. The light given off by them is of a bluish-white colour, and if traces of the culture be added to sea-water, it acquires the property of phosphorescence like that observed at sea. Closely allied to the foregoing is the Bacillus phos- phorescens iudicus, or Indian luminous bacillus, which was also found in sea-water by Fischer, and which forms small, thick, energetically motile rods. Round, sharply-defined colonies develop on the gelatine plate about the third day, and are also of a sea-green colour with a rosy shimmer, but later turn dirty yellow. The thrust- culture shows on the fourth day a cavity filled with air on the site of the puncture, and later the loss of substance increases, the liquefied gelatine being covered by a dirty yellow pellicle. A white layer de- velops on potato. Boiled fish and p(o meat furnish a good medium for the growth of the micro-organism, which is checked at temperatures below 10° C. The fact of its growing on potato distinguishes it from the indigenous species. In the presence of air and moisture it shows a luminosity in the dark, best at a temperature of 25° to 30° C. Both bacilli admit of being photographed by their own light. They grow best on herring gelatine, which is prepared in a manner similar to ordinary gelatine. Bacillus ramosus, or root-bacillus (Wurzelbacillus) , shows short rods with rounded ends and possessing slight power of movement, and is found in the water both of wells and rivers, and also on the surface of the ground. Small 134 BACTERIOLOGY colonies with ill-defined margins develop on the gelatine plate, and later on send out shoots resembling the filaments Liquefied portion Processes extending radially from the needle-track FIG. 47.— THBUST-CCLTUKE ix GELATINE OF BACILLUS EAMOSUS (THIRD DAY). of mould. When, after growing for some time, the fibres have become entangled with one another, they form an interlacing root-like network (fig. 46), an appearance which BACILLUS RAMOSUS 135 is seen still more characteristically in the thrust-culture, pro- cesses springing out radially from the needle-track, after the manner of fibres from a tap-root. The aspect of the culture has been aptly compared to that of an inverted pine-tree (fig. 47) . Older cultures are completely liquefied, and carry a pellicle on the surface. The ramification is equally distinct upon agar, and spores are also formed on potato. On plovers' egg albumen there soon develop whitey-grey colonies growing in root form, which become continually more and more interlaced and entangled. The liquefaction of the albumen is very tardy. Bacillus aurantiacus. — This bacillus, found by P. Frank- land in deep wells, shows only a feeble motility. The rods are often arranged in pairs, and grow out into threads. Gelatine is not liquefied. Small raised colonies appear on the plate, which are at first dark-coloured but subsequently become bright orange-yellow, and this orange colour is also very distinct on the surface of thrust-cultures, whereas the track of the thrust shows no growth. On agar the deposit is also of an orange colour. On potato the growth is re- stricted to the spot inoculated. Bacillus aureus. — Adametz found the Bacillus aureus in water, and Unna upon the skin of persons suffering from sebnrrhoeic eczema. The rodlets are slender, have but slight motility, and do not liquefy gelatine. The colonies on the gelatine plate are irregular, coalesce with each other, and develop a golden-yellow pigment, which also occurs in the form of hemispheres on the surface of the streak-culture. On potato the golden -yellow colour soon changes to a brownish red. Bacillus bruneus. — The. brown bacillus described by Adametz and Wichmann shows small irregular rods. The colonies, which do not liquefy the gelatine in their growth, are rounded and whitish at first, later brown. The brown 136 BACTERIOLOGY colour is also developed along the whole length of the thrust- canal in the gelatine. Bacillus aquatilis, which was described by P. Frankland, exhibits short, feebly motile rods. On gelatine plates the colonies develop in the deeper parts, and spread thence to the surface, liquefying the gelatine. From the surface bundles of filaments extend to the periphery. In thrust- cultures a weak yellowish coloration first shows on the surface, and liquefaction takes place comparatively late. The growth on agar is restricted to the line of inoculation, and on potato the colonies show a similarly meagre de- velopment. The bacillus was found in the water of deep wells sunk in a chalky soil. Bacillus aquatilis sulcatus, — Weichselbaum found four micro-organisms in the water of the high-level water-works of Vienna at a time when water from the Schwarza was laid on,1 and these he designates as Bacillus aquatilis sulcatus 1-4. The first three forms have motile elements, those of the fourth variety are shorter, and do not move. On the gelatine plates colonies develop which are thicker in the centre than at the periphery, and possess a distinctly coloured border, while on their surface furrows can be seen with a low power crossing one another at the most varying angles, and this network of wrinkles is denser in the centre than at the periphery. The points of distinction of these varieties are limited to variations of temperature and differences of odour, which is like that of whey in the first form, resembles urine in the second, and is exceedingly unpleasant in the third, while the cultures of the fourth variety give off no smell. A superficial growth appears 1 The drinking-water supplied to Vienna is brought from the Schneeberg , a distance of 100 kilometers. In summer, however, sufficient cannot be obtained from this source, so that other water must be laid on in addition, and this is derived from the Schwarza, a little river in the vicinity of the Scemmering. BACILLUS UK.MIJIIANACKUS AM KTIIYST1XUS 137 in thrust-cultures. The fourth form does not grow on potato. Bacillus aquatilis radiatus. — Zimrnermann found this bacillus in the water of the Chemnitz water- works. The rods are motile and form irregular colonies on the gelatine plate, with root-like processes arranged in a radiating manner. The gelatine is liquefied with tolerable rapidity, and the mass of bacteria is collected in the centre of the fluid colony, while all round it extends a yellowish-coloured ring which is environed by a cloudy yellowish mass. Thrust - cultures show a circular pellicle on the surface when lique- faction has taken place. A layer forms on agar which appears bluish by direct, yellowish by transmitted light. Bacterium Ziirnianum, described by Adametz, displays short, immotile rods. The colonies, which do not liquefy gelatine, form masses like clusters of grapes, and the surface of thrust-cultures has also a similar appearance. It grows on potatoes at 25° to 30° C. Bacillus membranaceus amethystinus. — Eisenberg and Jolles have discovered this bacillus in the spring-water of Spalato. It has the power of liquefying gelatine with de- velopment of a dark violet pigment, and its rods are short and immotile. Small, dark violet colonies develop on gelatine plates and liquefy slowly, while a violet pellicle appears on the surface of the liquefied mass, resembling a membrane stained with gentian violet. A deposit with serrated edges develops on thrust-cultures and acquires a violet colour in about a fortnight, slowly liquefying the gelatine. The liquid mass is covered with a violet pellicle in this case also. A whitish layer, which later becomes violet, forms upon agar; the deposit on potato is of a dirty olive-green, while a violet sediment and a violet pellicle develop in bouillon. Bacillus indigoferus. —The bacillus of this name, detected 138 BACTERIOLOGY by Claessen in the water of the Spree, has slender rods with rounded ends and showing vigorous motility, but which often adhere together and form entire clumps, and seem to possess a jelly-like envelope. The colonies, which do not develop on the gelatine plate until the third day, contain an indigo-blue pigment as early as the day following. In thrust-cultures a punctiform mass of a deep indigo blue appears round the site of inoculation even in twenty-four hours, and the gelatine is not liquefied. On agar an indigo- blue layer develops over the extent of the line of inocula- tion, its surface being covered with a shining pellicle. On potato an intensely deep indigo blue develops in three or four days, but only when the reaction is acid. The formation of pigment does not seem to depend on the presence of light. Bacillus ianthinus was found by Zopf in the Chemnitz water-supply. It consists of short rods with active motile power, which liquefy gelatine, and form a bluish-violet pigment. Bacillus ochraceus. — Zimmermann obtained from the same source as the foregoing a bacillus with ochre-yellow pigment, which has been described as the Bacillus ochraceus. Gelatine is slowly liquefied. The rods show little motility. Bacillus gracilis, — Zimmermann described a micro-or- ganism to which this name has been given on account of the form of the rods, which are slender and fine and possess an oscillating rotatory motion. Gelatine is liquefied. On agar a bluish-white pigment appears. There is but little growth on potato. It has the property of thriving better in the substance of the solid media than on the surface ; hence it will also develop under the mica plate (see p. 61). Bacillus sulphydrogenus. — Miquel found not only in drinking-water, but in rain and canal water, an anaerobic bacillus possessing the peculiar property of decomposing albumen with the formation of sulphuretted hydrogen. Its BACILLUS OF CHOLERA 139 cells are very short motile rods, which increase in length when grown artificially. Carbon dioxide and hydrogen are liberated from a nutrient medium which contains no sulphur. 1 grm. of sulphur is decomposed in forty-eight hours in a bouillon mixed with ammonium tartrate and with sulphur in excess. Other bacteria, however, also generate sulphuretted hy- drogen, to prove the presence of which it is only necessary to hang strips of paper saturated with lead acetate in the test-tubes containing the cultures, when a blackening of the paper will be found with most water and air bacilli. According to Mace this reaction becomes more distinct if some flowers of sulphur be added to the culture medium. Bacillus of Asiatic cholera, and allied micro-organisms.— Koch discovered a comma- bacillus in the evacuations and the intestines of cholera patients, and adduced evi- FIG^-CHOLERA BACILLI, FRO! A PIKE CULTURE. (After Jaksch.) dence proving that this micro- organism is the cause of the disease. He found it also in the water of a tank in the Neighbourhood of Calcutta. Ex- periments have shown that the comma-bacilli can multiply very wrell in sterilised water, whereas in ordinary water they are soon overgrown by the water-bacteria ; and they can tolerate just as little the concurrence of other bacteria, and consequently perish in decomposing liquids, although Gruber has obtained pure cultures from alvine evacuations which had been allowed to putrefy for a week. The pathogenesis of this bacillus has, however, recently been rendered doubtful by the experiments (performed on themselves) of Pettenkofer and Emmerich at Munich, and Hasterlik in Vienna ; and of Dr. A. J. Wall, of H.M. 140 BACTERIOLOGY Indian Army, in London, who swallowed cultures of the bacilli. Cholera bacilli appear as plump rods curved in the direction of their long axis so as to resemble a comma in figure, hence the name ' comma-bacillus ' (fig. 48) . They have a twist in addition to this curve, so that they represent a kind of spiral bacteria, and on this account have been described as Vibrio or Spirillum cholera. The bacteria lie connected in chains forming a tolerably steep spiral, and when examined in the hanging drop show an exceedingly lively motion, so that they might not inaptly be likened to a swarm of midges dancing all over the field. Loftier found a flagellum at one end of the cell. Koch was unable to detect spores in them, but Fliigge, on the other hand, has succeeded in demonstrating the formation of arthrospores. They offer but small resistance to the influence of chemical substances, and are destroyed by the acid of the gastric juices ; and they refuse to grow upon feebly acid gela- tine. They also perish at temperatures above 50° C., but grow at room temperature as well as in the incubator. Drying causes speedy loss of the power of development. They stain in about ten minutes in diluted alcoholic solu- tions of the aniline dyes, but the process is accelerated by heating the solution, which also increases the intensity of the stain. Special importance is to be attached to the fact that they are decolorised if Gram's process be employed. Staining is done as follows : An alcoholic solution of fuchsine or methyl violet is diluted with water sufficiently to form a fairly strong aqueous staining fluid, which is poured into a watch-glass. A trace of the pure culture or a minute flake from the intestinal contents is next placed upon a cover- glass and spread out by rubbing lightly with another laid over it, after which the two glasses are drawn apart, dried in air, grasped with a forceps and passed several times through a STATXIXG OF CHOLERA BACILLI 141 flame to fix the micro-organisms, and then laid on the surface of the staining solution with the infected side down- wards. The solution is allowed to act for ten minutes on the preparations, with, or even without, slight warming, and it need hardly be said that rare must be taken to exclude all substances liable to decompose the stain. The cover-glasses are next taken from the solution with the forceps, washed in water, and dried with the prepared surface uppermost, after which a drop of Canada balsam is laid upon the prepared face of the glass, which is in this way cemented to the slide. Microscopic examination is carried out with the help of the oil-immersion lens and Abbe's condenser. AVhen a sample of the material to be examined for cholera bacilli is diffused through gelatine, and a plate- culture made, colonies are soon obtained possessing in- Non-liquefled marginal portion FIG. 49.— ISLET OF BACILLUS CHOLHUS ASIATICS ox A GELATINE PLATE, IN PROCESS OK LIQUEFACTION. dented and bowed margins, and presenting, when magnified 100 diameters, an appearance as if strewn with bits of glass. After a time, small round excavations appear, corresponding to the colonies, and soon give the entire gelatine the aspect of pock-marked skin, the funnel-shaped cavities being due to evaporation of water from the slowly- liquefying gelatine. The liquefaction does not extend very far at first (fig. 49). In thrust -cultures also the gelatine liquefies very slowly, the liquefaction being chiefly seen on the surface, and in this case too evaporation of the fluid occurs, so that a bubble communicating with the external air appears in the upper part of the funnel-shaped excavation. From this bubble a thin prolongation runs down along the track of 142 BACTERIOLOGY inoculation, and comes to resemble a capillary tube as the liquefaction advances downwards and the evaporation pro- Superficial air-bubble Xeedle-track FlG. 50. — THKUST-CULTUBE IN GELATINE OF THE CHOLERA BACILLUS (THIRD DAY). gresses. When liquefaction has gone still further the bacilli sink in the needle-track, and the coherent mass of bacteria forms a loose thread which is sometimes thicker GROWTH OF CHOLERA BACILLI 143 and sometimes thinner, and which extends to the bottom of the funnel-shaped liquid cavity (fig. 50). In four weeks liquefaction has made such progress that the entire mass of gelatine is fluid. Cholera bacilli grow with a fair degree of luxuriance upon other media also, forming on culture bouillon a wrinkled, much-folded membrane, while the bouillon itself remains tolerably clear. Only on shaking do the masses rise from the bottom and render it cloudy. They grow also in sterilised milk, but in milk which has not been freed from germs they undergo speedy destruction, owing to the occurrence of acid fermentation. In sterilised water they grow rather actively and main- tain themselves for a considerable time, so that cholera bacilli find in many places the conditions required for their increase. On agar the bacilli grow in the form of a whitish la}rer spreading out from the line of inoculation. On potato they thrive even when the surface shows a slightly acid reaction, but only at 30° to 40° C. In the region surrounding the site of inoculation there develops a greyish- brown layer, which gradually spreads and presents an appearance almost identical with that exhibited by cultiva- tions of the gliimli-rx bacillus. Blood serum is slowly liquefied by the growth of cholera bacilli. The inoculated streak in the case of plovers' egg albumen (Von Hovorka and F. Winkler) becomes covered with a coating which reflects more light than the surrounding mass of transparent white of egg, and consequently looks lighter. If examined with a lens the streak, which slowly widens, exhibits closely-packed, distorted colonies with a grey sheen, partly united with one another. There is no liquefaction of the nutrient medium. 144 BACTERIOLOGY During the progress of the disease the bacilli are found principally in the mucous membrane and contents of the intestinal canal, but not in the blood. To examine cholera stools for bacilli, the evacuation is mixed with an equal bulk of alkaline meat -bouillon and let stand in an open glass for twelve hours at a temperature of 30° to 40° C. An abundant development on the surface results, and preparations are obtained by this method of in- vestigation which consist of cholera bacilli only. To re- cognise the presence of the bacilli even without the micro- scope, a good way is to add 10 per cent, hydrochloric acid, which after only a few minutes stains the cholera cultures a rose-violet colour, forming the cholera reaction. The introduction of pure cultures in bouillon into the stomach of guinea-pigs re- sulted in Koch's hands in an unmistakable infection, when FIG. 51.— FINKLER-PRIOR BACILLI FROM A Combined with the admillis- PURE CULTURE. (After Jaksch.) tration of sodium bicarbonate and opium. To avoid the detrimental action of the gastric juice, and at the same time to diminish intestinal peri- stalsis, Nicati and Eietsch ligatured the ductus choledochus and made the injection directly into the duodenum. Intro- duction of cholera cultures into the circulation also causes the death of the animals, and in that case the bacilli can be detected in all the organs. The problem frequently arises, to determine with cer- tainty whether in a given case it is the bacilli of cholera Asiatica that have to be dealt with, or other micro-organ- isms resembling them in form. One of these is the Vibrio proteus, which was discovered by Tinkler and Prior in the evacuations of persons suffering from cholera nostras, and FIMvLFJM'IlinK I5.VCILLUS 145 which may possibly be identical with the comma-bacillus found by W. D. Miller in carious teeth. The Finkler-Prior bacillus, or Vibrio proteus, is somewhat larger and thicker than the bacillus of Koch, but the spi- rilla formed by it are never so long as the cholera spirilla (fig. 51). The culture on a gelatine plate liquefies so rapidly and extensively that the difference between it and a culture of cholera bacillus must at once become apparent. If two gelatine tubes are inoculated by making in them two thrusts parallel with one another, the one with the bacillus of cholera Asiatica, and the other in the same way with Vil>ri<> ]»-<>t<>us, speedy liquefaction will be observed in the latter, whereas with cholera Asiatica it occurs very slowly and shows at the surface an excavation occupied by a bubble of air. Furthermore the liquefaction of Vibrio proteus ex- tends widely into the surrounding parts, the masses of bacilli sink to the bottom, and there is obtained in the liquefied area corresponding to both thrusts the form of an empty stocking-leg (the so-called ' tronscr-ler<>t< ti* grows on potato even at ordinary temperatures, whereas the cholera bacillus only grows at that of the incubator. On plovers' egg albumen a very distinct difference quickly becomes apparent, liquefaction beginning in the case of Vibrio proteus as early as the second day, and an intense yellow coloration of the entire nutrient mass soon setting in, whereas the culture of cholera Asiatica shows neither the one nor the other. The nutrient mass runs round from the sides of the test-tube towards the bottom, where it gathers in a comparatively thick stratum (Von Hovorka and F. Winkler). L 146 BACTERIOLOGY Deneke's comma-bacillus is more difficult to distinguish from that of cholera. It was grown by Deneke from old Iffl^lH^ -Expanded part of the funnel- shaped area of liquefaction Air-bubble Liquefied gelatine 'Constricted part of the liquid funnel FIG. 52.— THRUST-CULTURE IN GELATINE OF THE FlNKLER-PRIOR BACILLUS (THIRD DAY). — Mass of bacilli at the bottom of the liquid funnel FIG. 53.— THRUST-CULTURE ix GELATINE OFDENEKE'S COMMA-BACILLUS. BEGIN- NING OP THE THIRD DAY. (After Baum- garten.) cheese, and scarcely differs from the cholera bacillus in its morphological relations. It is, however, distinguished by YII5RIU MKTSrilNlKnFFI 147 its speedier liquefaction on the gelatine plate, and the yellowish colour of the colonies, which appear irregular under the microscope, and are surrounded by a thick ram- part. Like the cholera bacillus, it shows an air-bubble in thrust-cultures at the uppermost part of the funnel-shaped excavation, but the bubble is larger than in the former case (fig. 53). A delicate yellowish coat, composed of beautifully- formed spirilla, appears on potatoes at incubation tempera- ture. Like the VU>rn> j>n>t,'ns, this bacterium possesses pathogenic properties. The differential diagnosis of the Bacillnx nrdjioHttiniia (p. 120), regarded by Emmerich as the originator of cholera, presents no difficulties, as its elements are short immotile rods and the gelatine is not liquefied by their growth, which takes place more chiefly upon its surface. The Vibrio Metschnikoffi, which was found by Gamaleia in the intestinal contents in a Eussian disease of poultry, is a curved bacterium forming screw-shaped spirilla of con- siderable length, but which is shorter and thicker than the cholera bacillus. Its accurate identification is often difficult, as, although the gelatine of plate-cultures becomes fluid at times as rapidly as with Vibrio j>r<>f<'iis, the liquefaction is sometimes greatly protracted. The thrust-culture shows a distinct air-bubble, which gradually enlarges, and disappears when liquefaction is complete. It grows on potato as a brown deposit, but only at the temperature of the incubator. A peculiarly characteristic property of the cholera bacillus lies in the cholera reaction already alluded to, which was discovered by Bujwid and Dunham. Cultures of the bacilli in media containing peptone (bouillon or gelatine) give a reddish-violet or purple-red colour in a short time when treated with pure hydrochloric or sulphuric acid, in which process a definite pigment, cholera red, is developed. Salkowski explains this as an indol reaction, since that sub- L 2 148 BACTERIOLOGY stance gives a red coloration with nitrous acid, the theory being that the cholera bacilli split off indol from the peptone of the nutrient medium, and at the same time develop nitrites which are decomposed by the addition of a strong acid. Of all the other morphologically and biologically similar micro-organisms, Vibrio Mctschnikqffi alone gives this cholera reaction. According to Gruber and Schottelius, a still more trust- worthy point of difference is the fact that the cholera bacilli grow with particular luxuriance in a very dilute bouillon, and develop a wrinkled membrane on the surface in a few hours, , A., ,- ,'- /icM&3&f£tt ) ->:^V; tfrtN'* -i?iVi»-- vfcVflfKSsJS^SiSs** FIG. 54.— TYPHOID BACILLI FROM A PURE CULTURE. (After Jaksch.) FIG. 55.— TYPHOID BACILLI ('SPIDER- CELLS'). Magnified 1,100 times. (After Lbffler.) 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.1 Bacillus of typhoid fever. — The bacilli of typlioid or en- teric fever (the Typhus abdominalis of the Germans) are met 1 [For an account of recent researches on cholera vaccination, see Appendix]— TK. BACILLUS OF TYPHOID FEVER 149 with in water as well as in the faeces and organs of patients suffering from the disease. They were described by E berth and Gaffky, and have been more thoroughly studied by Klebs and Eppinger. The bacilli are short plump rods with rounded ends, the length of which is three times as great as their breadth, and which sometimes unite to form what are apparently threads of considerable length (fig. 54). Accord- ing to Gaffky and Birch-Hirschfeld they develop spores. The rodlets are distinguished by a high degree of motility, dependent, as Loffler has found, on the possession of flagella, which are present in such abundance that the bacilli, when subjected to the proper staining processes, take on the appearance of spiders (fig. 55). They thrive whether oxygen is excluded or has free access, although in the latter case the growth is more vigorous, and they stain in watery solutions of the aniline dyes, yielding up their colour, however, very 'easily on application of different bleaching fluids, so that the demon- stration of them in tissues is beset with difficulties ; and under this head it is to be observed that they lose their colour completely when treated by Gram's method. On the gelatine plate there form whitish colonies lying superficially, and at first mere dots, which have an un- evenly indented margin. Sometimes the growths lie deeper, and take the form of a whetstone. They soon become yellowish-brown, particularly in the centre of the islets. The gelatine is not liquefied. Thrust-cultures show on the surface a thin growth, which also takes place along the entire inoculated track (fig. 56) . A white layer covering the whole surface develops on agar, blood-serum, and plover's egg albumen. According to Petruschky, the typhoid bacillus is one of those which form acids. When it is desirable to come to a positive decision 150 BACTERIOLOGY regarding fthe presence of the typhoid bacillus, the experi- ment of growing it on potato is indispensable. In three or Superficial coating upon the gelatine, which is not liquefied Needle-track FIG. 56. — THRUST-CULTURE IN G-ELATIXE OP THE BACILLUS OP 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 bat-Hills 01 ' Kntmcnrh, which forms a greasy yellowish layer upon the discs of potato. E. Friinkel and AH Cohen have given prominence to the fact that this growth only occurs upon slices of potato having an acid reaction, so that the reaction of the potatoes must always be previously tested. Frankel himself, however, as well as others, most recently Kamen, have drawn attention to an atypical growth of the typhoid bacilli upon potato, in which a yellowish layer, which later becomes brown, develops slowly out from the area of inoculation and spreads in a tongue-shaped figure, the potato assuming a violet tint after some days. As a further means of recognition, Chantemesse and Widal have mentioned a peculiarity of typhoid bacilli, namely, that they thrive on a nutrient gelatine which has been mixed with 2 per 1,000 of carbolic acid, whereas all other micro-organisms perish on this mass. 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 grms. carbolic acid to 100 grms. water), deposits them for twenty- four hours in the incubator, and then treats them with one to ten drops of the water under examination. If turbidity is visible after another twenty-four hours' standing in the incubator, it may be concluded with certainty that typhoid bacilli are present. Intravenous injections kill rabbits in about twenty-four hours, when the bacilli may be detected in the urine, blood, and excreta. Infection takes place principally by means of water contaminated with the bacilli, but also by contaminated milk and linen. The microbes are capable of effectually resisting the action of the gastric juice, and as soon as they reach the intestinal canal they penetrate into the lymphatic canals and are carried by the stream of lymph into the other organs, particularly the [mesenteric glands] spleen and liver. Eberth has shown that they may penetrate into the placenta, and in this way reach the foetus. [The typhoid bacilli have also been found in the blood, not only in the rose spots (Neuhauss) but in the general circulation, having been detected in blood from the finger.] Bacterium coli commune. — Eodet and Koux 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 IX 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 ^•rapi-Mi.uar. and then generates a gas consisting of hydrogen and carbon dioxide. Rabbits succumb to a subcutaneous injection in from one to three days, witli the symptoms of diarrhoea and collapse. According to Gasser, an agar medium tinted with fuchsine is decolorised only by this bacterium and the bacillus of typhoid fever, whilst a sufficient point of distinction between these two is, that the growth of Bacterium cull Flagella FIG. 57.— SPIRILLUM UXDULA, WITH FLAGELLA. Magnified 800 times. (After Loffler.) commune remains restricted to the strip inoculated, where- as that of the typlm'nl hat-illus 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- bareuSy Micrococcus flavus liquefaciens, Micrococcus desidens, Micrococcus flavus tardigradus, Micrococcus candicans, Micro- coccus viticidosus, Staphylococcus pyogenes aureus, Staphylo- coccus pyogenes albus, Staphylococcus cereus albus, Sarcina alba, Sarcina Candida, Bacillus subtilis, Bacillus midtipedicu- losus, &c., as well as some which decompose milk and will be described under that head. 155 CHAPTEK VII BACTERIOLOGICAL ANALYSIS OF EARTH AND OF PUTREFYING SUBSTANCES Micro-organisms in the soil. — The examination of soil proves that very many micro-organisms which thrive in the air and in water can also grow in earth. Moreover, in every putrefactive process on the surface of the ground there occurs an oxidation, or resolution of organic matter with the aid of atmospheric oxygen ; consequently, in all these processes a considerable number of micro-organisms are afforded the possibility of maintaining themselves and multiplying. In agriculture the land is manured with the view of enabling highly complex organic substances to undergo decomposition on the surface of the ground into simple combinations, capable of serving as nutriment for plants and of being assimilated by them in order that they may be once more converted into higher combinations. In these putrefactive processes the action of bacteria takes a very prominent part. That it is the surface of the soil which is so very rich in varieties of germs becomes evident from the fact that at so short a distance as one to two meters beneath the surface the amount of bacteria present rather suddenly decreases, and that further down, at a depth of three or four meters, the earth is found to be completely free from germs. The ground-water level of the soil is tolerably pure in this respect, and hence also only very few micro-organisms are found in the water of springs. For the same reason fountains fed by pipes, if properly con- 156 BACTERIOLOGY structed and kept clean, deliver water which is poor in germs, or entirely free from them (C. Frankel). According to Kirchner, the freedom of ground-water from germs is due to the filtering action of the soil, and therefore, where this is too coarse and porous, the filtering power fails, and the whole or a part of the germs met with in the upper stratum of earth pass unhindered into the ground-water. According to Keimers, the germs contained in the deeper part grow more slowly than those derived from superficial layers. Method of examination. — If it is desired to examine soil, or the dust of windows and rooms, for micro-organisms, a small sample is taken — freshly, if possible — and introduced into sterilised nutrient gelatine, which has been melted but is not too hot, in order to prepare a roll-culture by Von Esmarch's method. Cultures of this form are preferable to plates, because the small particles of earth do not sink to the bottom of the tube and get missed, as is liable to happen in pouring out the contents on the plate. Special instruments are employed for obtaining earth from different depths, of which a borer constructed by C. Frankel is that principally in use. When the individual islets have been isolated by means of the roll-culture, they are transferred to different plates, in order to obtain pure cultivations of the particular organisms. The examination of anaerobic micro-organisms is carried on by the methods detailed above. The 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). — Fliigge found, as a very frequent guest in the soil of fields and gardens, a micro-organism whose rods strongly resemble those of BACILLUS MYCOIDES 157 anthrax, but are distinguished from them by a lively motility. Gelatine is liquefied. On plate-cultures there Liquefied portion Processes from the needle-track FIG. 58.— TlIUUST-CULTURK IN GELATINE OF BACILLUS MYCOIDES (FoUUTH 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). Eamifications 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. — Luderitz found the Bacillus radiatus in the ground, and in the juice from the subcutaneous tissue of white mice which had been inoculated with garden mould. Its rodlets possess a ready motility and grow anaerobically, liquefying gelatine. Upon the gelatine plate, in ' high ' thrust-cultures, and on agar, there appears a tangle of anastomosing fibres, recalling the radiating forms of moulds. A very unpleasant -smelling gas is generated in cultures on serum or sugared gelatine. VARIOUS BACILLI IN THE SOIL 159 Bacillus spinosus was also found by Liideritz in the juice from the tissues of white mice inoculated with garden mould. Like the last, it can only develop anaerobically, and liquefies gelatine. In high cultures there are visible in two days little punctiform fluid spots, from which radiating processes soon push out ; in later stages an expansion appears at the layers of gelatine above and below the track of inocula- tion, giving the slimy, liquefied mass the form of a sand- glass. This bacillus also generates a gas, which has an odour resembling cheese in growths on gelatine containing sugar. Bacillus liquefaciens magnus. — In the juice of the sub- cutaneous tissue of animals inoculated with garden mould, Liideritz also found the bacillus of this name, which consists of large rods rounded at the ends, and endowed with an active motility. It is also an anaerobe, and its growth resembles that of the moulds and liquefies gelatine, the liquefaction in thrust-cultures not taking an hour-glass-, but a more cylindrical sausage-like, form. Mossy colonies develop on agar. The gas generated by it smells very unpleasantly, recalling the odour of onions. Bacillus scissus. — This bacillus was discovered in the earth by Percy Frankland; It displays short, thick, immotile rods, and does not liquefy gelatine. The colonies on the gelatine plate develop superficially, and in thrust-cultures no growth takes place along the puncture canal, but an ir- regular deposit with smooth edges forms on the surface. A slight greenish colour is imparted to the gelatine. Besides the above there exists in the earth an entire series of different micro-organisms, amongst which especially the Bacillus rmnosus and Bacillus siibtilis are met with. They possess, however, no significance, so far as research has shown up to the present. Clostridium foetidum. — 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 foetidum is, according to Liborius, an uncompromising anaerobe, and displays rods of various lengths endowed with active motility. It admits of being easily cultivated artificially, if the care is taken to fulfil the conditions necessary for the growth of anaerobes. The gelatine is liquefied in the form of roundish globular cloudinesses occurring in its substance. Surface-cultures on agar show little collections with short processes, and colonies with irregular ramifications form in like manner beneath the surface of serum. Development of a gas takes place in the cultures, the evil odour of which has given the micro-organism its name. Bacillus cedematis maligni. — As long ago as the year 1872, Coze and Feltz found in their researches on septicaemia a micro-organism which was more fully described by Pasteur, and which obtained the name of vibrion septique, or Bacillus septicus. Koch called it the Bacillus cedematis 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. Yan Cott found them also in unprepared musk- sacs, and from this explained the circumstance that patients are occasionally attacked by malignant oedema after injection of tincture of musk. The best mode of carrying out an investigation is to introduce as much earth as will lie on the point of a knife beneath the skin of the abdomen of a rabbit or guinea-pig. The animal dies in from twenty-four to twenty-eight hours, and the bacilli are found in the cedeniatous fluid and on the surface of the organs, but not in the blood-vessels, whereas I'.ACILLl S <>r MALIGNANT (KlHl.MA 161 the bacilli of antltnu- 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 K. Pfeiffer has found, upon flagella, and form central spores. The bacilli soon lose their motility in the hanging drop, access of oxygen being fatal to them, as they are strictly anaerobic. Growth takes place either at the temperature of the room or at that of the incubator. They stain quickly in aniline dyes, but the colour is easily discharged by ap- plying Gram's method. Consequently, the points on which reliance is to be placed in distinguishing between the bacilli of malignant oedema and those of anthrax are the form, motility, distribution in the organs, manner of staining, and relation to oxygen. In gelatine cultures, which must be made with a regard to their strict anaerobiosis, small colonies occur, the contents of which soon liquefy, so that each forms a liquid globule in the interior of the gelatine. In the high cultures there is soon seen, as Liborius pointed out, an extensive decom- position of the nutrient medium, which is changed into a turbid fluid with simultaneous disengagement of abundant bubbles of gas (fig. 59). The addition of grape sugar to the gelatine may prove advantageous. Dull, cloudy, indistinctly- defined colonies of a shaggy appearance show themselves on agar after eight hours. They consist of a closely-woven network of finely granulated fibres, and develop lenticular gas-bubbles ; indeed, this development of gas is so abundant that thick layers of agar are thrown towards the upper part of the test-tube, while a considerable quantity of a cloudy, whitish liquid condenses and gathers at the bottom. A. network of bacilli forms on potato at incubating heat. The temperature most suitable for their growth lies between 37° M 162 BACTERIOLOGY and 39° C. ; under 16° C. no development takes place. The spores lie at one end of the rods, and are very resistent. Gas bubbles in the colonies Fluid globules (colonies) FIG. 59. — ANAEROBIC CULTURE OF THE BACILLUS OF MALIGNANT (EDEMA 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 TETAM S 163 mould, but, although anaerobic, it may be distinguished by the fact that its rods are not endowed with motility, and are of a different shape. An oedema containing a reddish fluid which swarms with bacilli, and is charged with bubbles of gas, is found on making post-mortem examinations of the infected animals, and bacilli are also encountered in great numbers in the peritoneal fluid. Cultures in bouillon retain their power of infection for a long time, even for several months. In the case of the mouse the bacilli effect an entrance from the tissues into the circulation, probably by penetrat- ing the walls of the blood-vessels. FII;. tin. :IIK i;.\< n.i.i - ••<• M M,II;- FK;. 01.— TKTANTS Kvi-iu.i WITH TKH- N AN I < Hi I KM A ( IlatillUS SfJJi I MINAI, Si' <:KI.ATIXK PLATE. (After Mace.) According to Penzo the bacilli of malignant O3clema, notwithstanding their strict anaerobiosis, develop in ordi- nary cultures from which oxygen is not excluded, provided these are simultaneously inoculated with Bacillus prodi'jio- siis or Protrtix ndf/aris. The oedema bacilli break up albumen, according to Kerry, producing the ordinary putrefactive processes, and, in addition, an exceedingly poisonous oily body, which is formed by the oxidation of valerianic acid. Bacillus of tetanus. — The tetanus bacillus was discovered by Nicolaier in garden mould, and in pus from the wounds of patients who had died of the disease; but Carle and Rattone had already established the fact that tetanus is M 2 164 BACTEEIOLOGY communicable. The experiments proved that these bacilli are slender bristle-like rods, having circular spores at one end and possessing but little power of automatic movement, which very often range themselves in chains or clumps (fig. 61). The tetanus bacillus is rigidly anaerobic and occurs very often in conjunction with other anaerobes, so that the disease has been supposed to be due to the united action of several of these anaerobic micro-organisms. This is described as FIG. 62. — ANAEROBIC GELA- (After Fraeukel Pfciffer.) and The bacilli stand a tolerably high tem- perature— about 80° C. — without losing their pathogenic power, but growth takes place best at incubating heat. This peculiarity, viz. that the spores can be subjected to a high temperature without losing their vitality, enabled Kitasato to obtain pure cultures of the tetanus ba- cillus, since the other bacilli cultivated along with them are destroyed at a tem- perature of 80° C., so that it is then not difficult to procure a pure culture of the tetanus bacilli, which remain alive. A trace of pus from a patient suffering from tetanus is smeared upon serum solidified in the slanting position, or upon agar, and the cultures so made are then deposited in the incubator for some days. They are next transferred for from half an hour to an hour to a water- bath heated to 80° C., in order to kill the micro- organisms which have grown along with STREPTOCOCCUS SEFTICUS 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 Strcfttocorniif wptirnx 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 anthrax bacilli — Spaces between the rods of a chain Bacilli containing spores FIG. 63. — BACILLI OF ANTHRAX. the heating must not be carried too far, otherwise the protoplasm inside the limiting membrane of the cell will undergo fine granulation. Carbolic fuchsine or carbolic methyl blue are also used for rapid staining. The cells sometimes have their ends thickened into knobs and with shallow excavations, so that an oval light spot appears between the individual cells, or, owing to the thickened nodes, they assume the figure of a bamboo rod. This latter appearance is well brought out by double staining with Bismarck brown and methyl blue. When the anthrax bacillus is grown in bouillon, long fibres are obtained which are felted together in the form of a tress of hair, an appearance which becomes visible in IJACILLUS OF AXTIIKAX 167 twenty-four hours. On the gelatine plate little white dots appear in ;i 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 (tig. 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 Kiln-mis processes at the margin Hair-like arrange- ment (if tililV- Fio. 64.— COLON v I.K THI. ANTHRAX BACILLUS ox A GKI.ATINK L'I.ATK, KI>I:MI:LIXG TI:K.-SKS OF HAIR (THIRD DAY). from the inoculated track delicate filaments push out into the gelatine (fig. 65). When liquefaction is further ad- vanced the bacilli sink to the bottom of the funnel-shaped excavation, but no skin forms on the surface, and from the deepest parts of the liquefied area processes push into the still solid gelatine. Superficial cultures on agar form a layer which can be easily raised with the platinum needle. Serum is slowly liquefied, and a dry white coating develops on potato, with considerable formation of spores. Disinfected silk threads are often saturated with such spores, dried, and kept for experimental purposes. 168 BACTERIOLOGY Infection experiments performed on different animals by inoculation and inhalation, as well as by admission through the digestive track after previously neutralising Liquefaction Air-bubble Canal of inoculation with fibrous processes FIG. 65.— THRUST-CULTURE IN GELATINE OF THE ANTHRAX BACILLI'S (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 MALA11LE 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 iiKili'/nant jni*tn1<'. According to Paltauf and Eiselsberg, the ' rag-picker's disease,' which affects persons engaged in sorting rags, especially in paper factories, is identical with anthrax of the lungs. Owing to lack of oxygen no- spores are formed by it in the body, but it is possible that they may develop on the surface of the ground where there is free access of the gas, and this renders great care necessary in disposing of the bodies of persons or animals dead of anthrax. When it is wished to make an experiment on animals the most convenient for the purpose are white mice, which are inoculated by a pocket made under the skin near the tail. The animal dies within forty-eight hours, when the spleen is found greatly enlarged, and abundant bacilli can be detected in it as well as in the blood. A trace of the blood, or of the spleen pulp, is now used to make a gelatine plate, on which the characteristic islets are found in a few days, and a pure culture can be prepared from them. Hankin obtained an excessively poisonous albuminoid body from cultures of anthrax, while Martin ascribes its virulence to an alkaloid. Plasmodium malariae. — The protozoa bearing this name, whose connexion with malaria has been established, stand in close relationship to the soil. The names associated with their discovery are those of Laveran, Marchiafava, Celli, Golgi, and Guarnieri. If a drop of blood is taken from a patient suffering from intermittent fever, at the beginning of the fit, there are found within the red corpuscles small, roundish amceboid bodies, difficult to distinguish from the corpuscular protoplasm (fig. 66). They are 170 BACTERIOLOG Y rendered more easily visible if the blood be smeared on a cover-glass with the edge of another, allowed to dry, and stained with watery solution of methyl blue ; or, according to Celli and Guarnieri, a good method is to dis- solve the methyl blue in serum or ascitic fluid, and let it run in from one edge upon a preparation of the blood, which should not be previously dried. During the attack Amoeboid figure iu a red corpuscle J *- Remnant of the red corpuscle IB&*) ^^ Other red corpuscles FIG. 66.— PLASMODIUM OF MALARIA IN HUMAN BLOOD, AT THK PERIOD OF APYKKXIA. of fever the plasmodia induce alterations in the corpuscle, causing a conversion of the haemoglobin into melanine, so that if the blood is examined after the attack the corpuscles are found to be paler, and show in their interior clumps consisting of minute granules of black pigment (melancemia) . This formation of pigment advances so far that the blood corpuscles are totally destroyed, and, according to Golgi, it Pigment Segmented plasmodium Hi iniipik TJjp HH|p : Red corpuscles FIG. 67. — PLASMODIUM OF MALARIA AT THE STAGE CORRESPONDING TO THK TIME OF ONSET OF THE FEVER. goes on between the attacks. Multiplication of plasmodia takes place by segmentation, and the new plasmodia at first adhere to the edge of the blood-corpuscle, then become free and subsequently penetrate again into other corpuscles (fig. 67). According to Golgi, the process of segmentation takes two days with some plasmodia, and with others three, before the stage is reached at which the segmented portions LA\ KUAN'S SICKLES 171 become free, and in this way he explains the occurrence of tertian and quartan fever respectively, while quotidian fever is dependent on the presence of both varieties, the new spores of one kind being set free on the first day, and those of the other on the second. In malarial cachexia there are found, besides the plas- niodia, sickle-shaped bodies inside the blood-corpuscles (' Litn-rttn'x strides '), and forms provided with flagella are also occasionally observed (fig. 68). In the individual plasmodia an outer highly-refractive part which easily takes the stain (ectoplasm) and an inner part hard to colour (cntoplasm) are distinguished, the latter Vli:. 68. — SKMII.fNAi: OK M< KU-:-SHAPKH MuDIKS, AMI I'KKK liupIKS I'KoVIDED WITH Fi.AiiKi.LA. (After Jaksch.) being enclosed by the former as by a ring. An excentrically- placed nucleus containing nucleoli is found in the entoplasm according to Mannaberg and other investigators. Artificial cultivation has not as yet been successful. It has only been possible to preserve the plasmodia in the living leech, in the digestive canal of which, as Eosenbach found, they retain their vitality for at least forty-eight hours. The simplest mode of staining is that of Grassi and Feletti, and is as follows : — A small drop of malarial blood is placed upon a cover-glass, which is inverted and laid upon a slide carrying a drop of aqueous solution of methyl blue or fuchsine; raising one side of the cover-glass a little and letting it fall again suffices to effect mixture of the blood 172 BACTERIOLOGY and the staining fluid. By this method the nucleus appears stained darker than the rest of the body of the parasite. Mannaberg has introduced the following method in order to study the order of events in the life-history of the malaria parasite : — The preparation after being dried in air is left for twelve to twenty-four hours in a mixture of equal parts concentrated solution of picric acid and water, to which from 3 to 5 per cent, of glacial acetic acid has been added. It is then deposited in absolute alcohol until com- pletely decolorised, double- stained with alum hsematoxyline solution, and differentiated in 25 per cent, hydrochloric acid in alcohol, and weak ammonia alcohol. The preparations show the red corpuscles and the protoplasm of the leucocytes unstained, but the nuclei of the leucocytes and the plasmodia strongly coloured. Malachowski lays the cover-glass preparations in alcoho and stains them in a mixture of equal parts of a 1 per cent, solution of eosine and dilute aqueous solution of borax and methyl blue (-J grm. each of borax and methyl blue in 100 c.cm. water), by which method the red corpuscles show yellowish-red, the nuclei of the leucocytes violet, and the plasmodia blue. Aldehoff recommends that the blood should be spread out on a cover-glass in the thinnest possible layer, dried in an exsiccator, and subjected for ten or twelve hours to a temperature of 120° C. in the hot-air sterilising chamber. The cover-glass is now left in a concentrated alcoholic solu- tion of eosine for half an hour, or for two or three minutes if warmed at the same time, rinsed in distilled water, double- stained by dipping it several times in a concentrated alco- holic solution of methyl blue, and rinsed repeatedly in water. The blood must be examined immediately after being taken, to avoid mistaking blood-plates for plasmodia. Other micro-organisms of the soil. — Besides the micro- ANALYSIS OF PI TIIK1 YIXG SUBSTANCES 173 organisms here described, a large number of others have been found which also occur in water or air and have been described under those heads, e.g. litn-ilhis raim^-ux, 7 >'m /'////. s- subtilis, Staphylococcus j>i/<>nnHfii<>sn*, which, however, causes only a very slow sepa- ration out of the caseine, and does not originate any further changes. Sarcina rosea. — Menge has found that the agent pro- ducing the red tint may be a sarcina to which he has given the above name. In does not, however, appear to be identical with the micro-organism of the same name de- scribed by Schrdter (see p. 109). In two days small translucent, perfectly circular colonies form on the gelatine plate, and soon assume the figure of a rosette having a red nodule in the centre surrounded by concentric rings. In the thrust-culture the surface is covered with a thin rose- red coat with jagged edges, whilst the needle-track remains colourless. Liquefaction takes place very late. On agar a coherent growth develops which is white at first but becomes coloured on the third day ; in the incubator, however, pig- mentation fails altogether. On potato which has been rendered alkaline the sarcina grows excellently. The pig- ment forms in milk independently of light. The Micrococcus of bovine mastitis. — In different diseases originated in the lacteal glands of animals by the action of micro-organisms the latter can be found in the milk also, and amongst these is the micrococcus of mastitis in the cow described by Kitt, which is met with in the partly milky and partly purulent contents of the udder of cows suffering from this complaint. On the gelatine plate small puncti- form colonies of the size of a pin's head develop, and a thrust 188 BACTERIOLOGY in the same medium produces a nail-culture without lique- faction. A wax-like layer appears on potato. Other pathogenic bacteria in milk. — In the milk of animals suffering from tuberculosis bacilli may be found, ac- cording to Bollinger, even before the udders become diseased. Typhoid bacilli may also find their way into milk along with water, and may develop abundantly there. The bacteria of cholera, too, are similarly capable of copious multiplication in milk without perceptible alteration, but they are destroyed when lactic acid fermentation sets in. In milk from a cow suffering from inflamed udder, the Staphylococcus pyogenes aureus has been detected by Kriiger. Saccharomyces ruber. — Demme has found a yeast occur- ring in milk and cheese which produces in the latter red punctiform accumulations of pigment, and in the milk a red sediment. This yeast he has described as Saccharomyces ruber. Gelatine is not liquefied by it, and it develops on the plate colonies of the size of a grain of millet-seed, which show no red colour until a week has elapsed. In thrust- cultures the growth is chiefly on the surface. Discs of potato are covered with a coating of the colour of raspberries in from eight to twelve days. Bacillus Caucasicus (Dispora Caucasica, or Kephir bacillus). —The name of kephir grains is given to maeses of micro- organisms in a zooglcea, which are used in the Caucasian districts for the preparation of the drink known as ' kephir ' from milk. In this process the caseine passes into solu- tion, while the milk-sugar is affected after the manner of a lactic acid and alcohol fermentation (Adametz). The granules contain yeast and bacteria capable of peptonising caseine, viz. the Bacillus acidi lactici, Bacillus butyricu*, Bacillus subtilis, and in addition to these a micro-organism to which Kern ascribes the principal role, and which bears the name Bacillus caucasicus. This consists of short BACILLUS MEGATERIUM 189 cylindrical rods which are destitute of movement so long as they are in zoogloea, but when isolated possess an extra- ordinarily lively motility. The name Dispora was assigned to the micro-organism from the fact that the protoplasm is withdrawn to the ends, giving the rod the appearance of being divided into two spores. By its vital action milk- sugar is changed into glucose, upon which the yeast can then begin to work. EXAMINATION OF OTHER ARTICLES OF DIET Bacillus megaterium. — This bacillus was found by De Bary on boiled cabbage-leaves. It consists of long, thick, somewhat curved rods with rounded angles, frequently connected in chains, and possessing slightly granular cell- contents. The motion of the rods recalls the amoeboid movements of cellular protoplasm. During involution the shape of the rods loses its distinctness and malformations occur, which, however, regain their normal outline on Chain with links containing spores Fio. 71.— ISLET OF BACILLI'S Fiu. 70.— BACILLUS MEGATEKICM WITH MK'.ATKHH.-M ox A GELA- SPORES. TIXE PLATK. transference to fresh nutrient substances. Their sporu- lation is particularly suitable for purposes of study, and the spores also stain easily (fig. 70). On the gelatine plate there form minute roundish colonies consisting of undulat- ing filaments, which liquefy the medium slowly, and possess a uniform or semilunar outline (fig. 71). A funnel-shaped liquefaction appears in thrust-cultures, in which the bac- teria sink to the bottom (fig. 72). On agar grey deposits 190 BACTERIOLOGY develop, which adhere firmly to the mass, and on potato there grows a dirty whitish-grey layer containing many involution forms. Funnel-shaped area of liquefaction Needle-track FIG. 72.— THRUST-CULTURE IN GELATINE OF THE BACILLUS MEGATERIUM. Besides the above, the Bacillus prodiyiostis, Bacillus sub- tilis, Bacillus amylobacter, and Bacillus batyricus are met BACILLUS ACETI 191 with on plants. F. Winkler and Von Schrotter found the Stophylococcu* />//"//''/"'* ^///v//.s- in rotten portions of apples, and the Bacillus melochloros in the excreta of cater- pillars in worm-eaten specimens of the same fruit. Bacillus aceti. — As early as 186i Pasteur brought for- ward proof that the oxidation of alcohol and its alteration into acetic acid are connected with the vinegar ferment, which could only develop in the presence of oxygen. This ferment consists of short thick rods often uniting in curved chains, and forming a mass of zooglcea which is thick and glutinous, the [so-called] ' mother of vine- gar.' Mace found another bacterium of vinegar, the zooglcea mass of which is thick, white or slightly rose-tinted, never wrinkled, and feels almost like cartilage. Numerous rods, sometimes isolated, sometimes joined in pairs or in threes, lie in a colourless interstitial substance ; they are without movement in the membrane, but when free in fluids possess a slow motility. Gelatine is not liquefied, and a thick, undulating, rather hard layer develops on its surface. A less hard, but smooth and non- undulating, layer forms upon agar. The bacteria of vinegar are very widely diffused through nature, according to Duclaux, who assigns an important part in transmission of the ferment to a kind of fly, the Mnxm (rllaris, which is met with everywhere. Bacillus indigogenus was found by Alvarez in infusion of indigo leaves. It consists of short motile rods surrounded by an envelope, and has the property of causing the ap- pearance of blue indigo in decoctions of the leaves of the Indigofera. Surface cultures on agar cause cleavage of the medium, with development of gas. Pediococcus cerevisiae. — Balcke found the Pediococcus in beer, in breweries, and in the washings from 192 BACTERIOLOGY them. It forms diplococci and tetracocci, and shows colonies on plate-cultures which are at first colourless and later brownish, and which do not liquefy the gelatine. In thrust-cultures a white, leaf-like layer is developed. An iridescent greyish-white coating occurs upon agar, while upon potato the growth is scanty. Some formation of lactic acid accompanies its development. Sarcina cerevisiae. — A number of sarcinae derived in part from the air are found in beer. Lintner encountered a variety in soured beer which, according to Adametz, occurs also in water, and which sets up lactic acid fermentation. Gelatine is not liquefied. The islets formed on plate-cultures are round, colourless, and smooth-edged, and a delicate fluorescent film develops gradually over the gelatine. Thrust-cultures show a smooth white coating, and on potato light granular islets form. Micrococcus viscosus. — A peculiar disease of wine and beer, called by the French la graisse, which consists in a clouding and inspissation of the liquor, so that it becomes stringy like white of egg, depends, according to Pasteur, upon the vital activity of the Micrococcus viscosus. The cells occur singly, or more usually arranged in diplococci or streptococci. Artificial cultivation succeeds best in solu- tions containing sugar. Bacillus viscosus cerevisiae. — In viscous beer Van Laer constantly found slender rods with no tendency to unite in groups. Gelatine is liquefied. Upon plates there develop round or oval colonies with uneven edges, which pro- ject somewhat above the surface. In thrust-cultures a white, irregularly-edged deposit is observed, while the track of inoculation also shows an abundant development of colonies. Growth on agar is very rapid, with the forma- tion of a broad white slimy layer. These bacteria also occur in milk and render it slimy. White, watery, and very tena- MOULDS OX ARTICLES OF FOOD 193 cious colonies develop on potato, and give off an odour of putrid fish. Bacillus viscosus sacchari. — Kramer assigns long rods with rounded corners, which are destitute of motility and frequently arranged in chains, as the cause of solutions of sugar becoming slimy. Gelatine is liquefied, a whitish film develops upon agar, and a firm mass upon potato. No growth takes place on acid media. Moulds on articles of food. — The moulds find abundant opportunity for development and propagation upon vege- table foods, and the three varieties, PemnUinm, Aspergillmt, and Mucor, are all represented. Pcnicillium yhiucum has already been described under the ' Bacteriological Analysis of Air' (see p. 103). The various kinds of Aspergillus flourish on bread and candied fruits. To obtain the Aspergillus niger, bread pap is prepared in an Erlenmeyer's flask. After some time stout fructifying hyphse are found, not ramified at the end like Penicillium, but swollen so as to resemble clubs. Upon these are arranged the sterigmata, at the upper ends of which the spores become segmented off and form aggre- gations which take the form of rounded, bulging black swel- lings. The entire flask soon becomes filled with fibres and grey points. Aspergillus albus and Aspergillus glaucus are similar, but grow better at incubating temperature (see fig. 3). The Aspergillus flavescens is distinguished by its well- marked fructifications and the greenish colour of the cul- tures. Aspergillus fumigatus bears very fine fructifications, and forms an ash-grey fur. Both grow luxuriantly on bread at incubation temperature. On gelatine plates fila- ments appear which spread rapidly into the surrounding parts with liquefaction of the medium. Of the Mucorineae, the Mucor mucedo, Mucor corymbifer, o 194 BACTERIOLOGY and Mu cor rltizopodiforinis are found upon food- stuffs, par- ticularly bread. They possess a branching mycelium, with spore-bearers which resemble flexible tubes, are unseg- mented, and stand up vertically from the mycelium. At the upper ends of these are the swollen sporangia, by bursting of which the spores are set free. In addition to this process, a kind of conjugation also takes place, in which two cells (zygospores), developed from the mycelium, coalesce with one another and form spores. The Mucor mucedo is one of the commonest moulds, and is frequently found in animal excreta, especially in horse- droppings. It grows on acid media, and is not pathogenic^ A dense fur with black fructifications as large as poppy- i seeds appears on the gelatine plate. The Mucor rhizopodiformis was described by Lichtheim. It grows very luxuriantly on bread gelatine, which it lique- fies. The fur is white and bears black fructification heads. The culture on bread is distinguished by the development of an aromatic odour. Mucor corymbifer was likewise described by Lichtheim. It forms a dense snow-white fur upon bread, resembling teased-out cotton-wool. The Mucor ramosus, described by Lindt, grows very well upon agar made with bread infusion, and upon potato. The fur is at first white, but soon assumes a greyish-brown tint. Both Mucor rhizopodiformis and Miicor corymbifer are pathogenic. Intravenous injection of fluid containing their spores causes a fatal disease in rabbits, in which the organs are always attacked in the following order : viz. kidneys, intestine, mesenteric glands, spleen. Mucor ramosus acts quickest, an acute hsemorrhagic disorder being originated by its injection. The Aspergillus fumigatus and Aspergillus flavescens in 1'ATIIOGENESIS OF MOULDS 195 like manner exhibit pathogenic properties. Lichtheim found that a peculiar disturbance of equilibrium occurred in rabbits and dogs 'after intravenous injection, and after death, which took place in twenty-four hours, the germina- tions were found especially in the myocardium and kidneys.1 1 [The three varieties of Aspergillus may grow in various parts of the body in man, notably the external ear, in which they produce the disease known as otomycosis. One or other form has also been found growing in the lungs, on the nasal mucous membrane, and on the cornea. Such occurrences are, however, very rare.] — TR. o 2 196 BACTERIOLOGY CHAPTEE IX BACTERIOLOGICAL EXAMINATION OF PUS Properties and composition of pus. — Pus shows an alkaline reaction and possesses a high specific gravity, consequently it furnishes an excellent nutrient medium for the most widely differing micro-organisms, and that whether it is derived from exudations or from the surface of wounds, or is formed in some other inflammation of tissue. Furthermore, pus may be coloured, the tint being greenish or brown-red, and it exhales a peculiar odour. In it are found white and red blood corpuscles, blood pigment, and crystals of hsematoidin, epithelial cells, drops of fat, fungi, micrococci, and bacilli. Suppuration may also be excited by bringing certain of the micro-organisms contained in pus into contact with the tissues, when their vital activity will start the process. Amongst these microbes is included more especially the Stapliylococcus pyogenes, which, as we have already remarked in the chapter on the ' Bacteriological Analysis of Air ' (see p. 110), is very widely distributed in nature. Kosenbach also found the Bacillus saprogenes III (see p. 176) in pus. Actinomyces. — A fungus has been detected compara- tively recently in almost all organs, in certain cases of chronic inflammation combined with suppuration, which has been named ^Actinomyces or ray -fungus. It had been discovered by Langenbeck as long ago as 1845, but it was ACTINOMYCES 197 not until 1878 that it was more thoroughly described by Israel. The disease caused by it is known as w//w/////o;.s/'x. E. Ullmann states, however, that the actual suppuration in this disorder is due to the specific organisms of pus, viz. the staphylococci and streptococci, the actinomyces fungus being, according to him, merely an accidental occurrence in the pus. The fungus forms minute sulphur-yellow nodules of the size of poppy- seeds, which, if a cover-glass is lightly pressed upon them and they are examined even with a very low power, show compact globules having a clustered arrangement. They were first discovered by Bollinger in cattle, and admit of being very easily transmitted to human beings. Under a higher power they are seen to be made up of filaments re- sembling hyphre, which for the most part radiate OUt FIG. 73.— Ax ACTINOMYCKS GRANULE. UNSTAINED PIIEPAUATION. (After Jaksch.) from a central point, each ray being thickened into an elongated club-shaped enlarge- ment at the periphery. R. Paltauf describes the actino- myces fungus as a bacterium, regarding the club-shaped figures as degenerative forms ; Israel and Wolff count it amongst the polymorphic fission fungi, on the grounds that thread-like figures and groups of globular bodies are visible in addition to the radiating forms ; Petroff and Florin ann found that the fibres are made up of granules alternating with small roundish gaps ; and Eabe is of opinion that a certain alga, Cladothrix canis, presents a very great similarity to actinomyces. The fungus takes aniline stains when they are allowed to act on it for some time, and does not discharge them during the application of 198 BACTERIOLOGY Gram's method ; but the simplest mode of recognising them in pus consists in examining the little yellow globular bodies without staining (figs. 73 and 74). Cultivations on glycerine agar show, according to Protopopoff and Hammer, a mass of closely-packed miliary nodules of the size of hemp-seed at largest, which have a yellow colour and adhere very firmly to the medium. The growth on potato is similar, except that the culture looks quite dry. In broth miliary nodules develop after a short time, and may attain the size of a hazel-nut. Gelatine is slowly liquefied. Growth takes place also in the absence of oxygen, in eggs, by Hueppe's method. FIG. 74. — ACTINOMYCES, STAINED BY GUAM'S METHOD. (After Jaksch.) F. Winkler has cultivated actinomyces on plover's egg albumen in the author's Institute. Formation of sulphur- yellow colonies as large as poppy-seeds takes place all over the surface of the medium, which is, however, not liquefied. Bacillus pyocyaneus. — This bacillus is the cause of the grey or blue colour sometimes seen in pus and in pieces of dressing saturated with it. The Bacillus pyocyaneus a, described by Gessard, consists of small slender rods provided with a flagellum, by means of which they move swiftly. Upon gelatine plates roundish islets of a yellow colour appear in the substance of the medium, to the whole of which they impart a greenish tint in about two days, causing at the same time slow lique- BACILLUS PYOCYANEUS 199 faction. In thrust-cultures the gelatine liquefies along the needle-track, the fluid mass assuming a greenish colour, while the part still solid displays a green fluorescence. A layer forms upon agar which shows a fluorescence, at first greenish, but subsequently dark green in tint ; while on potato the deposit appears coloured brown, turning red when treated with acids, blue with ammonia. According to Gessard, the generation of pigment depends on the nutrient medium. The green colouring matter formed, which is soluble in chloroform, has received the name of pyocya /////. Babbits die when injected into the peritoneal cavity with pure cultures of this micro-organism or with pyocyanin. The Bacillus pyocyaneus ft, described by Ernst, is not pathogenic, but is commonly met with in the company of the foregoing, and when isolated causes the blue coloration of pus. It liquefies gelatine e f ? f* much more speedily than the last, and shows **''**. •"/ a brown tint on potato which changes to * * grey on touching with the platinum wire FN-.. 75.— laouLTKD 0 * ELEMENTS OF (the chameleon phenomenon). The reaction STAPHYLOCOCCUS CEREUS FLAVOB. of the potato culture, the more rapid lique- faction, and the fact that injections produce no result, all serve to distinguish it from Bacillus pyocyaneus a. With reference to its physiological relations, Bacillus pyucyaneus produces coagulation of milk, rapidly peptonises albumen, and causes vigorous inversion of sugar with fer- mentation. Staphylococcus cereus. — Passet found in pus the Staphy- lococcus cereus albus and Staphylococcus cereus flavus, the cocci of which are arranged in minute clumps (fig. 75). The former produces upon the gelatine plate white dots which spread out superficially and do not liquefy the medium. In thrust-cultures a white deposit is likewise BACTERIOLOGY found which gives the surface the appearance of a drop of stearine (fig. 76). On agar a layer spreads out from the _ Superficial Deposits at the site deposit of inoculation Needle-track • Needle-track Fia. 76.— THRUST-CULTURE ix GELATINE OF STAPHYLOCOCCUS CEREUS PLAVUS. FIG. 77. — THRUST-CULTURE ix GELATINE OF STREPTOCOCCUS PYOGEXES. streak, in the neighbourhood of which further colonies are seen. The deposit on potato is similar, but soon becomes dirty grey. STREPTOCOCCUS rVnuKNES L'Ol Staphytocoeeut ••< r.'n* jhim* is differentiated by the yellow colour exhibited in its cultures. In addition to these, Von Schrotter and F. Winkler discovered also the Staphylococcus cereus aureus, distinguished from both the others by the orange-red colour of the colonies, and by its slower growth. These microbes are also constantly found in the nasal secretion in cases of coryza. The Staphylococau n'mix may also occur in the interior of pus corpuscles ; but the cells may then be arranged in diplococci, and in the pus of urethritis it may be confounded with the yonococcus. The latter, however, is bleached by Gram's method, whereas the Staphylococcu* m-ms retains its colour. Streptococcus pyogenes. — In phlegmonous suppurative processes the Streptococcus pyogencs described by Rosenbach is constantly present, and is probably identical with the Stn-jitiK-nrnix rnixijH-Jiiti* of Fehleisen (p. 113). It is found, however, not only in erysipelas but also in puerperal processes occurring in lying-in women. It does not liquefy gelatine, and forms fine punctiform colonies, while in thrust-cultures a thin delicate film appears around the puncture (fig. 77). On agar minute dots resembling dew- drops occur along the streak, and range themselves into a ribbon-like stripe. The elements so far alter upon potato that some individual cells appear larger, and others smaller, than usual. It grows better at incubation temperature than at that of ordinary rooms, but growth is usually slow at best. Transmission of cultures into the lymph-channels sets up a typical erysipelas, according to E. Fraenkel. The Micrococcus of gonorrhoea. — The infectious nature of gonorrhoeal secretion is due, as shown by Neisser, to cocci which have received the name of Gunown, and which can easily be recognised in the secretion owing to their arrange- ment in pairs. The individual cells are reniform, and may be found in the pus arranged round the nuclei in the interior of the cells, but not in groups (fig. 78), and are distinguished 202 BACTERIOLOGY from similar micrococci which occur in the rnethra by their being invariably decolorised by Gram's method, although otherwise they take the aniline stains very readily. If it is wished to demonstrate the gonococci in fresh gonorrhceal secretion, the following is the best mode of procedure : — A drop of the pus is placed upon a cover-glass and spread out lightly, so that the glass is covered with a thin cloudy film ; but the common method of laying two cover-glasses one over the other and then drawing them asunder must not be employed here. The cover-glass is now stame(i with a con- centrated alcoholic solu- tion of methyl blue, rinsed in water, dried, and mounted in Canada balsam. Neisser has recom- mended the following method of double stain- ing : — The cover - glass preparations are brought FIG. 78.-GoN'ococci FROM THE UKETHRAL for S0me HlinuteS illto a warm concentrated alcoholic solution of eosine, the superfluous dye is then soaked up with blotting-paper, the preparations laid for a quarter of a minute in concentrated alcoholic solution of methyl blue, and rinsed in water. Schiitz's method consists in preparing a filtered satu- rated solution of methyl blue in 5 per cent, aqueous carbolic acid, in which the preparations are stained for five to ten minutes in the cold. After rinsing in water they are laid for an instant in very dilute acetic acid, again rinsed in water, and double- stained in very dilute solution of safranine. The gonococci are blue and the pus-cells salmon-coloured. GOXOCOCCUS 203 To differentiate them from other diplococci, Stein- schneider. and Galewski recommend that the preparations should be left for half an hour in a solution of gentian violet in aniline water, rinsed, laid for five minutes in solution of potassium iodide, and left in alcohol until decolorised, after which they are again rinsed, dried, and double-stained in dilute alkaline solution of methyl blue. The gonococci are pale, the other diplococci stained blackish. In making a rapid examination of a series of secre- tions for the presence of gonococci, F. Winkler uses the following dryin^-on process : — A clean slide is prepared with a small drop of gonorrhoeal secretion, and immediately passed several times through the flame without being previously dried in air. The pus is thus desiccated in a thin film, over which a concentrated alkaline solution of methyl blue is next poured ; after a half a minute the preparation is washed in water, dried over the flame, and mounted in Canada balsam. The pus-cells appear pale blue, the gonococci dark blue. The ordinary nutrient materials which prove favourable to the growth of other micro-organisms fail us completely in the case of gonococcus, which grows neither on gelatine, agar, nor potato, though on the other hand, as Bumm has proved, it thrives on human blood-serum. According to him the cultures exhibit a greyish -yellow layer, resembling from its pointed prominences a group of mountains, and whose margins extend diffusely into the surrounding parts. The serum is not liquefied. The most suitable temperature lies between 33° and 27° C. Growth is very slow, and comes to an end in three days. Cultivation experiments on plover's egg albumen gave good results in the hands of Von Schrotter andF. Winkler, a thin, tolerably transparent, whitish deposit showing itself on the surface of the solidified mass of white of egg in as short a time as six hours at incubation tern- 204 BACTEEIOLOGY perature. The growth quickly extends, but ceases after the lapse of only a few days. Experiments have been carried on by Wertheim at the clinic of Professor F. Schauta in Vienna, on the cultivation of the gonococcus upon a medium consisting of a mixture of two or three parts peptone glycerine agar and one part human blood-serum. He obtains the latter from the blood of the maternal portion of the umbilical cord, which is severed but not ligatured, and the blood caught in sterilised flasks. In this way Wertheim was enabled to grow gonococci in plate-cultures, on which he was able to observe colonies visible to the naked eye even after twenty-four hours in the incubator. The microbes grow rapidly, displaying their characteristic forms and colour, and give the best evidence of their specific action by transmission to human beings. Wert- FIG. 79. — DEEP -LYING . . COLOXY OF GOXOCOC- heim s whole process vields astonishing cus ON A SERUM AGAR (After Wert" results, and according to his researches growth takes place decidedly quicker when deprived of oxygen than when the gas is admitted. The plate appears diffusely clouded in twenty -four hours, and assumes the appearance of a delicate flocculent layer of moss. The majority of the colonies develop in the substance of the medium, the deep ones appearing whitish - grey by direct, yellowish-brown by transmitted light, and in three days show a peculiar nodulation, which is so regular as to recall the appearance of a blackberry (fig. 79). The superficial colonies have a compact dot situated exactly in the centre, which, when examined with a low power, is found to correspond in structure with the deeper colonies, but is surrounded on all sides by a superficial film which, though at first very delicate, transparent, finely granulated, and colourless, develops in three days round the central IJACILLUS OF SYPHILIS 205 mass numerous minute accumulations of condensed matter having a brown colour (fig. 80). Bacillus of syphilis. — In tissues affected with syphilitic disease and in the layer of pus covering syphilitic ulcers Lustgarten found small comma-bacilli showing a slightly S-shaped curvature, but they have not as yet been cultivated outside the body. In the interior of the tissues they are not found in the interstices of fibrous bundles or other tissue elements, but are included in cells which are much larger than the white blood-corpuscles. The methods of staining are of particular interest. They are carried out in the case of tissues only with fine sections prepared from them ; but Finely granular superficial co:i- Condi ; agglomera- tions Central mass / FIG. HO.— srpKut-iciAL COLONY OP GONOCOCCUS ox A SERUM AGAK PLATE. V (After \Yertheim.) in the case of secretions a portion is placed upon a cover-glass and spread out by rubbing. Staining is then done with Ehrlich's solution of gentian violet in aniline water, in which the preparations are allowed to remain for twenty-four hours at ordinary temperature, or for two hours at that of the incu- bator. They are then placed in absolute alcohol, and after that for ten seconds in a 1£ per cent, solution of potassium permanganate, which deprives the tissues of their colour while the bacilli retain their violet tint. To remove the precipitate of manganese dioxide which forms upon the specimens, they are immersed in an aqueous solution of sul- phurous acid. The preparation can be mounted in Canada 206 BACTERIOLOGY balsam in the ordinary way, and examined under the micro- scope. The stained bacilli are decolorised at once by treatment with glacial acetic acid ; other acids take longer. A better method than Lustgarten's is that described by De Giacomi, in which the preparations are treated with solution of iron perchloride after being stained in Ehrlich's solution of fuchsine in aniline water. Lewy recommends staining in carbolic fuchsine and decolorising with distilled water as the surest and most convenient method. That of Doutrelepont and Schiitz consists in staining the pre- parations for twenty-four hours in a one per cent, solution of methyl violet and decolorising for some seconds in dilute nitric acid, after which they are transferred for ten minutes to alcohol of about sixty per cent, strength. The sections are then double- stained by leaving them for a few minutes in a watery solution of safranine, and are thoroughly washed in sixty per cent, alcohol. By this process the bacilli appear blue and the nuclei and tissue light red. Marschalko recommends staining the sections of syphilitic tissue and cover-glass preparations of syphilitic secretion in Loffler's methyl blue for three to four hours at incubation temperature, or twelve to twenty-four at that of an ordinary room, washing in water, and double-staining for one to five minutes' in concentrated aqueous solution of vesuvine.1 Bacillus tuberculosis. — Tuberculosis was regarded as an infectious disease even by the older physicians ; indeed, this opinion may have preceded the teaching introduced in the course of time by the pathological anatomists (such as Virchow and Eokitansky), which was restricted to the description of the microscopic and naked-eye appearances 1 [Some authorities believe this to be identical with a bacillus found in normal smegma praeputiale, &c., which has a similar appearance and similar peculiarities of staining. The latter is said, however, not to stain by Doutrelepont's method.] — TB. TUBERCLE BACILLUS 207 produced by it. But it was Koch who first discovered the cause of the disease in the tubercle bacilli, recognisable in all such products of the bodies of men and animals as have undergone tubercular changes. These furnish a foundation for the doctrine that tuberculosis is transmissible, inasmuch as Koch was able to set up a typical tuberculosis experi- mentally in animals by means of pure cultures. Lortet and Despeignes are of opinion that the tubercle bacilli are frequently disseminated by earth-worms. Tubercle bacilli are fine rods having a length nearly equal to the diameter of a human blood-corpuscle, somewhat curved, and often united in groups of two or more, but seldom in very extensive combinations. They are destitute of motility. They form spores of an oval outline, both in artificial cultures and in the bodies of animals, and offer a high degree of resistance to drying, boiling, and the action of the gastric juice and of putrefaction. The presence of carbohydrates or of glycerine is, according to Hammer - schlag, essential for their growth. The following is the procedure to be followed when it is wished to obtain a pure culture of tubercle bacillus : — The sputum of phthisical patients is mixed with water, and in- jected into a subcutaneous sac made over the abdomen in several guinea-pigs. In three or four weeks the tuber- culosis will be so far advanced as to cause the death of one or other of the animals, and in the post-mortem examina- tion tubercular changes will be found in the various organs, particularly the omentum, spleen, and liver. Another of the animals is now killed by strangling, a window is cut in the thoracic wall with all "due precautions regarding disinfection, and a corner of the lung drawn out with the platinum wire. From this piece of lung some distinct tubercular nodules are now taken and rubbed between two slides. (Of course all the instruments used, such as knives, scissors, and slides, must be carefully sterilised.) The 208 BACTERIOLOGY glasses infected with the crushed tubercular masses are now laid upon serum which has been poured out into glass capsules and inspissated, the tubercular mass is smeared over the surface of the serum with a strong platinum wire, and the capsules are covered with glass plates and placed in the incubator. The colonies are perfectly formed in about three weeks, when further cultures can be made from them in test-tubes. When examined under a low power, the cultures show figures coiled in the shape of an S and thickened in the centre, which consist of bacilli massed together (fig. 81). The colonies form dry, white scales, having a dull surface and not larger than poppy-seeds, which adhere but loosely to the surface of the medium, never penetrate into its substance, and do not liquefy it (fig. 82). Pastor gives the following pro- cess for obtaining pure cultures of tubercle bacilli from sputum : — A patient is chosen whose sputum is FIG. 81. — IMPRESSION- PREPARA- TION FROM A CULTURE OF TU- very rich in bacilli and shows com- BERCLE BACILLUS ox SERUM. paratively little contamination with other micro-organisms, and he is made to rinse out his mouth and pharyngeal cavity repeatedly with distilled water, and then to expectorate into a sterilised test-glass. The sputum, or more properly the liquid contents of the pul- monary cavities, is shaken up with sterilised water and filtered through fine gauze to remove the coarser particles. A few drops of the filtrate are mixed with melted nutrient gelatine in such a manner as not to render it very turbid, and the mixture is poured out on plates which are left under bell-glasses at room temperature. In from three to four days the various colonies of bacteria contaminating the sputum form. The portions of gelatine between the colonies, and which still remain clear, are now sought out PREPARATION OF PlKi: ( I'LTURES 209 with a lens, carefully cut away with a sterilised knife, and transferred to the surface of serum which has been made to solidify in a slanting position. The following is Kitasato's method of preparing pure cultures from sputum :— The patient is requested to evacuate his morning sputum into sterilised double capsules. A flake derived from the deeper parts of the respiratory appa- ratus is isolated with sterilised instru- ments, and carefully washed in at least ten watch-glasses full of distilled water, one after the other, to remove the bacteria taken up in passing through the cavity of the mouth. The Hake is now transferred to glycerine agar or serum. After it has been kept for some fourteen days in the incubator the first colonies form, appearing as circular pure white transparent specks, which project above the surface of the medium. Ob- tained by this method the colonies are flat, shining, and smooth, whereas those obtained by the earlier method from tubercular organs are from the first dry, dull, and wrinkled.1 The difference, however, disappears as growth advances, and the whole of the medium becomes covered with a coat consisting of trains of bacilli many times coiled. * m Fns. 82. — Ctri/rrRK OP Hammerschlag obtains a luxuriant growth by the addition of mannite and 1 [The bacilli of tuberculosis in' birds form a permanent wrinkled mem- brane on the medium ; but they differ so much in other respects also that, 210 BACTERIOLOGY grape-sugar to the agar. Pawlowsky found that white detachable colonies appear on the surface of potatoes in twelve to twenty days, but the potato discs must be pro- tected from evaporation in air-tight glass tubes. The bacilli grow in the most luxuriant manner, according to Koch, in an infusion of veal rendered feebly alkaline, and to which an addition of 4 or 5 per cent, glycerine and 1 per cent, peptone has been made. Inoculation is done by floating a fairly large piece of the seed-culture upon the surface of the fluid, and when the cultivation has been kept for several weeks in the incubator, the surface becomes covered with a tolerably thick skin, dry above, and often wrinkled, which a few weeks later becomes moistened by the fluid, breaks up into single ragged pieces, and sinks to the bottom. Growth requires from six to eight weeks for its completion. With regard to the staining of tubercle bacilli, it is of considerable importance, according to Koch, that the colour- ing fluid should have an alkaline reaction, as these micro- organisms will only take .the aniline dyes when acted on simultaneously by alkalies. In order to protect the ob- server, Pampukes recommends that the tubercular sputa should be sterilised at 120° C. before being examined, as this does not impair their power of absorbing stain. For practical investigation, the observation of Dahme is of importance, that the flakes which lie at the bottom of the sputum contain the greatest number of bacilli (fig. 83). According to the method of Koch and Ehrlich, the sputum to be examined is poured upon a dark surface, and the tenacious yellowish particles are picked out. By means of a penholder containing a nib one half of which has been broken away the smallest possible lump of although identical in appearance and staining, they are now generally re- garded as a distinct variety.]— TK. STAINING OF TUBERCLE BACILLI 211 sputum is taken up, and is then rubbed between two cover- glasses. When the sputum is very viscid, the cover-glasses between which the lump is spread out are, before being drawn apart, laid on a hot plate at a temperature below 100° C. until a slight clouding takes place, betokening coagulation. The cover-glass preparation, after drying, is passed three times through the flame, and stained in a warm solution of fuchsirie or methyl violet in aniline water for a quarter to half an hour. To decolorise, the cover-glass is brought into an acid mixture containing one part nitric acid, two parts water, and two parts sulphanilic acid, or into I'li;. 83.— TUBERCLE BACILLI ix SI-CTI M. (After J;ik>t:Ii.) a solution of 3 per cent, of hydrochloric acid in 9 per cent, alcohol, after which it is rinsed in 60 per cent, alcohol and double-stained in methyl blue or malachite green if the tubercle bacilli have been stained red, or in vesuvine or Bismarck brown if they have been stained blue. It is then rinsed again, dried, and mounted in Canada balsam. Kaatzer has modified the process in the following manner : — The sputum — best that first expectorated in the morning — having been received into a completely empty spitting-cup, is spread out upon a black plate or a piece of black glazed paper. A very minute particle is now picked 212 BACTERIOLOGY up from a yellow purulent spot by means of a platinum needle with the point flattened like that of a scalpel, and which has previously been sterilised at a red heat ; this particle is rubbed between two cover-glasses, which are then passed three times through the flame. Staining is done with an aniline water solution of gentian violet which has first been warmed, and the preparations are decolorised by transferring them from the gentian violet, as soon as it has grown cold, to a hydrochloric acid alcohol containing 20 parts water and 2 parts hydrochloric acid to 100 parts alcohol. The preparations remain in this mixture for half a minute to a minute, are then transferred to concentrated alcohol for the same length of time, and afterwards rinsed in water. They are now dried and placed for a half to one minute in a filtered concentrated solution of vesuvine in water by way of double-staining, rinsed in 96 per cent, alcohol, and brought under the microscope in a drop of water. The tubercle bacilli appear of a dark violet colour, or even nearly black, as the aniline brown absorbs the blue part of the spectrum, so that a blue object must seem black in a brown solution. The other portions of the preparation are brown. The following is the method employed by Giinther: — The cover-glass, having been prepared with sputum and fixed in the flame, is deposited face downwards in a watch- glass filled with solution of fuchsine in aniline water, the centre of which is now heated over a very small flame while being kept moving vertically up and down. When the fluid begins to give off bubbles, heating is stopped, the watch-glass placed on the table, and let stand for a minute. The process of heating is repeated about five times with intervals of one minute's standing, after which the cover- glass is taken from the stain and laid with the film of sputum uppermost in a watch-glass containing 3 per cent. ZIEHL-NKELSKN METHOD 213 hydrochloric acid in alcohol, in which it is moved to and fro for one minute, and is then rinsed in water. Some drops of a dilute aqueous solution of methyl blue are now poured upon it, when, after again washing and drying, it is passed three times through the flame and mounted in Canada balsam. In the Ziehl-Neelsen process, the cover-glass with the sputum is seized in a forceps, covered with an alcoholic carbolic fuchsine solution, warmed over a feeble spirit flame until bubbles appear, washed in water, and flowed with a 5 per cent, solution of sulphuric acid. It is then rinsed in 70 per cent, alcohol, dried, and double-stained with aqueous solution of methyl blue or malachite green. According to Friedliinder's method, some sputum is smeared on two slides, which are drawn three times through the flame. The sputum is then covered with two or three drops of carbolic fuchsine, passed afresh through the flame, moistened with water and a few drops of nitric acid alcohol (3 per cent, of nitric acid in 90 per cent, alcohol), rinsed in water and watery solution of methyl blue, and examined without a cover-glass under the oil-immersion objective. Kiihne recommends shaking up the sputum thoroughly in a glass with a concentrated solution of borax, so as to render it fluid. The cover-glasses prepared with this are stained in carbolic fuchsine for five minutes, decolorised in 30 per cent, nitric a.cid, rinsed in water, dried, and examined in a drop of aniline oil coloured light yellow with picric acid, which is best done. by adding two or three drops of a con- centrated solution of picric acid in aniline oil to a watch- glass full of pure aniline oil. To obtain permanent pre- parations the double-staining should be done by transference for a few minutes to an aqueous solution of picric acid after decolorising with nitric, after which the specimen is dried and put up in balsam. An addition of 4 per cent, citric 214 BACTERIOLOGY acid is advisable, in order to increase the solubility of picric acid in water. In the process of B. Frankel, and in that of Gabbet, the staining and decolorisation are carried on simul- taneously. The former is done with fuchsine in aniline water, from which the preparation is directly transferred into a filtered mixture of 50 parts alcohol, 30 parts water, 20 parts nitric acid, and as much methyl blue as will dis- solve with repeated shaking. If aniline water gentian violet has been used for staining, the preparation is transferred to a vesuvine solution, consisting of a filtered mixture of 70 parts alcohol, 30 parts nitric acid, and as much vesuvine as will dissolve. Staining is finished in a short time — one or two minutes — and the preparation is rinsed in water or alcohol rendered feebly acid with acetic acid, and thoroughly dried. In Gabbet's modification, the Ziehl-Neelsen carbolic fuchsine is used instead of the aniline water solution, and as the second stain methyl blue in sulphuric acid, consisting of 1 or 2 parts methyl blue to 100 parts of 25 per cent, sulphuric acid. Arens pours three drops of absolute alcohol over a crystal of fuchsine about the size of a millet- seed in a watch- glass, so as to obtain a saturated alcoholic solution, which is mixed with 2 or 3 c.cm. chloroform. This causes a tur- bidity of the solution, which begins to clear with separation of the fuchsine in the form of flakes. Cover-glass preparations treated in the usual way are laid in the solution, when clear, for four to six minutes, and, after allowing the chloroform to evaporate, are decolorised in a watch-glass full of 96 per cent, alcohol, to which three drops of hydrochloric acid have been added. They are now rinsed in water and examined in the same medium, or finally double-stained with methyl blue. I5.\t AKJARTKX'S METHOD 215 Gibbes gave the following recipe : 2 grms. fuchsine and 1 grm. methyl blue are slowly introduced into a solution of 3 c.cm. aniline oil in 15 c.cm. absolute alcohol until they are completely dissolved, and 15 c.cm. water are then added. A few drops of this liquid are heated in a test-tube and poured out into a watch-glass, in which the cover-glass is laid for five minutes, being then washed in alcohol until no more colour is discharged. The bacilli are red on a blue ground. Further staining may be well done with a concentrated aqueous solution of cosine. Occasionally it is of advantage to subject the cover-glass preparations to different methods of examination. Kaatzer recommends staining the same preparations first with carbolic fuchsine after the Ziehl-Neelsen or B. Frankel's method, and examining them with the oil immersion lens, after which the cedar oil should be removed with blotting- paper and alcohol, the preparation dried in the flame, stained in hot aniline water gentian violet, decolorised in hydrochloric acid and alcohol, and double-stained in vesu- vine. The tubercle bacilli, which were red when first examined, are turned dark violet on a light-brown ground by the second staining. In cases where staining yields doubtful results, the pro- cess of Baumgarten may be employed as a control observa- tion. The preparation of sputum, having been dried in air and passed three times through the flame, is first laid for a few minutes in a watch-glass containing chloroform to free it from fat, rinsed in absolute alcohol, and, after this has evaporated, is placed on a slide in a drop of potash solution, prepared by adding two or three drops of a 33 per cent, solution of caustic potash to a watch-glass of water. If the microscopic examination (without oil im- mersion) reveals the presence of bacilli, the cover-glass is removed from the slide, dried in the air, passed three 216 BACTERIOLOGY times through the flame, moistened with a few drops of dilute aqueous solution of methyl violet, and examined at once under the microscope. The tubercle bacilli, if present in the preparation, remain completely colourless if the examination is not protracted beyond five or ten minutes, whereas all the other bacteria almost instantly assume a blue colour. In very doubtful cases in which the ordinary methods of staining are at fault, preparations must be made from the sediment. According to Stroschein's method, a table- spoonful of sputum is vigorously shaken in a test-glass with three tablespoonfuls of a mixture of 1 part concentrated solution of boric acid and 3 parts water until the sputum is liquefied, when it is poured into a glass ending in a point below. After standing for twenty-four hours, the clear supernatant liquid is poured off and a cover-glass prepara- tion made from the sediment at the bottom. Biedert recommends that a tablespoonful of sputum should be boiled with two tablespoonfuls of water and seven or eight of solution of caustic potash ; four more table- spoonfuls of water are then added, and boiling is repeated until the mass has become evenly fluid. This remains, standing for three or four days in a pointed glass covered over, and is then poured off until a depth of only five to eight millimeters is left. The sediment is energetically stirred up with some fresh white of egg, and some of the mixture is rubbed on a cover-glass, dried at a moderate heat, and stained by the Ziehl-Neelsen method. A small quantity of untreated sputum from the same source may be used instead of the white of egg for fixing the sediment to the cover-glass. Sedimentation is particularly important in the examina- tion of fluids which are poor in corpuscular elements, and is, most conveniently done by centrifuging. In the absence, TIT.KJICLK IJ.U'ILLI IN SECTIONS OF TISSCK 217 however, of an apparatus for this purpose, Des Vos mixes white of egg with four times its bulk of distilled water, which causes the globulines to sink to the bottom as a coarsely flocculent mass. Up to 10 c.cm. of the supernatant opales- cent liquor, which consists of dilute albumen, are now added to the fluid to be examined, and the whole is well shaken up and heated in the water-bath until the white of egg coagulates. In a short time, according to the quantity of white of egg added, a greater or less amount of a finely flocculent sediment forms, which is to be examined for bacilli. To examine milk for tubercle bacilli, the most con- venient mode is to place a drop of the suspected milk upon a cover-glass and add to it two or three drops of a 1 per cent, solution of sodium carbonate. The whole is well mixed with a platinum needle, and the cover-glass is then warmed carefully over a small flame until complete evapo- ration has taken place. The fat is in this way saponified, so that finally a thin film of soap remains behind on the glass, which is stained, &c., like an ordinary cover-glass preparation. Regarding the staining of tubercle bacilli in sections of tissue, the same methods hold good with suitable modifica- tion, for a more detailed description of which the reader may refer to the general section (see p. 84 et seq.). Here it is only necessary to remark that the usual method of stain- ing employed is that with Ziehl's solution, but the sections must be left lying in it for an hour or even longer. Bleach- ing is carried out in 10 per cent, nitric acid for a half to one minute until the red colour has vanished, after which the sections are transferred to 70 per cent, alcohol until they acquire a rose -red tint or become completely colourless. They are then double-stained in methyl blue, thoroughly washed in alcohol, and put up in Canada balsam. The 218 BACTERIOLOGY bacilli are distributed through the tissue, or lie in masses between the lymph cells, or are taken into their interior, and they are also found enclosed in the so-called giant- cells. In order to obtain the active principle tuberculine, Koch evaporated pure cultures of tubercle bacilli, made in glycerine and veal infusion, to a tenth part of their volume in a water-bath, and filtered the fluid through an earthen- ware filter. An albumose can be procured from tuberculine after the process of Klebs, which this observer has named tuber ciiloddinc. The tuberculine is treated with platinum chloride, or with the so-called alkaloid reagents, and the albumose remaining in the solution thus formed is precipi- tated out with alcohol. Bacillus of glanders. — This bacillus was discovered by Loftier and Schiitz to be the cause of the disease called glanders or malleus, which may be transmitted from horses and donkeys to human beings. The micro-organisms are also found in what are called glanders nodides, from which they penetrate into the surrounding parts and originate morbid phenomena, and which occur as a rule in the cavity of the nose, and form deep ulcers on the mucous membrane cover- ing the turbinated bones, on that of the larynx, and in the lungs. The neighbouring lymphatic glands are swollen and rendered hard, as well as the cutaneous lymphatic vessels, which often burst outwards, forming ulcers. The bacilli are slender rods with rounded ends. They are about the size of tubercle bacilli, are without, power of automatic movement, and grow upon our nutrient media only at incubation temperature. Development is suspended at a temperature above 42° C. In consequence of this peculiarity gelatine cannot well be used, as growth is very scanty at a lower temperature, but the micro-organisms thrive very well on glycerine agar at 37° C., showing pale BACILLUS OF GLANDERS 219 yellow or whitish round colonies on the plate as early as the second day, while along a streak of inoculation made on the same medium a shining coating appears in four to five days. On the surface of blood-serum there are de- veloped yellowish deposits which coalesce with one another and form a slimy covering. The cultivation on potato is particularly important : a deposit appears at incuhation temperature which is at first yellowish and transparent, but which gradually increases and assumes a darker tint. In about a week the colour changes to reddish -brown, and iinally passes into red. The bacilli readily take up the aniline dyes, but discharge them again when Gram's method is employed (fig. 84). «• o o -;. -BACILLI <>F <;LAM>I:K> IN HTMAX Bt,«><'i>. < After Jaksch.) The staining process of Loffler consists in colouring the ^over-glass preparations for five minutes in a hot alkaline solution of gentian violet or fuchsine in aniline water con- taining an addition of 1 per cent, of caustic soda solution. Bleaching is done in 1 per cent, acetic acid coloured to a wine yellow with a watery solution of tropseoline 00, in which the preparations remain for an hour, and are then rinsed in water. Kiihne treats the cover-glasses with hot carbolic fuchsine or carbolic methyl blue, and decolorises in water contain- ing two drops of hydrochloric acid per 100 grams. For staining glanders bacilli in sections, Kiihne has •devised the following method, which is reliable and easy in 220 JJACTERIOLOGY use : — The sections, having been stained with carbolic fuchsine and soaked in water, are quickly decolorised in an aqueous solution of hydrochloric acid (3 per cent.), well rinsed in water, and then either dipped lightly into alcohol or freed as far as possible from water by pressing them with blotting-paper Aniline oil mixed with 20 per cent, of oil of turpentine is now allowed to act on the sections in a watch-glass for eight to ten minutes, during which they remain adherent to the cover-glasses, and thence they are transferred to oil of turpentine, xylol, and balsam. Noniewicz recommends that the sections should be stained for a few minutes in alkaline methyl blue, rinsed in water, and decolorised in a mixture of 3 parts of i per cent, acetic acid, with 1 part of a J per cent, solution of tropseoline 00 in water. Thin sections are only dipped in quickly, but thick ones may remain in for two to five seconds or longer. They are then washed in water, spread out upon a slide, freed from moisture with blotting-paper, dried in air, cleared with xylol, and mounted in Canada balsam. The glanders bacilli appear black on a more or less blue ground. By this method the discovery was made that the characteristic bacilli appear in the acute form of the disease, whereas in the chronic form they are sparser, and round bodies, which stain intensely, appear in their stead in large numbers. The glanders bacilli are also found in the interior of dead cells. The vessels are free, and but few appear in the blood. Transmission experiments made with pure cultures have yielded successful results, the infection spreading most from wounds in the skin. The coramunicability of the disease is considerable, and the infecting power of the virus- is not altered by drying for three months. Other microbes of pus. — It need hardly be said that the pus from wounds in other specific infectious processes con- OTHER MICROBES OF PUS 221 tains the corresponding micro-organisms, e.g. nnthmt'ls and the bacillus of tetanus ; but besides these, a considerable number of other microbes have been found which have the power of originating suppurative processes. Thus Weichselbaum discovered the Micrococcus intracellu- laris meningitidis ; Neumann and Schaffer the Micrococcus meningitidis purulentae ; Becker the Micrococcus osteomyeli- tidis, and Heydenreich the micrococcus of Pende's ulcer (Micrococcus Biskra) ; while the Streptococcus contagiosae coryzae equorum was detected by Schiitz in suppuration occurring in animals, and Pfeiffer found in the abdominal cavity of a guinea-pig which died spontaneously a tenacious, puriform exudation consisting of a pure culture of Bacillus capsulatus. Some of the methods invented by Unna for staining micro-organisms in cutaneous abscesses (see p. 90) are excellently adapted for showing those in pus. The follow- ing process maybe especially recommended : — The cover- glass preparations are stained in carmine, transferred for two minutes to borax methyl blue (1 part each borax and methyl blue to 100 of water), rinsed in water, and placed in alcohol to which a few drops of Spiritus saponis kalini l have been added. They are finally brought once more into alcohol, and may then be submitted to examination. 1 [See p. 91, note.]- TR. 222 BACTERIOLOGY CHAPTER X BACTERIOLOGICAL EXAMINATION OF THE ORGANS AND CAVITIES OF THE BODY AND THEIR CONTENTS Micro-organisms of the living body. — In this section a series of micro-organisms will be dealt with, which find a congenial pabulum in the organs and cavities of the living individual. They are derived partly from the air, partly from water or other surrounding media, and may occur either as saprophytes or parasites. I. THE SKIN Micro-organisms of the skin. — Most of the micro-organ- isms met with on the skin in the cutaneous secretions are saprophytes, but many pathogenic varieties also occur in this situation. The space beneath the nails, in particular, is rich in forms, containing especially germs, according to Preindlsberger, which are found also on other regions of the cutaneous surface as well as often in the air, and hence may penetrate with it into the respiratory organs. Patho- genic germs are found amongst them. Penetration . of micro-organisms into the unbroken skin probably takes place by the openings of the glands, or perhaps by the agency of stinging insects. Amongst the cutaneous micro-organisms the bacteria of the female generative organs are also included. Straus and Toledo have found that microbes do not occur either in the parietes or secretion of the normal uterus, and it is only in the vagina, where there are accumulations of METHODS OF EXAMINATION 225 epithelial masses, that the hacteria find a favourable medium upon which to develop. In normal vaginal secretion the nnjinnl bucillna of Doderlein is regularly found, and the thrush fnmjHs and yeast cells are often met with, while Sta- jiJii/loroccus pyogenes albus, citreus, and aiireus are no rarities. Micro-organisms are also found in the sebaceous and sweat glands. Brunner discovered, for example, that strep- tococci may be detected in the sweat during suppurative processes, and Garre conversely was able to start a phlegmon by rubbing a culture of staphylococci into his unwounded arm. The appearance of coloured sweat is due to the presence of Bacillus jtyocyaticus ttfld Micrococau lia-matodes, whilst in the foul-smelling perspiration on the feet Eosen- bach found the Bacillus saprogenes II. (see p. 176). Preindlsberger isolated from the sub-ungual space the MicromrcHtt rr/v/fx dlbus and flams, the Diplococciis lique- lacii'ns albus and r/7/v//.s, Micmcoccus candicans, Sarcina alba &ndflava, and irhitc y<'i SYMPTOMATIC AXTIIKAX, KI;OM A CULTURE ox SERUM. Magnified 1,100 times. (After Loffler.) Lepra bacillus. — Hansen found in leprous tissue, and particularly in the lepra cells, small slender rods which have club-shaped ends and approximate nearly to tubercle bacilli in size. They are immotile, and are principally distinguished from the tubercle bacilli by their property of staining readily in aqueous solutions of the aniline dyes. Besides this, however, they also stain by the methods devised for tubercle bacilli, as well as by Gram's process, but staining succeeds with much greater ease with bacilli of lepra than with those of tubercle. By Baumgarten's process, cover-glass preparations are stained for six or seven minutes in a dilute alcoholic solution of fuchsine, decolorised in a mixture of one part of 230 BACTERIOLOGY nitric acid and ten of alcohol, rinsed in water, and double- stained in an aqueous solution of methyl blue. According to Lustgarten, cover-glass preparations, after staining in aniline water solution of fuchsine or gentian violet, are decolorised in one per cent, sodium hypochlorite and rinsed in water. Unna stains sections for twelve to twenty-four hours in aniline-water fuchsine, and decolorises in a ten to twenty per cent, aqueous solution of nitric acid until the preparations have acquired a yellow colour, after which they are transferred to dilute alcohol, and left there until the tint has turned red. The acid still present is now removed by immersion in a weak solution of am- monia in water, and the water is ab- sorbed up with blotting-paper, after which the preparations are mounted in balsam free from oil, dissolved in chloroform. FIG. 86.— ISLET OP LEPRA The Lutz-Unna method consists in BACILLUS ON A GELA- PLATE> (After staining the preparations in a warm aniline-water solution of gentian violet until they have become darkly-coloured, after which they are decolorised by successive immersion in solution of iodine and potassium iodide, in alcohol and nitric acid, and in plain alcohol. This procedure must be repeated until the preparations show a bluish slate-colour, after which they are mounted in balsam dissolved in oil of thyme or oil of cloves. Cultivation of the bacilli succeeded in the hands of Bordoni-Uffreduzzi upon peptone glycerine serum, which was inoculated with marrow from the bones of a person who had died of leprosy. From this it was possible to transfer the microbes to gelatine, on which they form rounded colonies at 20° to 25° C. without liquefying the IJAUILLUS SYCOSIFERUS FCETIDUS medium (fig. 86). A surface-culture on agar likewise shows roundish colonies, prominent in the centre. Those on serum are ribbon-like with jagged edges, and do not cause lique- faction. Cultivation experiments are best carried on at 37° C. The disease can be transmitted to men and animals. In the tissues, the bacilli are found in the cutaneous con- nective tissue, nerve-sheaths, spleen, and lymphatic glands, but do not occur in the blood. According to Unna they are found in the lymph-paths of the tissue, and he also believes the Irpni rrlls to be transverse sections of lymphatic vessels. The other methods of staining the micro-organisms of the epidermis, which can also be used for abscesses in the skin, have already been dealt with in the general section, to which the reader is referred (p. 90). Bacillus sycosiferus foetidus was found by Tommasoli on the hairs of the beard of persons suffering from *#. OF Tl;l< llnl'llVTuN TiiNSI l:\V-. 1 i:»M A IT UK (i i.it UK o\ Ixyusiox OK MALT. (After Leslie Roberts.) mentally that glucose, and not, as Grawitz supposed, a nitrogenous body, is the medium suitable for the tricho- phyton. The development, therefore, of the tonsurans fungus in the epithelium of the skin covering the body does not progress by means of spores, but merely by the swelling, constriction, and final separation of fibres (fig. 87). This fungus is also, according to H. von Hebra, the exciting cause of /////^////o contagiosa (an exanthem charac- terised by the formation of pustules), as well as of eczema marginatum, [and it is now agreed that tinea sycosis, tinea circinata (common ringworm), and onychomycosis (an affection of the nails) are due to it. (See also note at end of chapter.)] Gelatine is quickly and actively liquefied, according to 234 B ACTERIOLOG Y Grawitz, and the fungus fur floats upon the surface as a thick coating, white above and yellow underneath. The fungus of favus (Achorion Schoenleinii) . — In the scaly accumulations from persons suffering from favus, Schonlein discovered a fungus to which the name of Achorion Schcenleinii has been given. The favus crusts or scutula show the mycelium of the organ- ism, which is remarkable for the fact that the fibres grow up perpendicularly from the horny layer. The right angle formed by these fibres with the substratum is characteristic of the scutula of favus, since, in the horny scales permeated by the fungus of pityriasis versicolor or by the Trirho- phyton the fibres of the organism form acute angles, or run parallel with the cells of the horny layer. Unna differentiates three varieties of favus fungus — the Achorion euthythrix, with straight fibres and abundant formation of spores ; the Achorion dicroon, with forking hyphae ; and the Achorion atacton, the hypha3 of which run an irregular course and pos- sess peculiar knotty ramifications and many sharp angles. The first forms voluminous greyish-yellow scutula (favus griseus), the second sulphur-yellow scutula, which are slow in developing (favus sulphur eus tar- dus), and the third sulphur-yellow scutula also, which form more quickly (favus sul- IG. 88. — SURFACE vlmreus cclereus). Unna used for cultiva- CULTURE OP THE " ON : SERUM0™™ cS ti°n a medium composed of 4 per cent, of ?u>.°D(AfteNrEpiaut!) agar, 1 per cent, of peptone, and 5 per cent. Tin; FUNGUS <>r FAVUS of levulose or ^ per cent, of common salt. In order to obtain pure cultures dry media only must be employed, and they must accordingly have been kept in a warm place for a considerable time before use. All other micro-organisms which might spoil the pure culture of favus require a tolerably large amount of water, and soon cease to grow upon very dry soils. The Ac]i<>ri»n eitfhythrix forma dense wroolly white furs, half ft centimeter in height, upon the nutrient medium, and grows but a short way into its substance, whereas the growth of AcJ«>rl«n dn-nmn lies for the most part in the medium and only raises a thin coating of mycelium above the surface. Thu Adiornm atactnn occupies an inter- mediate position between the other two. Those varieties of favus fungus which are marked by their deep growth in artificial cultures grow also into the hair-follicles and arc more difficult to treat. Gelatine is liquefied very late and only very slightly by the growth of the favus fungi. On agar plates there develop in twenty-four hours at incubation heat, according to Plant, very small shining fibres resembling cotton wool, which are recognisable under the microscope as minute masses of conidia. In forty- eight hours the colonies have changed to round milk-white opaque discs possessing a border of very fine and quite short threads. On potato Plaut obtained, at incubation temperature, an irregular dis- coloration on the site of inoculation, from which the fun- gus grows into the substance of the potato. Similar coatings to that upon potato appear on boiled white of egg, .and on blood-serum a greyish-white, thin, coherent film develops below the surface and sends out long thin pro- -cesses downwards and parallel to it. The serum is neither liquefied nor discoloured (fig. 88). [Microsporon furfur is found in the yellowish or brownish scaly patches on the skin in pit t/ri axis rcrsicolor. When some of the scales are examined under the microscope in a 236 BACTERIOLOGY drop of potash solution, abundant mycelial threads are seen, together with spores, which are gathered in groups resembling clusters of grapes. Cultivation on solid media has not yet succeeded.] — NOTE BY TRANSLATOR. Certain observers, most recently M. Sabouraud, believe that Trichophyton tonsurans really includes several distinct varieties. The latter states as the result of an extended research that there are two principal kinds, distinguished by the size and arrangement of their spores and by their bio- logical characters. The first, called by him Trichophyton microsporon, has spores 3//, in diameter which lie in no- particular order, and is apparently destitute of mycelium, It occurs only in tinea tonsurans, causing about 60 per cent, of the cases, and is most difficult to treat, On beer-wort and agar it produces a growth which is at first white and downy, but from the fifteenth to the eighteenth day becomes dry, floury, and yellowish ; on potato it is dry and yellowish- brown from the first. The other variety, Trichophyton macro- or megalosporon, which causes all the remaining forms of tinea, is distinguished by spores Ip to 8/j, in diameter and arranged in lines in the branches of its mycelium. Cultures show a white downy growth, which appears a little later than in the case of the former variety, and does not alter. On potato a reddish- brown spot, like dried blood, appears ten days before the white down develops. The macrosporon variety, however,, seems to include many species distinguished by further dif- ferences in growth and producing distinct clinical phenomena. One such causes about half the cases of tinea circinata, the others are rare. The above-named varieties always reproduced the same species, and the former could not be made to grow on any part of the body except on the head. (See Ann. de Derm, et de Syph., vol. iii. Nov. 1892 ; also Brit. Journ. of Derm., vol. v. Jan. 1893, and Med. Week, vol. i. Feb. 24, 1893.)— Tn. CHAPTER XI THE ORGANS AND CAVITIES OF THE BODY AND THEIK CONTENTS (continual) II. THE DIGESTIVE TRACT The Camty of the Mouth Micro-organisms of the mouth and their examination.— A number of micro-organisms are found in the mouth, which are derived from the air and are taken into the digestive tract with the food. Their occurrence in the buccal cavity is so constant that in each examination of saliva cocci, bacilli, spirilla, and other fungi are encountered in large numbers. When saliva is freely smeared over the surface of a cover-glass, allowed to dry, fixed in the flame, and stained in a dilute alcoholic solution of an aniline dye, micro-organisms will be found which belong in part to the month fnn49 gelatine is very rapidly liquefied. Round yellow colonies with bulging edges form on the gelatine plate, being larger on the surface than in depth. In thrust-cultures the upper part of the liquefied mass is coloured brick-red, and on agar there develops a brick-red deposit with white edges. Serum is liquefied, sometimes with, sometimes without, pigmentation. On potato a brick-red deposit forms on the place of inoculation. Pigmentation depends upon the access of air. This bacillus is distinguished from the Bacillus prodiyitHHix, to which it has otherwise considerable resem- blance, by its pathogenic action on animals, which it kills with the symptoms of a severe gastro-enteritis. The Intestine Intestinal micro-organisms. — Passing from the stomach along with the chyme, the micro-organisms reach the internal surface of the intestine, an occurrence which, how- ever, only becomes possible from the fact that certain of them are capable of preserving their vitality notwithstanding the gastric juice, or that the reaction and other qualities of the juice itself are not such as to exert a prejudicial influence on the microbes. The internal surface of the intestinal mucous membrane contains very many micro-organisms, and seems as a rule to afford a very favourable environment for them, while the contents of the gut, whether liquid or firm, show microbes in such abundance even under normal •conditions that of all parts of the body they are richest in such organisms. The alkaline reaction of the intestinal secretion and the length of time which food takes to pass through the intes- tinal canal are important factors in favour of the growth of micro-organisms. Duclaux ascribes to them a certain amount of digestive significance, on the ground that their activity is capable of assisting the physiological digestive 250 BACTERIOLOGY function1, and he believes that the duty has fallen to them of carrying on a ' digestion bacterienne ' by their vital action. Under normal conditions the microbes lie on the inner surface of the intestine, but in pathological processes the parasites penetrate between the epithelia of the villi, thence into the interior of the villi, and eventually through the intestinal parietes to the peritoneum. Articles of food which contain bacteria increase the number of colonies obtained from the contents of the gut to a very considerable extent, while the ingestion of sterile food-stuffs or of red wine causes, according to Arnold, the appearance of a striking diminution in the number of germs. According to Duclaux, only those bacteria can develop luxuriantly in the intestine which have no stringent need of oxygen ; but Abelous sets up the hypothesis that although the oxygen necessary for the existence of aerobes occurring in this situation can, in point of fact, reach the intes- tine with the saliva, the intestinal micro-organisms have the power of accommodating themselves to a deficiency of it. Most of the bacteria living upon the mucous membrane are saprophytes ; the pathogenic varieties are present only in small numbers, and often perish by being overgrown by the former, while many pass away in the faeces. According to Nothnagel, a very large number of yeast- cells are found in the contents of the gut, so much so that Brefeld seeks the normal habitat of yeast in the large intestine. Micrococcus aerogenes. — Miller found in the digestive tract fairly large non-motile cocci of oval outline, which are distinguished for their marked power of resisting acids y and hence do not lose their vitality in acid digestive juices. Gelatine is slightly liquefied. The colonies on plate-cultures BACILLUS COPROGENES FCETIDUS 251 are round, and have on their surface several specks, which under a low power appear sometimes light and sometimes dark. In thrust-cultures the growth takes the form of a nail, and is of a greyish-yellow colour. A yellowish-white deposit develops upon a gar ; that on potato shows irregular prominences. Generation of gas takes place in the presence of carbohydrates. Bacillus putrificus coli. — Bienstock found a micro-organ- ism in the contents of the intestine which has the power of decomposing albuminous substances with formation of am- monia, a process which takes place whether air is excluded or is present. It consists of short motile rods, the growth of which does not liquefy gelatine. A yellowish coating develops upon agar after some time. Bacillus coprogenes foetidus.— In the intestine of pigs attacked by f/v/x/'/Wfl*, Schottelius very often found short immotile rods with rounded ends, which play no part in the erysipelas, but can very readily make their way into the blood and neighbouring organs in consequence of the intestinal ulcers. Gelatine is not liquefied, and small pale yellow islets develop in it, which coalesce and form a grey transparent coating. The culture gives off a very unpleasant smell. Bacterium Zopfi. — Kurt and Fliigge discovered in the intestine of chickens actively motile bacilli, the growth of which does not liquefy gelatine. Threads resembling mycelium appear on the plate even in a day, and in thrust- cultures abundant filaments pass out from the needle track, extending in a radial direction, but often crossing each other. No growth takes place on serum. The bacilli thrive best at a temperature between 20° and 30° C. ; higher than this their vitality is reduced. Bacterium aerogenes, Helicobacterium aerogenes, and Bacil- lus aerogenes. — These micro-organisms were found by Miller 252 BACTERIOLOGY in the intestinal tract. They are motile bacilli which do not liquefy gelatine by their growth, possess the peculiarity of offering resistance to moderately concentrated acids, and hence are able to pass through the stomach without injury to their vitality. Bacillus dysenteriae. — Chantemesse and Widal discovered short rods with rounded angles and but scanty power of movement in the contents and walls of the intestines, as well as in the spleen and abdominal glands, in cases of dysentery. They take up the aniline colours badly, and do not liquefy gelatine. On the plate there develop at first small white specks, which assume a yellow colour and unite with the neighbouring islets ; but in some days the yellow colour vanishes, and the colonies become white and granular. A dry yellow membrane develops on potatoes.1 Bacillus of fowl cholera. — Poultry typhoid ox fowl cholera is an epidemic disease accompanied by diarrhoea, often occurring amongst poultry, and which Perroncito first in- investigated more thoroughly. Pasteur describes it as ' cholera des poules,' and more recently Marchiafava, Celli, and Kitt have been occupied in bacteriological research concerning it. In the blood, in the capillaries of all the various organs, particularly the spleen and liver, and in especial abundance in the intestinal contents, there are found short plump rods which are somewhat constricted in the centre. A typical attack of the disease can be caused in hens by inoculation with these or by feeding the fowl with them, and other birds also, such as geese and pigeons, are very susceptible of infection, as are, moreover, mice and rabbits. Guinea- pigs, however, prove refractory. The growth of the bacilli 1 [Many cases of dysentery are, however, believed to be due to the action of a protozoon, named by Losch Amcsba coli, by Councilman and Lafleur Amoeba dysenteries, for an account of which the reader is referred to the Appendix.] — TB. BACILLUS OF FOWL CHOLERA 253 does not liquefy gelatine, and the colonies on the plate are roundish with uneven edges. When grown in thrust- cultures a delicate film appears on the surface, while a slight whitish-grey coating develops on agar. A dirty yellow7 fur appears on potato only at incubation temperature, and a shining whitish deposit upon serum. A characteristic point in the staining of these microbes is that the poles only are coloured by aniline dyes, the centre remaining unstained. They discharge the colour when treated by Gram's process. Other intestinal bacteria. — Besides the above, the Bacillus butyrirns, Vibrio rmjttla, and Bacterium coll commune are met with in the intestinal canal, as well as the cholera bacillus, typhoid bacillus, Vibrio proteus (Bacillus Finkler- Prior), Bacillus neapolitanus (Bacillus Emmerich), Proteus mlf/aris. Vibrio MrtxrJntikojft, and in many cases the thrush- fun a us. [Various protozoa have also been described.] 254 BACTERIOLOGY CHAPTEE XII THE ORGANS AND CAVITIES OF THE BODY AND THEIR CONTENTS (continued) III. THE F^CES AND URINE The Faces Composition and modes of examining. — The faeces must at this point be dealt with as a supplement to the study of the intestine, and they are very rich in micro-organisms. When the stools are examined we find constituents derived from the vegetable matters eaten, and remnants of un- digested pieces of animal food, especially larger or smaller pieces of striped muscle fibre, which show the transverse striation distinctly. Fat often occurs in normal stools in the form of droplets, and, in addition to these constituents, epithelial cells are also found (usually cylindrical), red and white blood-corpuscles, masses of detritus, crystals, and under pathological conditions still other appearances. Cer- tain varieties of bacteria can be detected in the meconium of new-born infants, as well as in the evacuations of adults. According to Nothnagel, micrococci are found in preponde- rating numbers in firm, and bacilli in fluid, stools. In the examination of faeces, forms are found almost constantly which stain blue with tincture of iodine, and which Nothnagel considers to be identical with the Clos- tridium butyricum. Other micro-organisms also assume a blue colour under application of tincture of iodine ; for example, Von Jaksch found rods in the faeces which recall REGAL MICRO-ORGANISMS 255 the appearance of Liptothrix bucculis. The number of microbes which stain with iodine is found to be particularly large in intestinal catarrhs. Fischer succeeded in growing the Lsptothrix fibres artificially. Furthermore, the different micro-organisms which live in the intestinal tract are also to be found in the faeces. When observing the faeces it is important to examine a prnall quantity in the hanging drop, when, besides the micro-organisms, a copious quantity of desquamated cylindrical epithelial cells are found. Even in such an investigation as this it is very often seen that, in the case of the majority of the different micro-organisms described up to this point, pure cultures of one or other are present, the other microbes either having been suppressed altogether or being present in such small numbers that no attention need be paid to them. Hence it happens, for example, that, besides the cylindrical epithelium, nothing but actively motile cholera bacilli are encountered on examining the rice-water stools of that disease. Yeast cells are constantly found in the faeces, especially in the acid stools of children (summer diarrhoea), and in acute catarrh of the small intestine in adults. These cells stain brown with iodine, which Von Jaksch connects with the glycogen they contain. Bacillus subtilis appears with tolerable frequency in normal as well as pathological alvine evacuations ; and besides these, in diseased conditions of the intestines the respective micro-organisms are met with in the faeces —for instance, the bacilli of cholera, typhoid fever, and tubcr- < ulnxis. Netter was also able to isolate the Staphylococcus pyogenes from the faeces.1 1 [Lambl, Losch, Koch, Pfeiffer, Kartulis, and others have also described protozoa occurring in the fasces in health as well as under various patho- logical conditions.] — TR. 256 BACTERIOLOGY Bacillus subtiliformis. — Bienstock found, as a constant inhabitant of human faeces, a bacillus the morphological characters of which strongly resemble those of Bacillus subtilis, except that motility is wanting and the rods are always connected in long filaments. A luxuriant fatty coating of a whitish-yellow colour develops upon agar. When spores form, the rods are distended in the centre to a spindle shape. Bacillus albuminis. — The same observer very often en- countered a micro-organism in faeces, which possesses the power of energetically decomposing albumin, and to which he gave, in consequence, the name Bacillus albuminis. The rods are fairly long and show a marked motility, that part of the bacillus from which the spore divides off being always foremost during movement. A whitish layer, with the lustre of mother-of-pearl, develops on agar. Bacillus cavicida. — Brieger isolated from faeces and putrid substances a bacillus which has the property of de- composing saccharine solutions and generating propionic acid. The rods are short, being but twice as long as their diameter, and liquefy gelatine into a viscid fluid. On plates there develop white colonies with beautiful concentric rings resembling the scales upon the back of a tortoise. A dirty yellow deposit forms on potatoes and on serum. Subcutaneous injections have an extremely poisonous action upon guinea-pigs, often causing death even within a few hours. Micrococcus tetragenus concentricus. — The author found in the liquid evacuations of a person suffering from gastric dilatation cocci which were commonly united in tetrad form. The elements are small, round and motile, stain readily with all the different aniline colours, and thrive upon the various nutrient media at present used in bacteriological research. Gelatine is not liquefied. On the plate several MICROCOCCUS TETRAGENUS CONCENTRICUS 257 round islets appear, which when inoculation has been exten- sive coalesce with one another, and by confluence of several Concentrically stratified superficial growth Concentric stratification in a fis- sure in the gelatine Deep portion of the needle-track PIG. 92.— THBffST-OULTURE IN GELATINH OP MlCROCOCCCS TETRAGENUS CONCENTRICUS. a larger roundish compound colony is formed, resembling a holoblastic ovum in process of segmentation. Thrust-cul- s 258 BACTERIOLOGY tures show a superficial deposit possessing concentric rings, which may be described as diurnal rings, since a difference exists between the growth in the dark and that in daylight, which expresses itself in this form (fig. 92). If the culture be kept in the dark the con- centric circles on the upper surface disappear. On agar there appears a deposit showing scroll-like tracings, and on potato growth takes place on the site of inoculation (fig. 93.) The microbe cannot be found to possess any pathogenic properties, subcutaneous inocula- tions upon mice having produced no results. The Urine. Micro-organisms of the urine. — In the normal state no micro-organisms are found in urine when freshly passed ; those which appear in it reach it from without, or are de- rived from diseased conditions of the urinary passages. If, however, the urine stands for some time until alkaline fer- mentation sets in, a multitude of yeasts, moulds, cocci, and bacilli make their appearance, which, reaching the urine probably from the air, and finding there a favourable environ- ment, set up a fermentation of the urinary sugar and cause a splitting up of the urea into ammonium carbonate ; in fact, an entire series of processes taking place in the urine are dependent on the vital activity of micro-organisms. A true bacteriuria has also been described in different quarters (Eoberts, Schottelius, Fischer), which appears to be of a morbid nature : in pathological processes in the different organs, but especially when bacteria get into the blood and therewith also into the renal circulation, bacilli may penetrate into the urine during its secretion and be expelled with it. URINARY MICROORGANISMS 259 In a case of endocarditis, Weichselbaum found the specific micro-organisms in the urine, and Von Jaksch detected in eri/xipclas abundant streptococci which he identified with the Strt'jttnmrrHa ]n/ofji'ni'n ; while in typhoid /;•/•>•/• Neumann found the corresponding micro-organisms in the urine, and ijlandt'i-x biu-HIi and tnli-rcl' lactlli have also been dis- covered in it. In other diseases, too, the respective bacteria are found in the urine — for instance, the bacillus of ant lira.r or the spirillum of relapsing f ever. In gonorrhceal processes gonococci are found in it, and in suppurative processes con- siderable numbers of staphylococci ; and tubercle bacilli appear in s-shaped groups (see fig. 81) in tubercular ulcerative disease of the urinary passages. The process of centrifuging is especially to be recommended when ex- amining the urine for micro-organisms. Bacteriuria may also be caused by the conveyance of micro-organisms into the urethra in the introduction of in- struments, when they may set up a decomposition of the urine in the interior of the bladder. A number of different micrococci found in the air, in water, and in the soil are also met with in the urine ; these include the Micrococcus urea and various species of sarcina. The majority of the micro-organisms occurring in urine are bacilli. Yeasts and moulds in urine. — The occurrence of any considerable number of yeast -cells is in all probability de- pendent upon the urine being rich in sugar. Moulds, too, only appear in saccharine urine, and then not until the alcoholic fermentation of the sugar has come to an end. Urobacteria.— A number of bacteria have the property of converting urea into ammonium carbonate. That most frequently found in the normal urine during fermentation is the Micrococcus urea (Pasteur, Van Tieghem, Leu be), which is a constant inhabitant of the atmosphere, and has s 2 260 BACTERIOLOGY consequently been already described in the chapter on the 'Bacteriological Analysis of Air.' It is usually found in longish chains upon the surface of fermenting urine (see fig. 34, p. 107). Lundstroem found that two kinds of staphylococcus are also endowed with this property, viz. the Staphylococcus ureae candidus and the Staphylococcus ureae liquefaciens. The former develops shining white deposits upon the gelatine without liquefying it, the latter liquefies it. Miquel encountered amongst the water bacteria numerous varieties possessing the same power. They are for the most part bacilli, only a few being micrococci ; amongst the latter are found three which liquefy gelatine. According to Leube, a variety of sarcina is also to be included with these micro-organisms. The Micrococcus ureae liquefaciens described by the last- named observer forms small chains and liquefies gelatine slowly. Leube isolated a Bacillus ureae from ammoniacal urine, consisting of short stout rods, the growth of which does not liquefy gelatine. The colonies on the gelatine plate are at first very transparent ; their coalescence gives the gela- tine on that spot the appearance of a slab of ground glass. The margins are indented in consequence of irregularity of growth. In thrust-cultures also grey processes appear, ex- tending out from the needle-track, and these grow at room temperature. Of other micro-organisms which decompose urea the Urobacillus Freudenreichii and the Urobacillus Maddoxii should also be mentioned. The former grows best on gela- tine at 20° C., forming a milk-white coating on the surface, while deeper a cavity filled with a turbid stringy fluid develops. Liquefaction of the gelatine progresses slowly, and the liquefied region gradually clears, a white slimy MICROCOCCUS OCHROLEUCUS 261 mass with the smell of ammonia settling down into the deeper part. If some urine he added to the gelatine a coronet of very fine crystals forms after some days around the white colonies. It also grows excellently upon bouillon and upon urine. The Vrnb.i-ii renders urine viscid and stringy. Gelatine is not liquefied, bouillon is very quickly rendered turbid, and in this case also crystals form round the cultures in gelatine mixed with urine. Micrococcus ochroleucus. — Prove found cocci in normal urine which are possessed of a niotility particularly marked when they are united in chains, and Legrain met with them also in the pus of uri'thritis. They are distinguished by the formation of endogenous spores, which progresses best at 36° C. The gelatine is not liquefied. After even one day colonies with raised edges appear upon the plate, and these later on become yellow and push out processes. In thrust-cultures a sulphur-yellow pigment forms upon the surface, which is soluble in alcohol and is destroyed by acids. A dirty white, creamy layer develops on agar, in which the inoculated streak is prominent as a yellow stripe. On potato there forms a warty elevation of a yellow colour. All cultures diffuse an intense sulphurous odour. Streptococcus giganteus urethrae. — Lustgarten and Manna- berg have discovered in normal urine and in the human urethra large cocci arranged in rows, which form wavy lines and often dense coils. No growth takes place on gelatine, and even on agar it is slow, and best, moreover, at incubation temperature. Besides these, the same observers described four varieties of bacilli and seven of cocci as constant inhabitants of the urethra, amongst them the Sfaphylococcu* pyoycncs aurcus and Micrococcus subfiavns. 262 BACTERIOLOGY Bacterium sulphureum. — Eosenheim found different bacteria in urine which form sulphuretted hydrogen therein and do not liquefy gelatine. Holschewnikoff isolated a micro-organism from mud, which generates the same gas, and is also, although rarely, to be met with in urine. It consists of fine rods with rounded ends, and endowed with a slow motility. Their development causes liquefaction of the gelatine. Upon plate-cultures there form small punctate colonies, which sink inwards in funnel form during liquefaction. In thrust-cultures the superficial colonies are white, the deep reddish-brown. A viscid grey layer forms rapidly on agar ; but on potato development takes place only when air is excluded, the growth being reddish-brown. Bacillus septicus vesicae. — In persons suffering from cystitis and pyelonephritis Clado found, amongst numerous other microbes, an organism showing motile rods, mostly isolated, and which develop ovoid spores. They take up aniline stains readily, and do not discharge their colour under Gram's process. Gelatine is not liquefied. Upon the plate there develop punctiform colonies, which, however, do not go beyond a pin's head in size. In thrust-cultures the deeply-seated colonies appear larger than the superficial, and the latter unite to form a delicate opalescent film. On agar there develops a delicate coating, and upon this minute circular shining milk-white colonies grow. Both gelatine and agar quickly become alkaline. Growth in bouillon is particularly abundant, and a dry, light-brown layer develops upon potato. ITrobacillus liquefaciens. — Schnitzler has found a bacillus in cystitis to which the above name has been given, and which is apparently identical with the Urobacillns liquefa- ciens septicus discovered by Krogius in cystitis and pyelo- nephritis. It consists of short motile rods rounded at the UROBACILLUS LIQUEFACIENS 263 ends, which give up the stain when treated by Gram's method. Gelatine is rapidly liquefied. The plate-culture shows in the centre of the liquefied colony a nodule of the size of hemp-seed, with fringed edges. There develops a thick greyish-white unwrinkled coating upon agar even in one day. On potato a brownish-yellow layer forms. The cultures generate ammonia and smell like decomposed urine. This bacillus seems to be nearly akin to the Proteus rulijaris (see p. 175). The Staphylococcus pyogenes albus and aureus are also very often found in cystitis. 264 BACTERIOLOGY CHAPTEK XIII THE OEGANS AND CAVITIES OF THE BODY AND THEIR CONTENTS (continued) IV. BACTERIOLOGICAL EXAMINATION OF THE BESPIRATORY TRACT; AND (V.) OF THE BLOOD The Nasal Secretion Micro-organisms in the nasal secretion. — The micro- organisms occurring in the vicinity of men and animals may very easily penetrate into the respiratory passages along with the current of air. They first reach the cavi- ties of the nose, and find in the alkaline viscid mucus there a medium possessing the conditions most suitable for their development. The secretion of the nose contains epithelial cells, both pavement and ciliated, leucocytes, and even in the normal state a considerable number of micro- organisms— cocci, bacilli, and spirilla. Under pathological conditions the nasal mucus becomes thin, and more pus- corpuscles are found, as well as the characteristic micro- organisms in definite local morbid conditions, the kinds of microbe depending upon the variety of disease. . Micrococcus cumulatus tenuis. — Yon Besser isolated from the normal nasal mucus rather large non-motile cocci, the growth of which does not liquefy gelatine. Upon agar a development of raised colonies appears, showing a thin zone with ragged edges around a central nucleus. Pota- toes are not favourable for its growth. Mirococcus tetrageirus subflavus shows non -motile cocci DIPLOCOCCUS COKY/.K 265 arranged in fours, and was discovered by Von Besser in the normal mucus of the nose. No growth takes place upon gelatine, but it develops well on agar and potato. The older cultures have a yellowish-brown colour. Micrococcns nasalis. — Hack encountered in the naso- pharyngeal cavity motile diplococci, the growth of which does not liquefy gelatine. The islets on the gelatine plate show a small undulating excavation in the centre, and in individual colonies it is easy to recognise a radiating arrangement or coiled form, frequently with a rayed margin. A concentric stratification of the superficial layer is often seen in thrust-cultures, together with development of nodules along the thrust-canal. A greyish-white shining deposit forms upon agar and potatoes. Diplococcus coryzae. — This diplococcus was described by Hajek, who found it in the nasal secretion. It consists of cocci which are somewhat elongated, so as to look like short bacilli. Gelatine is not liquefied by them. Flat superficial-lying colonies develop on the gelatine plate, and on thrust-cultures a deposit forms, which is at first raised but becomes continually more and more flattened out, a fact which serves to distinguish the culture of Diplococcus <-<>ri/-(C even by the naked eye from Friedliinder's Pncumo- la cill us, since no alteration takes place in the nail-culture formed by the latter. A coating develops upon agar. This diplococcus was at first regarded as the exciting cause of those changes in the mucous membrane which are characteristic of coryza. Experiments on animals, however, have yielded results which are negative in this respect. Staphylococcus cereus aureus. — This microbe resembles the Staphylococcus cereus flarus, only differing from it in the orange-red colour of its colonies, which is particularly marked in thrust-cultures. It was discovered by Von Schrotter and F. Winkler in the author's Institute, in the thin secretion 266 B ACTERIOLOG Y of a cold in the head, and in association with the Staphylo- coccus cereus flams (see fig. 76). Bacillus fcetidus ozaenae. — Hajek found in the secretion of patients suffering from oz&na short actively motile rods, during the growth of which the gelatine is liquefied. Round- ish colonies form at first upon the gelatine plate, which appear sunken like pock-marks while liquefaction is be- ginning, but later become confluent and liquefy the entire gelatine. In thrust-cultures the liquefaction shows itself both superficially and along the track of the needle, and if the culture is kept at 37° C. a strong odour of putrefaction is developed, which is not the case at the ordinary temperature. Upon agar a superficial coating forms, and in like manner becomes foul-smelling at incuba- tion temperature. A brownish deposit forms oh potato. If animals be injected subcutaneously with the cultures a violent inflammation results, in the course of which the tissues become necrosed. The bacilli completely discharge their colour when treated by Gram's method. Hajek particularly recommends staining with alkaline methyl blue to which some aniline water has been added. Diluted alcoholic solutions stain the bacilli only very slightly. Bacillus striatus albus et flavus. — Both bacilli were met with by Von Besser in the normal nasal mucus, but the Bacillus striatus albus is very rare. Gelatine is not liquefied. A fairly good growth appears on the different nutrient media. Upon potato a streaky layer forms. Bacillus striatus flavus develops a sulphur-yellow pigment. Bacillus of rhinoscleroma. — The bacilli of rhinoscleroma are short rods rounded at the ends, which are devoid of power of automatic movement and are enclosed in capsules. They are found in rhinoscleroma in the tissue of the tumours and in the juice from them, and are believed to be the cause of this form of disease. Yon Frisch, Paltauf, Yon Eiselsberg, BACILLUS OF RHINOSCLEROMA 267 Dittrich, Cornil, Alvarez and several other investigators have made researches concerning this micro-organism. The best solution to use for staining sections of the rhinoscleroma tumours when it is desired to examine them for the presence of the bacilli, is the methyl blue or methyl violet solution of Loftier. The sections remain one or two days in the stain, are then washed in water con- taining iodine, and finally decolorised for a rather long time (two or three days) in absolute alcohol. The bacilli do not lose their colour under Gram's process. If they be stained with gentian violet in aniline water, washed in water containing acetic acid, and double-stained with carbolic fuchsine, the capsules round the bacilli appear coloured; safranine may be used with advantage for double- staining instead of the carbolic fuchsine. Gelatine is not liquefied. Roundish colonies appear on the gelatine plate, and thrust-cultures take the nail form, but do not attain any very extensive development. Upon agar a widespread milk-white coating appears even in half a day, on potato there likewise forms a creamy mass which gradually spreads out from the site of inoculation, and on blood-serum also a white coat develops. In all these methods of cultivation the capsule is retained even in the later stages of development, but it is lost in bouillon cultures. Inocula- tions do not produce rhinoscleroma, even when the injections are made into the nasal mucous membrane, but according to Stepanow tumours of granulation- tissue develop when one is made into the eye of a guinea-pig, and these tumours are rich in bacilli. Injections into the pleura of these animals resulted in death. The bacillus of rhinoscleroma seems to be at least closely akin to Friedlander's PneumobaciUus, but is less virulent. The points of difference with reference to their morphology which might be cited would perhaps be the greater trans- 268 BACTERIOLOGY parency of gelatine cultures of the former, and the persistence of capsules on most of the culture media. Bacillus capsulatus mucosus. — At the Institute of Professor Klemensiewicz in Graz, Fasching discovered, in crusts removed from the naso-pharynx of a case in which this cavity was diseased, a micro-organism with short and rather thick rods lying singly or in pairs or fours in a common enveloping capsule. The bacilli give up the dye under Gram's process. It is easy to bring out the capsule successfully as a delicate rose-tinted area by protracted staining of the prepared cover-glass infuchsine, or by slightly warming after covering it with the stain. The bacilli are destitute of motility, and do not liquefy gelatine. On the plate colonies develop which have the appearance of drops of mucus of the size of pins' heads. In thrust-cultures there forms a typical nail-like figure, with active generation of gas, and upon agar and serum a thick moist creamy layer develops. A moist, viscid, white coating occurs on potato. Subcutaneous injection causes a genuine septicaemia in white mice. Fasching also found this micro-organism in the sputum of a phthisical patient. Vibrio nasalis. — Both in the nasal mucus and in the buccal cavity rods of considerable size are found which possess no power of automatic motion, and are arranged as vibrios. They were cultivated pure by Weibel. Gelatine is not liquefied, and the growth on the plate leads very slowly to the formation of round islets. In thrust-cultures a delicate white streak develops along the thrust-canal, resembling a string of mucus. The culture on agar is less transparent and thicker, and spirilla are found in it which show a large number of bends (over' thirty). No growth takes place on potato. When treated by Gram's process the spirilla lose their colour. Other nasal bacteria. — Keimann isolated a great number MICROBES IN THE RESPIRATORY PASSAGES 269 of bacteria from the nasal mucus, amongst which also the Spirochate dentium is often to be met with. In tubercular diseases of the cavity of the nose the pathognomonic tubercle bacilli are found in the nasal mucus, in glanders ulcers the Bacillus mallei, and thrush futi'jns and moulds frequently occur in the secretion. [See p. 195.] The Respiratory PasK x Micro-organisms of the respiratory passages. — Micro- organisms may also be very easily conveyed from the air into the bronchi by respiration. Straus and Dubreuil have ascertained that the expired air is almost free from organisms, and even when germs do occur the proportion of those in expired to those in inspired air is very small (1 : 600). Tubercle bacilli produce infection with greater ease, as Cadeac and Molet found, when the particles of dust to which they adhere are damp with aqueous vapour, than when they reach the lungs in a dry state. Owing to the discovery in recent years of a series of micro-organisms which excite morbid processes in the lungs, it is of very great importance to acquire a more intimate knowledge of the microbes occurring there, and this is most conveniently done by a thorough examination of the sputum. Certainly there are found in the sputum admixtures of matter from the naso-pharynx and the buccal and nasal cavities ; still, it is possible to exclude all the elements coming from other cavities, and to retain only those derived from the lungs. Pansini constantly found different varieties of strepto- cocci in the sputum both of healthy and diseased individuals, and next to these in frequency, various kinds of sarcina. A yellow, reddish, or green colour in sputum is caused by chromogenic bacteria. Pansini derives the yellow and reddish colour from the vital activity of Bacillus aureus, 270 BACTERIOLOGY Sarcina lutea, and Sarcina aurantiaca, while the green colour is caused by the Bacillus pyocyanens and Bacillus fluorescens non-liquefaciens. Virchow and Lichtheim not rarely found moulds in the sputum, and amongst these particularly Aspergillus fumigatus. The fungus of thrush and yeast cells may also be frequently met with, and moreover, Fasching found here his Bacillus capsulatiis mucosus. Sarcina pulmonum. — Even in earlier years different authors have described sarcinae in the sputum which they considered identical with Sarcina ventriculi. By the re- searches of Hauser, however, it was brought out that a special variety has here to be dealt with, distinguished at once from the last-named by its smaller size. No pathogenic properties can be ascribed to it, but it pos- sesses the faculty of energetically decomposing urine, a power shared by it with a sarcina discovered in that ex- cretion by Leube. Gelatine is not liquefied, and on the plate small white colonies develop which appear indented at the margin and concentrically stratified during their growth. A superficial moist coating forms in thrust- cultures. Upon potatoes the growth is very scanty. Sarcina aurea. — Mace isolated from the lung of a patient, who had died of pneumonia with purulent pie tiritis, a variety of sarcina, the elements of which possess a very lively oscillating motility. Some of them stain bluish- violet with iodine and sulphuric acid, a reaction indicating the presence of cellulose or starch in the sarcina. They develop a beautiful golden-yellow pigment which is soluble in absolute alcohol, and liquefy gelatine with tolerable rapidity. In thrust-cultures a thick friable membrane, which very readily falls apart into separate pieces, develops upon the surface of the funnel-shaped area of liquefaction ; on agar there forms a thick streak with warty surface and of a shining PNEUMOCOCCUS '2 7 1 golden-yellow colour, while a thick golden -yellow layer grows on potato. Diplococcus pneumoniae. — Klebs and Eberth long ago pointed out the presence of cocci in croupons pneumonia, while A. Frankel and Weichselbaum have studied them with the help of the modern methods of research, and ascertained that the cause of this affection is a micrococcus which Weich- selbaum detected in ninety-one cases out of a hundred. These cocci are round or sometimes elongated, and possess a jelly-like envelope of varying thickness. They are very often found arranged in pairs, and it is not unusua for several such diplococci to be connected in rows, one behind the other, and enclosed in a common cap- sule (fig. 94). Motility is absent. These cocci occur in croupous pneumonia not only in the sputum and in the diseased lung, but also Fu, 94._MiruoBES op pjmjMl)NIA in the blood, and they are met with in different other diseases. According to Weich- selbaum they are found in exudations in the cavum tympani and in the ethmoidal labyrinth, and Foa and Bordoni believe them to be the sole cause of cerebro-spinal meningitis. Klein, Biondi, A. Frankel and Miller found them also in the buccal and naso-pharyngeal cavities of healthy individuals, so that they can exist as it were in the portals of entrance to the respiratory system. They are iden- tical with the microbes of xpntuni septiccemia. Emmerich found them in the dust of a room occupied by pneumonic patients. Their growth begins to make good progress at a tern- 272 BACTERIOLOGY perature of 24° C., and gives the best results at incubation heat. On the gelatine plate there form roundish colonies of medium size, and in thrust-cultures little white granules appear along the needle-track, and a slight transparent prominence on the surface. The gelatine is not liquefied. A thin transparent film forms upon agar, and a thin transparent slimy coating upon serum. The cultures do not last very long, and lose their virulence if kept at a temperature of 42° C. even for one or two days. The same result is also attained, according to Frankel, by cultivation in milk. In order to preserve the virulence of the cocci, they must be inoculated from time to time upon animals. The diplococci cultivated upon artificial nutrient media show no capsules, and after losing them they become regu- larly round and range themselves in chains, in consequence of which they have been described by Garnaleia as Strepto- coccus lanceolatus. They exhibit capsules in the blood of animals infected with the cultures. The cocci stain in dilute alcoholic solutions of the aniline dyes, and can easily be displayed in preparations coloured by Gram's method, differing therein from the Pnenrnobacillus of Friedlander. The capsules remain un- stained by the ordinary methods : to colour them Kibbert employs a hot saturated solution of the capsules of dahlia violet in 100 parts water, 50 parts alcohol, and 12i parts acetic acid. Staining takes place very rapidly in this solution, and moreover it is necessary to wash in water only for a short time. The cocci appear dark blue, the capsules light blue. For transmission of pure cultures those on bouillon are the most suitable, and of these one or two cubic centimeters are used as a hypodermic injection. The cocci with their capsules are found in the blood and organs, but subcuta- neous injections fail to set up inflammatory symptoms in 1-TJ.EI) LADDER'S I'XKfMOBACILLUS 273 the lungs. When the pleura is infected, however, inflam- mations of it and the lungs do occur ; and when pure cultures are introduced into the trachea of rabbits a pneu- monia with all the characteristic symptoms follows. Pneumobacillus Friedlaenderi. — Friedlander found a micro-organism in the expectoration and in the lung- tissue, the elements of which are rods of different sizes, lying singly or joined together in pairs or bands. They possess a capsule in the form of a transparent surround- ing area, but this is wanting in artificial cultures. The bacilli are immotile. In contrast to the 7>/y>/m-mv//.s- jnn-ii- inonife, the pneumobacilli discharge the dye under Gram's process. They also grow at a lower temperature than the former. Gelatine is not liquefied. Upon the plate there appear roundish, sharply-defined colonies of granular texture, and in thrust-cultures a thick porcelain-like prominence forms on the surface, and the growth rapidly advances along the thrust, so that there appears the distinct figure of a nail — the ' nail-i'Hltnw ' (fig. 95). Older cultures become brownish. Upon agar a dense deposit forms, and a thick yellowish moist coating upon potato. Bubbles of gas are often seen in the cultures. Infections of mice by the hypodermic method speedily result in death, but guinea-pigs are less susceptible. Sub-pleural or infra-pulmonary injections set up a pneumonia. Croupout pneumonia seems, however, to be due to the pneumobacillus only in a small proportion of cases (9 times in 100 cases), according to Weichselbaum and C. Friinkel. Micrococcus tetragenus. — Koch and Gaffky found in the contents of a tubercular cavity small immotile cocci, which were, as a rule, united with one another in fours and surrounded by a capsule. They, are also found in the sputum, and are very frequent, according to Kar- 274 BACTERIOLOGY linski, in dental abscesses. Gelatine is not liquefied, and white punctate colonies form on the plate and display a Mi Superficial elevation -Needle-track Needle-track FIG. 95.— THRUST-CULTURE ix GELATINE OP FRIEDLANDER'S PXEUMOBACILLUS (' NAIL-CULTURE '). FIG. 9C.— THRUST-CULTURE IN ( : ML.VTIXE <>K MICROCOCCUS TETRAfiExus. (After Macfe.) shining, porcelain-like appearance on the surface of the medium. Isolated sharply-defined colonies appear along BACILLUS TFSSIS CONVULSIVE 275 the canal in thrust-cultures, lying one above the other like a pile of discs, the most superficial of which projects in a hemispherical form above the surface (fig. 96). Upon agar and serum, also, round well-defined colonies develop along the inoculated streak, and upon potato a slimy coating forms. White mice die within four weeks after infection. Bacillus aureus. — The elements of this bacillus are short rods showing but slight motility. They were found in water by Adametz, and also upon the human skin in some forms of eczema by Unna and Tommasoli. Gelatine is not liquefied. Upon the plate there develop punctiform colonies, which assume a yellow colour and become uneven, and in the thrust-culture a dark yellow deposit forms upon the surface. Their growth on potato takes the form of shining hemispheres, which coalesce and assume a colour varying from dark yellow to brownish-red. Tubercle bacillus and Actinomyces. — The micro-organism to which Koch gave the name of ' tnln-rrl,> ln («il< rIlusrnm conttKjiosiun, lying partly free and partly in the interior of horny cells, are held by many to be coccidia, and to be the exciting cause of the diseased growth. Others, however, believe them to be merely degenerated epithelial elements. Bodies of a similar nature have also been described (by Darier, Wickham, and others) in the form of eczema of the nipple known as Payees disease, which is associated with cancer of the mammary ducts, but Buffer and Walker 1 are of opinion that many at least of the appearances are degenerating epithelial cells, or due to endogenous cell formation. Wernicke has described coccidia in the granulomatous tumours of mycosis j'nnti<>i Apparatus, list of, 20 ; counting, 125 ; plate, 28, 55 ; spray, 93 Aquatilis, Bac., 136 ; Mic., 128 — radiatus, Bac., 137 — sulcatus, Bac., 136 Arborescens, Bac., 131 Arthrospores, 5 Ascobatillus citrcus, 231 Aspergillinew, 10 Aspergilhis albus, flavcsccns, fumiga- tus, glaucus, niger, 193 Attenuated vaccine, Haffkine's, 285 Attenuation, 6 Aurantiaca, Sarc., 108 Aurantiacus, Bac., 135 — Mic., 129 Aurea, Sarc., 270 Aureus, Bac., 135, 275 BACILLI, 2 Bacteria, varieties of, 2 ; multiplica- tion of, 4 Bacteriuria, 258 Basidia, 11, 103 BAI-MEYER, petroleum incubator, 26 JUrMGARTEN, staining of tubercle ba- cilli, 215 Beggiatoa, 12 Birds, tuberculosis in, 209 Biskra, Mic., 221 Blood, micro-organisms in, 276 ; modes of examining, 276 ; staining of, 277 — serum. See Serum 304 BACTERIOLOGY Borax methyl blue, method, 90 Bougies, cesophageal, 94 Bouillon, 35 ; meat, 36 ; meat extract, 37 Bread, 53 ; moulds on, 193 BBIEGER, extraction of toxines, 7 ' Brownian movement,' 3 Bruneus, Bac., 135 Buccalis maximus, Bac., 238 Butyri fluorescens, Bac., 183 — viscosus, Bac., 183 Butyricus, Bac., 182 CADAVERINE, 148 Candicans, Mic., 106 Candida, sore., 108 Capsulatus, Bac., 3, 221 — mucosus, Bac., 3, 268 Capsules, 3 Carcinoma, protozoa in, 282, 290 Carmine, alum, 302 Carneus, Mic., 129 Carragheen moss, nutrient jelly from, 47 Caucasicus, Bac., 188 Cavicida, Bac., 256 Celloidine, imbedding in, 82 Cells, inclusions in, 290 — epithelial, proliferation of, caused by coccidia, 287 Centrifuges. See Machines, centri- fugal Cereus, Stapn.,albus, &ndflavus, 199 ; aureus, 201, 264 Ceremsice, Sarc., 192 ' Chamber-land's candles,' 123 'Chameleon -phenomenon,' 199 CHENZYNSKY'S solution, 86, 278 Cholera, inoculation of , 94, 144; vacci- nation against, 283 ; reaction, 144, 147 Cholera Nostras, Bac. of, 144, 145 Cholera-red, 147 Cholerce Asiatics, Bac., Vibrio, or Spi- rillum, 139 ; effect of light on, 296 ; toxopeptone from, 7 Choline, 148 Cilia. See Flagella Cinabareus, Mic., 106 Citreus conglomerate, Dipl., 107 — liquefaciens, Dipl., 226 Cladothrix, 12 ; C. dichotoma, 13 ; C. canis, 197 Clearing agents, 85 Clostridium, 4, 159 ; C. butyricum, 181, 254 ; C.fcetidum, 159 Cocci, 2 Coccidia, 12, 295 Coccidium oviforme, 286 Coli commune, Bac., 152 ; effect of light on, 296 Colonies on gelatine plate, 56 Coloured images, 68 — light, action of, on micro-organ- isms, 297 Columella, 10 Comma-bacilli, 2, 140, 146 Concentricum, Spir., 176 Concentricus, Mic., 129 Condensation, water of, 45 Conidia, 11, 103 Conjugation, reproduction by, 194 Coprogenes fcetidus, Bac., 251 Coryza, Dipl., 265 — equorum contagiosce, Mic., 221 Counting apparatus, Wolffhiigel's, 125- Crates, wire, for test-tubes, 29 Crenoihrix Kitliniana, 11 Cultivation, methods of, 53-64 Cultures, anaerobic, 60 ; ' hanging drop,' 62 ; high, 62 ; permanent, 63 ^ plate, Koch's, 54 ; plate, serum and albumen, 60 ; roll, Esmarch's, 58 ; slide, 53 ; streak or surface, 57, 60 ; thrust, 57, 62 Cumulatus tennis, Mic., 264 > Cyanogenus, Bac., 185 1 Cystitis, Bac. in, 262 i DECOLORISING agents, 72 Decomposition, 173 Dehydration, 85 DENEKE, Bac., 146, 183 , Denticola, 242 Dentium, Spiroch&te, 242 | Diet, articles of, 178, 189 Digestive tract, the, 237; infection by, 93 Diphtheria, Bac., 240 Diplococci, 2, 4 Discontinuous sterilisation, 18 Disinfectants, chemical, 18 j Dispora Caucasica, 188 Double test-glasses, Schill's, 16 Drum-stick bacteria, 5 Drying-on processes, Unna's, 85 ; Kiihne's, Weigert's, 89 ; Winkler's,. 203 Dust, window and room, 156 Dysenteric, Amaba, 252, 288 - Bac., 252 EARTH, analysis of, 155 — bacillus, 156 INDEX 305 EBERTH, bacillus of typhoid fever, 148 Exogenous organisms, 1 Ectoplasm, 171 Eczema marginatum, 238 Eggs, birds', hens', 49, 50 ; plov< > EHBUCH-BlOMDl triple stain, 2'.i:> KHKI.K H. acid Ita'inatoxyline. :>"•_» Kmmerirli, I lac.. 120 KMMKKICH, method of air-analy-i-. '.t'.i Kndoi-di-ditis cliniy, 184 Fervidosus, Mic., 12'.i l-'i. watei -liartcria, 122 Frer/ing method, 711 ; in gum, 301 Fi:ii,i'i..\M>i:i;, pneumobacillus, 273; stain, 213 l-'ruchtliypheii, 103 Furuncles, staining of, '.m Fusctts limbatus, Bac., 174 — Mic., 128 , method of staining tubercle bacilli, 7-',, -2\ \ •'oi'inniix, l:'2 Hollowed slides, 29 Hot-air steriliser, 15 Hot-water filtering funnel, 27 Hyphffi, 10 Hyphomycetw, 10 lanthinus, Bac., 138 Imbedding, 81 ; in celloidine, 82 ; in glycerine jelly, 81 ; in gum arabic, 81 ; in paraffine, 83 Immunity, 6 Impetigo contagiosa, 233 Impression preparations, 78 Incubators, 20 ; petroleum, Bau- meyer's, 26 Indicus, Bac., 248 Indigoferus, Bac., 137 Indigogenus, Bac., 191 Infection, artificial, 92 ; by air-pas- sages, 93 ; by digestive canal, 93 ; into eye, 95 ; intraperitoneal, 94, 284 ; intravenous, 94 ; subcutaneous, 94 Influenza, Bac. of, 2, 278 Inoculation. See Infection Inspissator, serum, Koch's, 47, 48 Intestine, micro-organisms of, 249, 253 Iodine method, 89 lodococcus magnus, I. parvus, I. vagi- natus, 238 Islets, on plate cultures, 56 KAATZER, staining of tubercle bacilli, 211 Kephir, 188 Knob-moulds, 10 KOCH, comma-bacillus, 139 ; plate process, 54 ; serum-inspissator, 47 ; stain, 86 ; steam steriliser, 16 ; sy- ringe, 31 ; tubercle bacillus, 206 ; tu- berculine, 7, 8, 218 KOCH-EHRLJCH method of staining tubercle bacilli, 73 KUHNE, method of staining tubercle bacilli in sputum, 213 ; methyl blue, 86 Lactcus faviformis, Mic., 225 Lacticua, Bac., 179 Lactis aerogenes, Bac., 248 — erythrogenes, Bacter., 186 -fcstidus, Bac., 185 — pituitosi, Bac., 185 — viscosus, Bac., 184 j ' Laveran's sickles,' 171 Lepra, Bac. of, 229 - cells, 229 Leptothrix buccalis, 238, 25.") Levelling stand, for plate cultures, 28 Light, action of, upon micro-organisms, 295 ; coloured, 297 ; white, 295 ; electric, 297 ; mode of action of, 298 ; natural precautions against, 299 Liodermos, Bac., 117 Liquefaciens magnus, Bac., 159 Litmus agar, 45 ; gelatine, 41 LOFFLEK, diphtheria bacillus, 240 ; methyl blue, 86, 241, 277 LOSCH, amoeba of, 288 Lutea, sarc., 109 Luteus, Dipt., 130 - Mic., 128 MACHINES, centrifugal, 31 ; Csokor's, 34 ; Gartner's, 33 ; Stenbeck's, 31 Malaria, Plasmodium, 169 Malignant pustule, 169 Malleus, 218. See Glanders Mastitis, bovine, Mic. of, 187 Measurement, standards of, 65 Media, fixing, 84 — nutrient, 35 ; liquid, 35 ; solid, 37 ; turbidity in, 39 Megaterium, Bac., 189 Melanaemia, 170 Melanine, 170 Melochloros, Bac., 118 Membranaceus amethystinus, Bac., 137 Meningitidis intracellularis, Mic., 221 — 2)urulentcs, Mic., 221 Meningitis, pneumococcus in, 271 Merismopedia, 4 Mesentericus fuscus, ruber, mdcjatus, Bac., 116 Metabolism in bacteria, products of, 5 ; methods of extracting products (Brieger, Scholl, Koemer, Buchner, Koch), 7, 8, 218 Methyl blue, borax, Unna's, 90 ; car- bolic, 88; andeosine (Chenzynsky's), 86, 278 ; Kiihne's, 86 ; Loffler's, 86, 241, 277 ; Plehn's, 87 Metsclmikoffi, Vibrio, 147 IXDKX 307 Micra. Sec Micro-millimeters Micro-burners, 21 Micro-membrane filter, 123 Micro-millimeters, 65 Micro-organisms, classification of, 1,2; examination of, 12 ; examination in fresh state and hanging drop, 65 ; in sections of tissue, 78; M. in disease, 92 ; isolation of, 57 ; by Cohen's me- thod, 127 ; staining of, 67 et seq. ; transmission of, to animals, 91 .1 / icrosjioron fn rfn >\ 2 M ~. Milk, methods of examining, 171) ; micro-organisms in, 188 Milk-rice, 52 :! Miller. Spirillum. I'll' Minn ..i., method of air-analysis, 98 Moist chambers, 28, 56 Molecular movement, 3 Molluscum contagiosum, protozoa in, 295 M on ilia Candida, '2\'.'> Monococci, 2 M(»rd;mts. r.'.i • Mother-of- vinegar,' 191 Mould, brown. KM. l-'.t; Moulds, 1), 193 ; pathogenesis of , 194-5 Mouse septicaemia, 280 Mouth, the, micro-organisms of, 237, 244 Mucor corymbifcr, 194; M. mucedo, '.I, 111 J ; M. raniosus, M. rhizopodi- formis, 194 Mucorinete, 10, 193 Multipcdiculosus, Bac., 118 Multiplication, modes of, 4, 194 M/irisepticus, Bac., 280 Mycelium, 10, 103, 233 Mt/coderma albicans, 243 Mycoides, Bac., 156 Mycosis fungoides, coccidia in, 295 NAIL, linger, 223 • Nail-culture,' 186, 248, 273 Nasal bacteria, 264, 268 ; secretion, 264 Nasalis, Mic., 265 - Vibrio, 268 Neapolitan-its, Bac., 120, 147 NICOLLE, method of staining, 302 N trails, Bac., 130 XIIMKWK /, method of staining glan- ders bacilli, 220 Ocliraceus, Bac., 138 Ochroleucus, Mic., 261 (Edematis maligni, Bac., 160 Oiidiaceae, 11 Outturn albicans, 243 - lactis, 10, 181 Onychomycosis, 223, 233 Organs and cavities of body, 222 ) Osteomyelitidis, Mic., 221 , Otomycosis, 195 1 Ozaena, 266 1'A. KKT-COCCI, 4 FACET'S disease, protozoa in, 295 Pap, bread, 53 ; potato, 52 Parafnne, imbedding in, 83 Parasites, 1, 92, 286 Pediococcus cerevisia;, 191 Pellicles, 4 Pemphigus acutus, Dipl., 227 Pencil mould, 11 ' Pende's ulcer,' 221 Penicilliaceae, 11, 103, 193 Penicillium glaucum, 10, 103 • Perlsucht,' 178 Permanent cultures, 63 PETRI, method of air-analysis, 102 ; capsules, 28, 58 Phlogosine, 113 Phosphorescence, 6, 133 Phosphor escens, Bac., 132; indigenus, 132 ; indicus, 133 Picrocarmine, 302 Pigment-forming bacteria, 5 ; effect of light on, 295, 299 Plasmodia, 12, 169, 282 Plate process, Koch's, 54; apparatus for, 28, 55; modifications of, 58, 59, 61 Platinum wires, 30 Plugs, cotton-wool, 16 Pneumobacillus Friedlcendcri, 8, 273 Pneumonifc, Diploc., 271 Pneumosepticus, Bac., 275 Polymorphic bacteria, 197 Potato bacillus, 116 Potatoes, preparation of, for culture- media, 51 Poultry typhoid, 252 Prodigiosus, Bac., 114 Proteines, 7 Proteus, 174 ; P. capsulatus, P. ho- minis,P. mirabilis,P. Zcnkeri, 175 ; P. vulgaris, 175, 181 Protozoa, 12; in blood, 282; in car- cinoma, 290 ; in molluscuni conta- giosum, 295 ; in mycosis fungoides, 295; in Paget's disease, 295; in sarcoma, 294 ; parasitic, 286 ; patho- genesis of, 286 ; staining of, 293. See also under Amoeba, Coccidium, Plasmodium, &c. x 2 308 BACTERIOLOGY Pseudodiphtheritic Bac., 242 Pseudonavicella, 12 Pseudopodia, 12 Psorosperms, 12 Ptomaines, 6, 173 Pulmonum, Sarc., 270 Pus, 196; microbes of, 196, 220; staining of, 221 et passim Putrefactive processes, differences in, 173 Putrefying substances, analysis of, 173 Putrescine, 148 Putrificus coli, Bac., 251 Pyocyaneus, Bac., 8 ; a and £, 198-9, 223 ; effect of light on, 296 Pyocyanine, 199 Pyogenes, Staph., 109 ; aureus, 110 ; albus, citreus, 112 - Strept., 113, 201 Radiatm, Bac., 158 ; Mic., 105 ' Bag-picker's disease,' 169 Ramosus, Bac., 133 ' Kauschbrand,' 228 ' Kay fungus,' 196. See Actinomyces Keagents, bacteriological, 29 Eespiratory passages, micro organisms of, 269 ; E. tract, 264 ; infection through, 93 Khinoscleroma, Bac. of, 266 Rice, milk, 52 Ringworm, 233 Roll cultures, Esmarch's, 58 Root bacillus, 133 Rosea, Sarc., 109, 187 Rosens, Mic., 107 Rubrum, Spi-r., 176 Rugula, Vibrio, 242 RUSSELL, method of staining, 293 Saccharomyces cerevisice, 11, 105 ; S. ruber, 188 Saffranine, staining with, 301 Saliva, 237, 238 Salivarius septicus, Bac., 238 ; Mic., 238 Saprogenes, Bac., I., II., III., 175 Saprophytes, 1, 92 Sarcina, 2 ; S. alba, S. aurantiaca, 108 ; S. aurea, 270 ; S. Candida, 108 ; S. cerevisice, 192 ; S. lutea, 109 ; S. pulmonum, 270 ; S. rosea, 109, 187 ; S. ventriculi, 246 Sarcoma, protozoa in, 294 Schizomycetes, 2, 4 Scissus, Bac., 159 Section-stainer, 82 Sections, cutting of, 79 et seq., 301 ; examination of micro-organisms in, 68 ; staining of, 84 Sedimentation, methods of, 127, 216 Seed-organ, in moulds, 10 Segmentation, 170 Septicaemia, mouse, 280 ; puerperal, 238 Septicus, Bac., 160 — vesicce, Bac., 262 Serial preparations, 83, 84 Serum, blood, 47 ; modifications of, 48 ; sterilisation of, 19, 47 Skin, abscesses in, staining of, 90 ; micro-organisms of, 222 ; modes of staining, 223-4 Small-pox, protozoa in, 282 Smear preparations, 67 Smegma Bac., 206 Soil, examination of, 156 ; filtering action of, 156 ; micro-organisms of, 155, 172 ' Soorpilz,' 243. See Thrush-fungus SOYKA'S plates, 29 Spectrum, experiments with, 297 Sphcerococcus acidi lactici, 181 ' Spider cells,' 148-9 Spinosus, Bac., 159 Spirilla, 2 ; atmospheric, 121 ; water, 153 Spiritus saponatus kalinus, 91 Spirochffitas, 2 Spirochcete dentium, 242 — Obermeieri, 281 Sporangium, 10 Spores, 4, 9, 103, 189, 291 ; effect of light on, 297, 299 ; staining of, 71 ; swarm, 12 ; terminal, 5, 163 Sporocysts, 12 Sporozoa, 12, 287 Sporulation, 4, 9, 291 ; arthrogenous, 5 ; endogenous, 5 Spring-water, 124 Sputum, 269 ; staining of, for tubercle bacilli, 210 ; pure cultures of tu- bercle bacilli from, 207 Staining, 67, 301 ; combination, 86 ; cover-glass preparations, 67 ; sec- tions, 84. See also under Tubercle bacillus, Gonococcus, &c. &c. — processes, Arens, 76, 214 ; Ehrlich, 74; Ehrlich-Biondi, 293; Frankel, Gabbet, 75 ; Gibbes, 215 ; Gram, 76 ; Giinther, 74, 212, 277; Koch and Ehrlich, 73, 210 ; Loffler, 86, 241, INDEX 309 277 ; Nicolle, 302 ; Pittion, Pfuhl and IVtri, 7~>; \\Virlisclbaum, 7-") ; Weigert, 78, 80 ; Ziehl-Neelsen, 74, 213 Stains, 30; preparation of, ('»'.» Staphyloc-occi, '2, 4, 109 Staphylococctu cercus, 199 ; S. pyo- gencs, 100 Steam generator (Budenberg's), 17 — steriliser, Koch's. HI Sterigraata, 11, 103 Sterilisation, 15 ; by chemical agents, 18 ; by combined methods, 19 ; by heat, 15 ; by steam, 16, 18 ; discon- tinuous or fractional, 18 ; of hands, •20 ; of instruments, 19 Steriliser, hot-air, 15 ; Koch's steam, 16 Stomach, micro-organisms of the, 245 STKAI >s and WURX, method of ana- lysing air, 101 Streptococci, 2, 4, 113 Streptococcus crysipelatis, 113 ; S. gi- gantcus urethra, 261 ; S. pyogcnes, 113, 201 ; S. septicus, 165 ti trial us albus, Bac., S. flavus, Bac., 266 Sti-uctural images, 68 Styrone method, 'I'l \ Subflavus, Dipl., 'I'l \ Subtiliformis, Bac., 256 Subtilis, Bac., 113 Subungual space, micro-organisms in, 298 Sulphureum, Bact., 262 Sulphydrogenus, Bac., 138 Sweat, 223 Swine erysipelas, Bac. of, 279 Sycosis, 231. 233 Sycosiferus fcetidus, Bac., 231 Symbiosis, 164 Symptomatic anthrax, Bac. of, 228 Syncyanus, Bac., 185 Syphilis, Bac., 205 Syringes, hypodermic, 31 TABLET-COCCI, 4 Temperatures, 21 Test-tubes, charging of, 40 Tetanus Bac., 163 Tetracocci, 2, 4 Tetragemts, Mic., 273 — concentricus, Mic., 256 — mobilis ventriculi, Mic., 248 — subflavus, Mic., 264 Texas fever, 282 Thallus, 10 Thermo-regulators, 21 ; Altmann's, 2"> ; Bunst-n's. 21 ; (fiirtner's, L'-'I ; Meyer's, 24; Schcnk's, 22 Thrush-fungus, 213 Tinea circinata, T. sycosis, 233; T. tonsurans, 232 ; T. versicolor, 234, 235 Tissues, the, influence of bacteria on, 6 Toxalbumins, 6 Toxines, 6 Toxopeptone, 7 Trachoma, Mic. of, 227 Transmission, 91 ; by air-passages, 93; digestive canal, 93; skin. III ; peritoneum, 94 Trichophyton macrosporon (mcgalo- sporon), T.microsporon, 236 ; T. ton- surans, 223, 232, 236 ' Trouser-leg culture,' 145 Tuberculine, Koch's, 7, 8, 218 Tuberculocidine, 218 Tuberculosis, Bac., 206, 275; action of light on, 296 ; in birds, 209 ; pre- paring pure cultures of, 207-9 ; staining of, 73, 210 ; in sections, 217 Tubular buds, in moulds, 10 Tussis convulsive, Bac., 275 Tympanum, micro-organisms of, 244 TYNDALL, method of air-analysis, 102 ; of sterilisation, 18 Typhoid fever, Bac. of, 148; action of light on, 296 Typhus abdominalis, 148. See Typhoid Tyrogenum, Spirillum, 183 Ulna, Bac., 239 Undula, Spirillum, 153 UXXA, examination of skin for micro- organisms, 90, 224 Urea, Bac., 260 ; Mic., 107 — candidus, Staph., U. liquefaciens, Staph., 2(50 Urine, micro-organisms of, 258 ; yeasts and moulds in, 259 Urobacillus Freudenreichii, U. Mad- doxii, 260 Urobacteria, 259 VACCIXATION against cholera, Haff- kine's, 283 ; principle of, 283 ; re- sults of, 284 Vaccines, anti-cholera, preparation of, 283 ; attenuated, 285 ; exalted, 284 310 BACTERIOLOGY Vacuoles in cells, 12, 288 Vagina, micro-organisms of, 222-3 Vaginalis, Bac., 223, 228 Ventriculi, Sarcina, 246 Versicolor, Mic., 105 ' Vibrion septuple,' 160 Vibrios, 2, 4 • — aureus, 121 ; V. cholera, 139 ; F. flavescens, F. flavus, 121 ; F. .Mie£- schnikoffi, 147 ; F. proteus, 145 Vienna, water-supply of, 136 Vinegar ferment, 191 Violaceus, Bac., 132 Virulence, 6 Virus, exalted, 283 Viscosus, Mic., 192 — butyri, Bac., 183 ; F. cerevisice, Bac., 192 ; F. lactis, Bac., 184 ; F. sacchari, Bac., 193 Viticulosus, Mic., 106 WAFEKS, cultivation on, 53 WARD, researches on the action of light on bacteria, 297-300 Warm chamber, 20. 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